pFind Studio: a computational solution for mass spectrometry-based proteomics

Identification and mechanism of G protein-biased ligands for chemokine receptor CCR1

Nature chemical biology. 2022. Shao, ZH et al. Zhejiang Univ, MOE Frontier Sci Ctr Brain Res & Brain Machine In, Sch Med, Hangzhou, Peoples R China; Zhejiang Univ, Affiliated Hosp 2, Dept Pharmacol, Key Lab Resp Dis Zhejiang Prov,Sch Med, Hangzhou, Peoples R China; Zhejiang Univ, Dept Pathol, Sir Run Run Shaw Hosp, Sch Med, Hangzhou, Peoples R China; Zhejiang Univ, Affiliated Hosp 2, Dept Resp & Crit Care Med, Key Lab Resp Dis Zhejiang Prov,Sch Med, Hangzhou, Peoples R China; Zhejiang Prov Key Lab Immun & Inflammatory Dis, Hangzhou, Peoples R China; Zhejiang Univ, Key Lab Resp Dis Zhejiang Prov, Dept Resp & Crit Care Med, Affiliated Hosp 2,Sch Med, Hangzhou, Peoples R China; State Key Lab Resp Dis, Guangzhou, Peoples R China; Zhejiang Univ, Liangzhu Lab, Med Ctr, Hangzhou, Peoples R China; Zhejiang Univ, Int Inst Med, Sch Med, Affiliated Hosp 4, Yiwu, Peoples R China; Zhejiang Univ, Dept Biophys, Sir Run Run Shaw Hosp, Sch Med, Hangzhou, Peoples R China

ABSTRACT: Biased signaling of G protein-coupled receptors describes an ability of different ligands that preferentially activate an alternative downstream signaling pathway. In this work, we identified and characterized different N-terminal truncations of endogenous chemokine CCL15 as balanced or biased agonists targeting CCR1, and presented three cryogenic-electron microscopy structures of the CCR1-G(i) complex in the ligand-free form or bound to different CCL15 truncations with a resolution of 2.6-2.9 angstrom, illustrating the structural basis of natural biased signaling that initiates an inflammation response.

Complemented with pharmacological and computational studies, these structures revealed it was the conformational change of Tyr291 (Y291(7.)(43)) in CCR1 that triggered its polar network rearrangement in the orthosteric binding pocket and allosterically regulated the activation of beta-arrestin signaling. Our structure of CCL15-bound CCR1 also exhibited a critical site for ligand binding distinct from many other chemokine-receptor complexes, providing new insights into the mode of chemokine recognition.

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MStoCIRC: A powerful tool for downstream analysis of MS/MS data to predict translatable circRNAs

Frontiers in Molecular Biosciences. 2022. Cao, Zhou et al. Shaanxi Normal Univ, Coll Life Sci, Key Lab Minist Educ Med Plant Resource & Nat Pharm, Natl Engn Lab Resource Dev Endangered Crude Drugs, Xian, Peoples R China

ABSTRACT: CircRNAs are formed by a non-canonical splicing method and appear circular in nature. CircRNAs are widely distributed in organisms and have the features of time- and tissue-specific expressions. CircRNAs have attracted increasing interest from scientists because of their non-negligible effects on the growth and development of organisms. The translation capability of circRNAs is a novel and valuable direction in the functional research of circRNAs. To explore the translation potential of circRNAs, some progress has been made in both experimental identification and computational prediction.

For computational prediction, both CircCode and CircPro are ribosome profiling-based software applications for predicting translatable circRNAs, and the online databases riboCIRC and TransCirc analyze as many pieces of evidence as possible and list the predicted translatable circRNAs of high confidence. Simultaneously, mass spectrometry in proteomics is often recognized as an efficient method to support the identification of protein and peptide sequences from diverse complex templates. However, few applications fully utilize mass spectrometry to predict translatable circRNAs. Therefore, this research aims to build up a scientific analysis pipeline with two salient features: 1) it starts with the data analysis of raw tandem mass spectrometry data; and 2) it also incorporates other translation evidence such as IRES. The pipeline has been packaged into an analysis tool called mass spectrometry to translatable circRNAs (MStoCIRC). MStoCIRC is mainly implemented by Python3 language programming and could be downloaded from GitHub (). The tool contains a main program and several small, independent function modules, making it more multifunctional. MStoCIRC can process data efficiently and has obtained hundreds of translatable circRNAs in humans and Arabidopsis thaliana.

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A Hybrid Spectral Library and Protein Sequence Database Search Strategy for Bottom-Up and Top-Down Proteomic Data Analysis

Journal of Proteome Research. 2022. Dai, YL et al. Univ Wisconsin, Dept Chem, Madison, WI 53706 USA

ABSTRACT: Tandem mass spectrometry (MS/MS) is widely employed for the analysis of complex proteomic samples. While protein sequence database searching and spectral library searching are both well-established peptide identification methods, each has shortcomings. Protein sequence databases lack fragment peak intensity information, which can result in poor discrimination between correct and incorrect spectrum assignments. Spectral libraries usually contain fewer peptides than protein sequence databases, which limits the number of peptides that can be identified.

Notably, few post-translationally modified peptides are represented in spectral libraries. This is because few search engines can both identify a broad spectrum of PTMs and create corresponding spectral libraries. Also, programs that generate spectral libraries using deep learning approaches are not yet able to accurately predict spectra for the vast majority of PTMs. Here, we address these limitations through use of a hybrid search strategy that combines protein sequence database and spectral library searches to improve identification success rates and sensitivity. This software uses Global PTM Discovery (G-PTM-D) to produce spectral libraries for a wide variety of different PTMs. These features, along with a new spectrum annotation and visualization tool, have been integrated into the freely available and open-source search engine MetaMorpheus.

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High-throughput proteomic sample preparation using pressure cycling technology

Nature protocols. 2022. Cai, X et al. Westlake Univ, Sch Life Sci, Westlake Lab Life Sci & Biomed, Key Lab Struct Biol Zhejiang Prov, Hangzhou, Peoples R China; Westlake Inst Adv Study, Inst Basic Med Sci, Hangzhou, Peoples R China

ABSTRACT: High-throughput lysis and proteolytic digestion of biopsy-level tissue specimens is a major bottleneck for clinical proteomics. Here we describe a detailed protocol of pressure cycling technology (PCT)-assisted sample preparation for proteomic analysis of biopsy tissues. A piece of fresh frozen or formalin-fixed paraffin-embedded tissue weighing similar to 0.1-2 mg is placed in a 150 mu L pressure-resistant tube called a PCT-MicroTube with proper lysis buffer. After closing with a PCT-MicroPestle, a batch of 16 PCT-MicroTubes are placed in a Barocycler, which imposes oscillating pressure to the samples from one atmosphere to up to similar to 3,000 times atmospheric pressure.

The pressure cycling schemes are optimized for tissue lysis and protein digestion, and can be programmed in the Barocycler to allow reproducible, robust and efficient protein extraction and proteolysis digestion for mass spectrometry-based proteomics. This method allows effective preparation of not only fresh frozen and formalin-fixed paraffin-embedded tissue, but also cells, feces and tear strips. It takes similar to 3 h to process 16 samples in one batch. The resulting peptides can be analyzed by various mass spectrometry-based proteomics methods. We demonstrate the applications of this protocol with mouse kidney tissue and eight types of human tumors.

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Probing strigolactone perception mechanisms with rationally designed small-molecule agonists stimulating germination of root parasitic weeds

Nature communications. 2022. Wang, DW et al. Hunan Univ, State Key Lab Chemo Biosensing & Chemometr, Hunan Prov Key Lab Plant Funct Genom & Dev Regula, Coll Biol, Changsha 410082, Peoples R China; Nankai Univ, Collaborat Innovat Ctr Chem Sci & Engn, Natl Pesticide Engn Res Ctr, Coll Chem,Dept Chem Biol, Tianjin 300071, Peoples R China; Univ Amsterdam, Swammerdam Inst Life Sci SILS, Sci Pk 904, NL-1098 XH Amsterdam, Netherlands; Nankai Univ, State Key Lab Elementoorgan Chem, Collaborat Innovat Ctr Chem Sci & Engn, Coll Chem,Natl Pesticide Engn Res Ctr, Tianjin 300071, Peoples R China

ABSTRACT: The development of potent strigolactone (SL) agonists as suicidal germination inducers could be a useful strategy for controlling root parasitic weeds, but uncertainty about the SL perception mechanism impedes real progress. Here we describe small-molecule agonists that efficiently stimulate Phelipanchce aegyptiaca, and Striga hermonthica, germination in concentrations as low as 10(-8) to 10(-17) M. We show that full efficiency of synthetic SL agonists in triggering signaling through the Striga SL receptor, ShHTL7, depends on the receptor-catalyzed hydrolytic reaction of the agonists.

Additionally, we reveal that the stereochemistry of synthetic SL analogs affects the hydrolytic ability of ShHTL7 by influencing the probability of the privileged conformations of ShHTL7. Importantly, an alternative ShHTL7-mediated hydrolysis mechanism, proceeding via nucleophilic attack of the NE2 atom of H246 to the 2 ' C of the D-ring, is reported. Together, our findings provide insight into SL hydrolysis and structure-perception mechanisms, and potent suicide germination stimulants, which would contribute to the elimination of the noxious parasitic weeds.Strigolactone agonists could potentially help control noxious weeds by promoting suicidal germination. Here the authors describe a series of small molecule agonists that stimulate germination via the Striga ShHTL7 receptor and show that stereochemistry and hydrolysis-independent signalling mediate potency.

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Characterization of protein unfolding by fast cross-linking mass spectrometry using di-ortho-phthalaldehyde cross-linkers

Nature communications. 2022. Wang, JH et al. Natl Inst Biol Sci NIBS, Beijing 102206, Peoples R China; Tsinghua Univ, Tsinghua Inst Multidisciplinary Biomed Res, Beijing 102206, Peoples R China; Peking Univ, Coll Chem & Mol Engn, Peking Tsinghua Ctr Life Sci,Minist Educ, Beijing Natl Lab Mol Sci,Key Lab Bioorgan Chem &, Beijing 100871, Peoples R China; Chinese Acad Sci, Innovat Acad Precis Measurement Sci & Technol, Wuhan 430071, Peoples R China

ABSTRACT: Conformations sampled by a protein while it unfolds are difficult to visualize. Here, the authors develop di-ortho-phthalaldehyde cross-linkers for rapid chemical cross-linking mass spectrometry analysis and demonstrate that this method captures the conformations of protein unfolding intermediates.Chemical cross-linking of proteins coupled with mass spectrometry is widely used in protein structural analysis. In this study we develop a class of non-hydrolyzable amine-selective di-ortho-phthalaldehyde (DOPA) cross-linkers, one of which is called DOPA2.

Cross-linking of proteins with DOPA2 is 60-120 times faster than that with the N-hydroxysuccinimide ester cross-linker DSS. Compared with DSS cross-links, DOPA2 cross-links show better agreement with the crystal structures of tested proteins. More importantly, DOPA2 has unique advantages when working at low pH, low temperature, or in the presence of denaturants. Using staphylococcal nuclease, bovine serum albumin, and bovine pancreatic ribonuclease A, we demonstrate that DOPA2 cross-linking provides abundant spatial information about the conformations of progressively denatured forms of these proteins. Furthermore, DOPA2 cross-linking allows time-course analysis of protein conformational changes during denaturant-induced unfolding.

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Deep coverage proteome analysis of hair shaft for forensic individual identification

Forensic Science International: Genetics. 2022. Wu, JL et al. Chinese Acad Sci, Natl Chromatog Res & Anal Ctr, Dalian Inst Chem Phys, CAS Key Lab Separat Sci Analyt Chem, 457 Zhongshan Rd, Dalian 116023, Peoples R China; Peoples Publ Secur Univ China, Grad Sch, 1 Muxidi Nanli, Beijing 100038, Peoples R China; Inst Forens Sci, Natl Engn Lab Forens Sci, Key Lab Forens Genet, Minist Publ Secur, 17 Muxidi Nanli, Beijing 100038, Peoples R China

ABSTRACT: Hair shaft is one of the most common biological evidence found at crime scenes. However, due to the biogenic degradation of nuclear DNA in hair shaft, it is difficult to achieve individual identification through routine DNA analysis. In contrast, the proteins in hair shaft are stable and contain genetic polymorphisms in the form of single amino acid polymorphisms (SAPs), translated from non-synonymous single nucleotide polymorphisms (nsSNPs) in the genome. However, the number of SAPs detected still cannot meet the requirements of practical applications.

This paper developed a deep coverage proteome analysis method by combining a three-step sequential ionic liquid-based protein extraction and 2D-RPLC-MS/MS with high and low pH to identify both variant and reference SAPs from 2-cm-long hair shafts. We identified 632 +/- 243 protein groups from 10 individuals, with the average number of SAPs reaching 167 +/- 21/person. These were further used to calculate random match probabilities (RMPs), a widely accepted forensic statistical term for human identification. The RMPs ranged from 6.53 x 10(-4) to 3.10 x 10(-14) (median = 2.62 x 10(-8)) when calculated with frequency of matching nsSNP genotype data from exomes, and ranged from 2.62 x 10(-3) to 2.07 x 10(-10) (median = 4.88 x 10(-6)) with SAP genotype frequency. All these results indicate that the deep coverage proteomics method is beneficial for improving SAP-based forensic individual identification in hair shaft, with great potential in crime investigation.

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Efficient detection of the alternative spliced human proteome using translatome sequencing

Frontiers in Molecular Biosciences. 2022. Wu, Chun et al. Jinan Univ, Inst Life & Hlth Engn, MOE Key Lab Tumor Mol Biol, Guangzhou, Peoples R China; Jinan Univ, Key Lab Funct Prot Res Guangdong Higher Educ Inst, Inst Life & Hlth Engn, Guangzhou, Peoples R China

ABSTRACT: Alternative splicing (AS) isoforms create numerous proteoforms, expanding the complexity of the genome. Highly similar sequences, incomplete reference databases and the insufficient sequence coverage of mass spectrometry limit the identification of AS proteoforms. Here, we demonstrated full-length translating mRNAs (ribosome nascent-chain complex-bound mRNAs, RNC-mRNAs) sequencing (RNC-seq) strategy to sequence the entire translating mRNA using next-generation sequencing, including short-read and long-read technologies, to construct a protein database containing all translating AS isoforms.

Taking the advantage of read length, short-read RNC-seq identified up to 15,289 genes and 15,906 AS isoforms in a single human cell line, much more than the Ribo-seq. The single-molecule long-read RNC-seq supplemented 4,429 annotated AS isoforms that were not identified by short-read datasets, and 4,525 novel AS isoforms that were not included in the public databases. Using such RNC-seq-guided database, we identified 6,766 annotated protein isoforms and 50 novel protein isoforms in mass spectrometry datasets. These results demonstrated the potential of full-length RNC-seq in investigating the proteome of AS isoforms.

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Comprehensive identification of protein orthologs in the family Ascoviridae facilitates an understanding of phylogenomics, protein conservation, and phosphorylation

Archives of Virology. 2022. Shi, YH et al. Weifang Med Univ, Sch Life Sci & Technol, Weifang 261053, Peoples R China; Hunan Agr Univ, Hunan Prov Key Lab Biol & Control Plant Dis & Ins, Changsha 410128, Hunan, Peoples R China

ABSTRACT: Analysis of orthology is important for understanding protein conservation, function, and phylogenomics. In this study, we performed a comprehensive analysis of gene orthology in the family Ascoviridae based on identification of 366 protein homologue groups and phylogenetic analysis of 34 non-single-copy proteins. Our findings revealed 90 newly annotated proteins, five newly identified core proteins for the family Ascoviridae, and 14 core proteins for the genus Ascovirus. A phylogenomic tree of 11 Ascoviridae members was constructed based on a concatenation of 35 of the 45 ortholog groups.

In combination with phosphoproteomic results and conservation estimations, 30 conserved phosphorylation sites on 17 phosphoproteins were identified from a total of 176 phosphosites on 57 phosphoproteins from Heliothis virescens ascovirus 3h (HvAV-3h), providing potential research targets for investigating the role of these protein in the regulation of viral infection. This study will facilitate genome annotation and comparison of further Ascoviridae members as well as functional genomic investigations.

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A mechanism for oxidative damage repair at gene regulatory elements

Nature. 2022. Ray, S et al. Univ Sheffield, Hlth Lifespan & Neurosci Inst, Sheffield, S Yorkshire, England; Univ Bradford, Sch Pharm & Med Sci, Inst Canc Therapeut, Bradford, W Yorkshire, England; Univ Sheffield, Sch Biosci, Sheffield, S Yorkshire, England

ABSTRACT: Oxidative genome damage is an unavoidable consequence of cellular metabolism. It arises at gene regulatory elements by epigenetic demethylation during transcriptional activation(1,2). Here we show that promoters are protected from oxidative damage via a process mediated by the nuclear mitotic apparatus protein NuMA (also known as NUMA1). NuMA exhibits genomic occupancy approximately 100 bp around transcription start sites. It binds the initiating form of RNA polymerase II, pause-release factors and single-strand break repair (SSBR) components such as TDP1.

The binding is increased on chromatin following oxidative damage, and TDP1 enrichment at damaged chromatin is facilitated by NuMA. Depletion of NuMA increases oxidative damage at promoters. NuMA promotes transcription by limiting the polyADP-ribosylation of RNA polymerase II, increasing its availability and release from pausing at promoters. Metabolic labelling of nascent RNA identifies genes that depend on NuMA for transcription including immediate-early response genes. Complementation of NuMA-deficient cells with a mutant that mediates binding to SSBR, or a mitotic separation-of-function mutant, restores SSBR defects. These findings underscore the importance of oxidative DNA damage repair at gene regulatory elements and describe a process that fulfils this function.

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Mechanistic Insights into the Functioning of a Two-Subunit GMP Synthetase, an Allosterically Regulated, Ammonia Channeling Enzyme

BIOCHEMISTRY. 2022. Shivakumaraswamy, S et al. Jawaharlal Nehru Ctr Adv Sci Res, Mol Biol & Genet Unit, Bengaluru 560064, India

ABSTRACT: Guanosine 5 & PRIME;-monophosphate (GMP) synthetases, enzymes that catalyze the conversion of xanthosine 5 & PRIME;-monophosphate (XMP) to GMP, are composed of two different catalytic units, which are either two domains of a polypeptide chain or two subunits that associate to form a complex. The glutamine amidotransferase (GATase) unit hydrolyzes glutamine generating ammonia, and the ATP pyrophosphatase (ATPPase) unit catalyzes the formation of an AMP-XMP intermediate. The substrate-bound ATPPase allosterically activates GATase, and the ammonia thus generated is tunneled to the ATPPase active site where it reacts with AMP-XMP generating GMP.

In ammonia channeling enzymes reported thus far, a tight complex of the two subunits is observed, while the interaction of the two subunits of Methanocaldococcus jannaschii GMP synthetase (MjGMPS) is transient with the underlying mechanism of allostery and substrate channeling largely unclear. Here, we present a mechanistic model encompassing the various steps in the catalytic cycle of MjGMPS based on biochemical experiments, crystal structure, and cross-linking mass spectrometry guided integrative modeling. pH dependence of enzyme kinetics establishes that ammonia is tunneled across the subunits with the lifetime of the complex being & LE;0.5 s. The crystal structure of the XMP-bound ATPPase subunit reported herein highlights the role of conformationally dynamic loops in enabling catalysis. The structure of MjGMPS derived using restraints obtained from cross-linking mass spectrometry has enabled the visualization of subunit interactions that enable allostery under catalytic conditions. We integrate the results and propose a functional mechanism for MjGMPS detailing the various steps involved in catalysis.

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Itaconate and itaconate derivatives target JAK1 to suppress alternative activation of macrophages

Cell Metabolism. 2022. Runtsch, MC et al. Trinity Coll Dublin, Sch Biochem & Immunol, Trinity Biomed Sci Inst, 152-160 Pearse St, Dublin D02 R590, Ireland

ABSTRACT: The Krebs cycle-derived metabolite itaconate and its derivatives suppress the inflammatory response in pro-inflammatory "M1" macrophages. However, alternatively activated "M2" macrophages can take up itaconate. We therefore examined the effect of itaconate and 4-octyl itaconate (OI) on M2 macrophage activation. We demonstrate that itaconate and OI inhibit M2 polarization and metabolic remodeling. Examination of IL-4 signaling revealed inhibition of JAK1 and STAT6 phosphorylation by both itaconate and OI.

JAK1 activation was also inhibited by OI in response to IL-13, interferon-beta, and interferon-gamma in macrophages and in T helper 2 (Th2) cells. Importantly, JAK1 was directly modified by itaconate derivatives at multiple residues, including cysteines 715, 816, 943, and 1130. Itaconate and OI also inhibited JAK1 kinase activity. Finally, OI treatment suppressed M2 macrophage polarization and JAK1 phosphorylation in vivo. We therefore identify itaconate and OI as JAK1 inhibitors, suggesting a new strategy to inhibit JAK1 in M2 macrophage-driven diseases.

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AlphaPeptDeep: a modular deep learning framework to predict peptide properties for proteomics

Nature Communications. 2022. Zeng, Wen-Feng et al. Univ Copenhagen, Fac Hlth Sci, NNF Ctr Prot Res, Prote Program, Copenhagen, Denmark; Max Planck Inst Biochem, Dept Prote & Signal Transduct, Martinsried, Germany

ABSTRACT: Machine learning and in particular deep learning (DL) are increasingly important in mass spectrometry (MS)-based proteomics. Recent DL models can predict the retention time, ion mobility and fragment intensities of a peptide just from the amino acid sequence with good accuracy. However, DL is a very rapidly developing field with new neural network architectures frequently appearing, which are challenging to incorporate for proteomics researchers. Here we introduce AlphaPeptDeep, a modular Python framework built on the PyTorch DL library that learns and predicts the properties of peptides (https://github.com/MannLabs/alphapeptdeep).

It features a model shop that enables non-specialists to create models in just a few lines of code. AlphaPeptDeep represents post-translational modifications in a generic manner, even if only the chemical composition is known. Extensive use of transfer learning obviates the need for large data sets to refine models for particular experimental conditions. The AlphaPeptDeep models for predicting retention time, collisional cross sections and fragment intensities are at least on par with existing tools. Additional sequence-based properties can also be predicted by AlphaPeptDeep, as demonstrated with a HLA peptide prediction model to improve HLA peptide identification for data-independent acquisition (https://github.com/MannLabs/PeptDeep-HLA).

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Mucus sialylation determines intestinal host-commensal homeostasis

Cell. 2022. Yao, YK et al. NIAID, Mol Dev Immune Syst Sect, Lab Immune Syst Biol & Clin Genom Program, NIH, Bethesda, MD 20892 USA; NCI, Expt Immunol Branch, NIH, Bethesda, MD 20892 USA

ABSTRACT: Intestinal mucus forms the first line of defense against bacterial invasion while providing nutrition to support microbial symbiosis. How the host controls mucus barrier integrity and commensalism is unclear. We show that terminal sialylation of glycans on intestinal mucus by ST6GALNAC1 (ST6), the dominant sialyltransferase specifically expressed in goblet cells and induced by microbial pathogen-associated molecular patterns, is essential for mucus integrity and protecting against excessive bacterial proteolytic degradation.

Glycoproteomic profiling and biochemical analysis of ST6 mutations identified in patients show that decreased sialylation causes defective mucus proteins and congenital inflammatory bowel disease (IBD). Mice harboring a patient ST6 mutation have compromised mucus barriers, dysbiosis, and susceptibility to intestinal inflammation. Based on our understanding of the ST6 regulatory network, we show that treatment with sialylated mucin or a Foxo3 inhibitor can ameliorate IBD.

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HRS phosphorylation drives immunosuppressive exosome secretion and restricts CD8+ T-cell infiltration into tumors

Nature communications. 2022. Guan, L et al. Univ Penn, Sch Arts & Sci, Dept Biol, Philadelphia, PA 19104 USA

ABSTRACT: The lack of tumor infiltration by CD8(+) T cells is associated with poor patient response to anti-PD-1 therapy. Understanding how tumor infiltration is regulated is key to improving treatment efficacy. Here, we report that phosphorylation of HRS, a pivotal component of the ESCRT complex involved in exosome biogenesis, restricts tumor infiltration of cytolytic CD8(+) T cells. Following ERK-mediated phosphorylation, HRS interacts with and mediates the selective loading of PD-L1 to exosomes, which inhibits the migration of CD8(+) T cells into tumors.

In tissue samples from patients with melanoma, CD8(+) T cells are excluded from the regions where tumor cells contain high levels of phosphorylated HRS. In murine tumor models, overexpression of phosphorylated HRS increases resistance to anti-PD-1 treatment, whereas inhibition of HRS phosphorylation enhances treatment efficacy. Our study reveals a mechanism by which phosphorylation of HRS in tumor cells regulates anti-tumor immunity by inducing PD-L1(+) immunosuppressive exosomes, and suggests HRS phosphorylation blockade as a potential strategy to improve the efficacy of cancer immunotherapy.Lack of CD8(+) T-cell infiltration into solid tumors is associated with poor responsiveness to immune checkpoint therapy (ICT). Here, the authors show that blocking the phosphorylation of HRS to reduce the induction of immunosuppressive exosomes promotes CD8(+) T-cell infiltration into tumors and enhances the efficacy of ICT in mouse melanoma models.

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Glyco-Decipher enables glycan database-independent peptide matching and in-depth characterization of site-specific N-glycosylation

Nature Communications. 2022. Fang, Z et al. Chinese Acad Sci, Dalian Inst Chem Phys, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Peoples R China; Univ Chinese Acad Sci, Beijing 100049, Peoples R China; Dalian Univ Technol, Sch Bioengn, Dalian 116024, Peoples R China

ABSTRACT: Glycopeptides with unusual glycans or poor peptide backbone fragmentation in tandem mass spectrometry are unaccounted for in typical site-specific glycoproteomics analysis and thus remain unidentified. Here, we develop a glycoproteomics tool, Glyco-Decipher, to address these issues. Glyco-Decipher conducts glycan database-independent peptide matching and exploits the fragmentation pattern of shared peptide backbones in glycopeptides to improve the spectrum interpretation. We benchmark Glyco-Decipher on several large-scale datasets, demonstrating that it identifies more peptide-spectrum matches than Byonic, MSFragger-Glyco, StrucGP and pGlyco 3.0, with a 33.5%-178.5% increase in the number of identified glycopeptide spectra.

The database-independent and unbiased profiling of attached glycans enables the discovery of 164 modified glycans in mouse tissues, including glycans with chemical or biological modifications. By enabling in-depth characterization of site-specific protein glycosylation, Glyco-Decipher is a promising tool for advancing glycoproteomics analysis in biological research.Poor peptide fragmentation and unusual glycan structures limit mass spectrometry-based analysis of intact N-glycopeptides. Here, the authors develop Glyco-Decipher, a glycan-independent peptide search tool, to tackle these issues and improve the coverage of site-specific glycan analysis.

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Cryo-EM structure of the human CST–Polα/primase complex in a recruitment state

Nature Structural & Molecular Biology. 2022. Cai, Sarah W. et al. Rockefeller Univ, Lab Mol Electron Microscopy, New York, NY 10065 USA; Rockefeller Univ, Lab Cell Biol & Genet, New York, NY 10065 USA

ABSTRACT: The CST-Pol alpha/primase complex is essential for telomere maintenance and functions to counteract resection at double-strand breaks. We report a 4.6-angstrom resolution cryo-EM structure of human CST-Pol alpha/primase, captured prior to catalysis in a recruitment state stabilized by chemical cross-linking. Our structure reveals an evolutionarily conserved interaction between the C-terminal domain of the catalytic POLA1 subunit and an N-terminal expansion in metazoan CTC1. Cross-linking mass spectrometry and negative-stain EM analysis provide insight into CST binding by the flexible POLA1 N-terminus.

Finally, Coats plus syndrome disease mutations previously characterized to disrupt formation of the CST-Pol alpha/primase complex map to protein-protein interfaces observed in the recruitment state. Together, our results shed light on the architecture and stoichiometry of the metazoan fill-in machinery.

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Crucial role and mechanism of transcription-coupled DNA repair in bacteria

Nature. 2022. Bharati, BK et al. NYU, Sch Med, Dept Biochem & Mol Pharmacol, New York, NY 10012 USA; NYU, Sch Med, Howard Hughes Med Inst, New York, NY 10012 USA; Chinese Acad Sci, Inst Plant Physiol & Ecol, Ctr Excellence Mol Plant Sci, Key Lab Synthet Biol, Shanghai, Peoples R China

ABSTRACT: Transcription-coupled DNA repair (TCR) is presumed to be a minor sub-pathway of nucleotide excision repair (NER) in bacteria. Global genomic repair is thought to perform the bulk of repair independently of transcription. TCR is also believed to be mediated exclusively by Mfd-a DNA translocase of a marginal NER phenotype(1-3). Here we combined in cellulo cross-linking mass spectrometry with structural, biochemical and genetic approaches to map the interactions within the TCR complex (TCRC) and to determine the actual sequence of events that leads to NER in vivo.

We show that RNA polymerase (RNAP) serves as the primary sensor of DNA damage and acts as a platform for the recruitment of NER enzymes. UvrA and UvrD associate with RNAP continuously, forming a surveillance pre-TCRC. In response to DNA damage, pre-TCRC recruits a second UvrD monomer to form a helicase-competent UvrD dimer that promotes backtracking of the TCRC. The weakening of UvrD-RNAP interactions renders cells sensitive to genotoxic stress. TCRC then recruits a second UvrA molecule and UvrB to initiate the repair process. Contrary to the conventional view, we show that TCR accounts for the vast majority of chromosomal repair events; that is, TCR thoroughly dominates over global genomic repair. We also show that TCR is largely independent of Mfd. We propose that Mfd has an indirect role in this process: it participates in removing obstructive RNAPs in front of TCRCs and also in recovering TCRCs from backtracking after repair has been completed.

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Shelterin is a dimeric complex with extensive structural heterogeneity

PNAS. 2022. Zinder, John C. et al. Rockefeller Univ, Lab Cell Biol & Genet, New York, NY 10065 USA; Rockefeller Univ, Lab Mol Elect Microscopy, New York, NY 10065 USA

ABSTRACT: Human shelterin is a six-subunit complex-composed of TRF1, TRF2, Rap1, TIN2, TPP1, and POT1-that binds telomeres, protects them from the DNA-damage response, and regulates the maintenance of telomeric DNA. Although high-resolution structures have been generated of the individual structured domains within shelterin, the architecture and stoichiometry of the full complex are currently unknown. Here, we report the purification of shelterin subcomplexes and reconstitution of the entire complex using full-length, recombinant subunits.

By combining negative-stain electron microscopy (EM), cross-linking mass spectrometry (XLMS), AlphaFold modeling, mass photometry, and native mass spectrometry (MS), we obtain stoichiometries as well as domain-scale architectures of shelterin subcomplexes and determine that they feature extensive conformational heterogeneity. For POT1/TPP1 and POT1/TPP1/TIN2, we observe high variability in the positioning of the POT1 DNA-binding domain, the TPP1 oligonucleotide/oligosaccharide-binding (OB) fold, and the TIN2 TRFH domain with respect to the C-terminal domains of POT1. Truncation of unstructured linker regions in TIN2, TPP1, and POT1 did not reduce the conformational variability of the heterotrimer. Shelterin and TRF1-containing subcomplexes form fully dimeric stoichiometries, even in the absence of DNA substrates. Shelterin and its subcomplexes showed extensive conformational variability, regardless of the presence of DNA substrates. We conclude that shelterin adopts a multitude of conformations and argue that its unusual architectural variability is beneficial for its many functions at telomeres.

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Histone deacetylase 3 contributes to the antiviral innate immunity of macrophages by interacting with FOXK1 to regulate STAT1/2 transcription

Cell Reports. 2022. Yang, LP et al. Zhejiang Univ, Affiliated Hosp 2, Dept Infect Dis, Sch Med, Hangzhou 310009, Peoples R China; Chinese Acad Med Sci & Peking Union Med Coll, Inst Syst Med, Beijing 100005, Peoples R China; Suzhou Inst Syst Med, Suzhou 215123, Peoples R China; Univ Calif Los Angeles, Dept Microbiol Immunol & Mol Genet, Los Angeles, CA 90095 USA

ABSTRACT: It is well known that interferon (IFN)-alpha/-beta activates the JAK/STAT signaling pathway and suppresses viral replication through the induction of IFN stimulated genes (ISGs). Here, we report that knockout of HDAC3 from macrophages results in the decreased expression of STAT1 and STAT2, leading to defective antiviral immunity in cells and mice. Further studies show that HDAC3 interacts with a conserved transcription factor Forkhead Box K1 (FOXK1), co-localizes with FOXK1 at the promoter of STAT1 and STAT2, and is required for protecting FOXK1 from lysosomal system-mediated degradation, FOXK1 -deficient macrophages also show low STAT1 and STAT2 expression with defective responses to viruses.

Thus, our studies uncover the biological importance of HDAC3 in regulating the antiviral immunity of macrophages through interacting with FOXK1 to regulate the expression of STAT1 and STAT2.

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Protein posttranslational signatures identified in COVID-19 patient plasma

Frontiers in Cell and Developmental Biology. 2022. Vedula, Pavan et al. Univ Penn, Sch Vet Med, Dept Biomed Sci, Philadelphia, PA 19104 USA

ABSTRACT: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a highly contagious virus of the coronavirus family that causes coronavirus disease-19 (COVID-19) in humans and a number of animal species. COVID-19 has rapidly propagated in the world in the past 2 years, causing a global pandemic. Here, we performed proteomic analysis of plasma samples from COVID-19 patients compared to healthy control donors in an exploratory study to gain insights into protein-level changes in the patients caused by SARS-CoV-2 infection and to identify potential proteomic and posttranslational signatures of this disease.

Our results suggest a global change in protein processing and regulation that occurs in response to SARS-CoV-2, and the existence of a posttranslational COVID-19 signature that includes an elevation in threonine phosphorylation, a change in glycosylation, and a decrease in arginylation, an emerging posttranslational modification not previously implicated in infectious disease. This study provides a resource for COVID-19 researchers and, longer term, and will inform our understanding of this disease and its treatment.

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Iso-seco-tanapartholide activates Nrf2 signaling pathway through Keap1 modification and oligomerization to exert anti-inflammatory effects

Free Radical Biology and Medicine. 2022. Zhu, DR et al. China Pharmaceut Univ, Nanjing 210009, Peoples R China

ABSTRACT: Covalent modification of Keap1 results in reducing ubiquitination and the accumulation of Nrf2, which subsequently initiates the transcription of cellular anti-oxidant and anti-inflammatory genes. Iso-seco-tanapartholide (IST), a sesquiterpene isolated from the traditional Chinese medicine Artemisia argyi, had been reported to possess NF-Kappa B inhibitory activity. However, its deep anti-inflammatory effects and direct target have never been reported. Here we show that IST activated Nrf2 and increased its target gene expression.

In particular, LPScaused inflammation in vitro and in vivo was mitigated by IST-induced Nrf2 activation but aggravated by Nrf2 inhibition. Mechanically, IST targeted Keap1 proteins via alkylating its cysteine residues 151, 273, 288, and so on. Subsequently, the modifying agent IST was displaced by intermolecular sulfhydryl disulfide interchange to lead to a disulfide dimer of Keap1. The resulting conformational change of Keap1 liberated Nrf2 from sequestration and allowed it translocation to the nucleus to activate the transcriptional program. Further studies demonstrated that Keap1 dimer formation contributed to the anti-inflammatory effects of IST. Taken together, our findings reveal a new mechanism for Nrf2 activation and provide a potential lead compound to treat inflammatory diseases through targeting Keap1.

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Orally efficacious lead of the AVG inhibitor series targeting a dynamic interface in the respiratory syncytial virus polymerase

Science Advances. 2022. Sourimant, J et al. Georgia State Univ, Ctr Translat Antiviral Res, Inst Biomed Sci, Atlanta, GA 30303 USA

ABSTRACT: Respiratory syncytial virus (RSV) is a leading cause of lower respiratory infections in infants and the immuno-compromised, yet no efficient therapeutic exists. We have identified the AVG class of allosteric inhibitors of RSV RNA synthesis. Here, we demonstrate through biolayer interferometry and in vitro RNA-dependent RNA polymerase (RdRP) assays that AVG compounds bind to the viral polymerase, stalling the polymerase in initiation conformation. Resistance profiling revealed a unique escape pattern, suggesting a discrete docking pose.

Affinity mapping using photoreactive AVG analogs identified the interface of polymerase core, capping, and connector domains as a molecular target site. A first-generation lead showed nanomolar potency against RSV in human airway epithelium organoids but lacked in vivo efficacy. Docking pose-informed synthetic optimization generated orally efficacious AVG-388, which showed potent efficacy in the RSV mouse model when administered therapeutically. This study maps a druggable target in the RSV RdRP and establishes clinical potential of the AVG chemotype against RSV disease.

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Deephos: predicted spectral database search for TMT-labeled phosphopeptides and its false discovery rate estimation

Bioinformatics. 2022. Na, S et al. Hanyang Univ, Inst Artificial Intelligence Res, Seoul 04763, South Korea; Hanyang Univ, Dept Comp Sci, Seoul 04763, South Korea

ABSTRACT: Motivation: Tandem mass tag (TMT)-based tandem mass spectrometry (MS/MS) has become the method of choice for the quantification of post-translational modifications in complex mixtures. Many cancer proteogenomic studies have highlighted the importance of large-scale phosphopeptide quantification coupled with TMT labeling. Herein, we propose a predicted Spectral DataBase (pSDB) search strategy called Deephos that can improve both sensitivity and specificity in identifying MS/MS spectra of TMT-labeled phosphopeptides.Results: With deep learning-based fragment ion prediction, we compiled a pSDB of TMT-labeled phosphopeptides generated from similar to 8000 human phosphoproteins annotated in UniProt.

Deep learning could successfully recognize the fragmentation patterns altered by both TMT labeling and phosphorylation. In addition, we discuss the decoy spectra for false discovery rate (FDR) estimation in the pSDB search. We show that FDR could be inaccurately estimated by the existing decoy spectra generation methods and propose an innovative method to generate decoy spectra for more accurate FDR estimation. The utilities of Deephos were demonstrated in multi-stage analyses (coupled with database searches) of glioblastoma, acute myeloid leukemia and breast cancer phosphoproteomes.

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AlphaFold and structural mass spectrometry enable interrogations on the intrinsically disordered regions in cyanobacterial light-harvesting complex phycobilisome

JOURNAL OF MOLECULAR BIOLOGY. 2022. Liu, Haijun et al. Washington Univ St Louis, Dept Biol, One Brookings Dr,POB 1137, St Louis, MO 63130 USA

ABSTRACT: Intrinsically disordered proteins/regions (IDPRs) are a very large and functionally important class of pro-teins that participate in weak multivalent interactions in protein complexes. They are recalcitrant for inter-rogations using X-ray crystallography and cryo-EM. The IDPRs observed at the interface of the photosynthetic pigment protein complexes (PPCs) remain much less clear, e.g., the major cyanobacterial light-harvesting complex (PBS) contains an unstructured PB-loop insertion in the phycocyanobilin domain (PB domain) of ApcE (the largest polypeptide in PBS).

Here, a joint platform is built to probe such struc-tural domains. This platform is characterized by two-round progressive justifications of in silico models by using the structural mass spectrometry data. First, the AlphaFold-generated 3D structure of the PB domain (containing PB-loop) was justified in the context of PBS. Second, docking the AlphaFold-generated ApcG (a ligand) into the first-step justified structure (a receptor). The final ligand-receptor com-plex was then subjected to a second-round justification, again, by using unequivocal isotopically-encoded cross-links identified in LC-MS/MS. This work reveals a full-length PB-loop structure modelled in the PBS basal cylinder, free from any spatial conflicts against the other subunits in PBS. The structure of PB domain highlights the close associations of the intrinsically disordered PB-loop with its binding partners in PBS, including ApcG, another IDPR. The PB-loop region involved in the binding of photosystem II (PSII) is also discussed in the context of excitation energy transfer regulation. This work calls attention to the highly disordered, yet interrogatable interface between the light-harvesting antenna complexes and the reaction centers.(c) 2022 Elsevier Ltd. All rights reserved.

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Identification of microproteins in Hep3B cells at different cell cycle stages

Journal of Proteome Research. 2022. Li, B et al. Cent China Normal Univ, Sch Life Sci, Wuhan 430079, Hubei, Peoples R China; Cent China Normal Univ, Hubei Key Lab Genet Regulat & Integrat Biol, Wuhan 430079, Hubei, Peoples R China

ABSTRACT: Microproteins are generated from small open reading frames andturn out to play various vital biological functions. As an essential biological event ofeukaryotic cells, the cell cycle is involved in cell replication and division. For such ahighly regulated event, microproteins associated with cell cycle regulation remainedunclarified. Utilizing a combination of bottom-up and top-down proteomics, weanalyzed microproteins at specific cell cycle stages of Hep3B cells. A total of 657microproteins were identified under three cell cycle stages, including 151 in the G0/G1 stage, 163 in the S stage, and 132 in the G2/M stage.

The annotation of these microproteins showed their cell cycle-specific functions, such as translation, nuclear assembly,chromatin organization, and the G2/M transition of the mitotic cell cycle. Meanwhile, more than 50% of identified microproteinswere ncRNA-encoded. These nonannotated novel microproteins contain several function domains, such as the nucleosidediphosphate kinase domain, the high mobility group domain, and the DNA-binding domain. This suggested the potential functionsof these novel microproteins in specific cell cycle stages. This study presented a large-scale profile of microproteins at different cellcycle stages from Hep3B and may provide new perspectives on the regulation mechanism of the cell cycle. Liquid chromatography-mass spectrometry data were deposited to ProteomeXchange using the identifier PXD030286.

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EGLN1 prolyl hydroxylation of hypoxia-induced transcription factor HIF1α is repressed by SET7-catalyzed lysine methylation

Journal of Biological Chemistry. 2022. Tang, JH et al. Univ Chinese Acad Sci, Beijing, Peoples R China; Chinese Acad Sci, Innovat Seed Design, Wuhan, Peoples R China; Hubei Hongshan Lab, Wuhan, Peoples R China; Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan, Peoples R China

ABSTRACT: Egg-laying defective nine 1 (EGLN1) functions as an oxygen sensor to catalyze prolyl hydroxylation of the transcription factor hypoxia-inducible factor-1 alpha under normoxia conditions, leading to its proteasomal degradation. Thus, EGLN1 plays a central role in the hypoxia-inducible factor-mediated hypoxia signaling pathway; however, the posttranslational modifications that control EGLN1 function remain largely unknown. Here, we identified that a lysine monomethylase, SET7, catalyzes EGLN1 methylation on lysine 297, resulting in the repression of EGLN1 activity in catalyzing prolyl hydroxylation of hypoxia-inducible factor-1 alpha.

Notably, we demonstrate that the methylation mimic mutant of EGLN1 loses the capability to suppress the hypoxia signaling pathway, leading to the enhancement of cell proliferation and the oxygen consumption rate. Collectively, our data identify a novel modification of EGLN1 that is critical for inhibiting its enzymatic activity and which may benefit cellular adaptation to conditions of hypoxia.

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Targeting tumor endothelial hyperglycolysis enhances immunotherapy through remodeling tumor microenvironment

Acta Pharmaceutica Sinica B. 2022. Shan, YL et al. China Pharmaceut Univ, Key Lab Drug Metab & Pharmacokinet, State Key Lab Nat Med, Nanjing 210009, Peoples R China; Nanjing Med Univ, Dept Hematol & Oncol, Affiliated Huaian 1 Peoples Hosp, Huaian 223300, Peoples R China

ABSTRACT: Vascular abnormality isa hallmark of most solid tumors and facilitates immune evasion. Targeting the abnormal metabolism of tumor endothelial cells (TECs) may provide an opportunity to improve the outcome of immunotherapy. Here, in comparison to vascular endothelial cells from adjacent peritumoral tissues in patients with colorectal cancer (CRC), TECs presented enhanced glycolysis with higher glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. Then an unbiased screening identified that osimertinib could modify the GAPDH and thus inhibit its activity in TECs.

Low-dose osimertinib treatment caused tumor regression with vascular normalization and increased infiltration of immune effector cells in tumor, which was due to the reduced secretion of lactate from TECs by osimertinib through the inhibition of GAPDH. Moreover, osimertinib and anti-PD-1 blockade synergistically retarded tumor growth. This study provides a potential strategy to enhance immunotherapy by targeting the abnormal metabolism of TECs.

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Accurate discrimination of leucine and isoleucine residues by combining continuous digestion with multiple MS3 spectra integration in protein sequence

Talanta. 2022. Zhang, Weijie et al. Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog R&A Ctr, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Liaoning, Peoples R China

ABSTRACT: Protein de novo sequencing based on tandem mass spectrometry is a crucial technology that enables the identification of peptides without searching databases and assembling unknown sequence proteins, especially for monoclonal antibodies (mAbs). However, the discrimination of leucine (Leu) and isoleucine (Ile) residues in the target protein sequence is still challenging. Herein, we developed an accurate method by continuous digestion with MS3-based fragmentation and multiple spectra integration (evaluated by combined verification score, CVS) to distinguish Leu and Ile residues.

Continuous digestion promotes the diversity of peptides in order to expose more Leu and Ile at the N-terminal. CVS integrates multiple MS3 spectra to reduce the interference from noise and co-fragmented ions and improve accuracy. This method successfully resolved all 75 Leu/Ile in bovine serum albumin, especially 3 consecutive Leu/Ile. We further applied the method to analyze trastuzumab and 67 out of the 68 Leu/Ile from the light chain and heavy chain were accurately discriminated, demonstrating the great potential in mAbs sequencing.

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Mirror proteases of Ac-Trypsin and Ac-LysargiNase precisely improve novel event identifications in Mycolicibacterium smegmatis MC2 155 by proteogenomic …

FRONTIERS IN MICROBIOLOGY. 2022. Songhao Jiang et al. Guangzhou Univ Chinese Med, Clin Med Coll 2, Guangzhou Higher Educ Mega Ctr, Guangzhou, Peoples R China; Chinese Acad Med Sci, Inst Life, Beijing Proteome Res Ctr, Natl Ctr Prot Sci Beijing,State Key Lab Prote,Res, Beijing, Peoples R China; Chinese Acad Med Sci & Peking Union Med Coll, Inst Med Biotechnol, Res Unit Prote & Res & Dev New Drug, Beijing, Peoples R China; Hebei Univ, Sch Life Sci, Key Lab Microbial Divers Res & Applicat Hebei, Baoding, Peoples R China

ABSTRACT: Accurate identification of novel peptides remains challenging because of the lack of evaluation criteria in large-scale proteogenomic studies. Mirror proteases of trypsin and lysargiNase can generate complementary b/y ion series, providing the opportunity to efficiently assess authentic novel peptides in experiments other than filter potential targets by different false discovery rates (FDRs) ranking. In this study, a pair of in-house developed acetylated mirror proteases, Ac-Trypsin and Ac-LysargiNase, were used in Mycolicibacterium smegmatis MC2 155 for proteogenomic analysis.

The mirror proteases accurately identified 368 novel peptides, exhibiting 75-80% b and y ion coverages against 65-68% y or b ion coverages of Ac-Trypsin (38.9% b and 68.3% y) or Ac-LysargiNase (65.5% b and 39.6% y) as annotated peptides from M. smegmatis MC2 155. The complementary b and y ion series largely increased the reliability of overlapped sequences derived from novel peptides. Among these novel peptides, 311 peptides were annotated in other public M. smegmatis strains, and 57 novel peptides with more continuous b and y pairs were obtained for further analysis after spectral quality assessment. This enabled mirror proteases to successfully correct six annotated proteins' N-termini and detect 17 new coding open reading frames (ORFs). We believe that mirror proteases will be an effective strategy for novel peptide detection in both prokaryotic and eukaryotic proteogenomics.

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Deep N-terminomics of Mycobacterium tuberculosis H37Rv extensively correct annotated encoding genes

Genomics. 2022. Shi, JH et al. Chinese Acad Med Sci, Beijing Proteome Res Ctr, Natl Ctr Prot Sci Beijing,State Key Lab Proteom, Res Unit Prote & Res & Dev New Drug,Inst Lifeom, Beijing 102206, Peoples R China

ABSTRACT: Mycobacterium tuberculosis (MTB) is a severe causing agent of tuberculosis (TB). Although H37Rv, the type strain of M. tuberculosis was sequenced in 1998, annotation errors of encoding genes have been frequently reported in hundreds of papers. This phenomenon is particularly severe at the 5 ' end of the genes. Here, we applied a TMPP [(N-Succinimidyloxycarbonylmethyl) tris (2,4,6-trimethoxyphenyl) phosphonium bromide] labeling combined with StageTip separating strategy on M. tuberculosis H37Rv to characterize the N-terminal start sites of its annotated encoding genes.

Totally, 1047 proteins were identified with 2058 TMPP labeled N-terminal peptides from all the 2625 mass spectrometer (MS) sequenced proteins. Comparative genomics analysis allowed the reannotation of 43 proteins' N-termini in H37Rv and 762 proteins in Mycobacteriaceae. All revised N-termini start sites were distributed in 5'-UTR of annotated genes due to over-annotation of previous N-terminal initiation codon, especially the ATG. In addition, we identified and verified a novel gene Rv1078A in +3 frame different from the annotated gene Rv1078 in +2 frame. Altogether, our findings contribute to the better understanding of N-terminal of H37Rv and other species from Mycobacteriaceae that can assist future studies on biological study.

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Ac-LysargiNase efficiently helps genome reannotation of Mycolicibacterium smegmatis MC2 155

Journal of Proteomics. 2022. Zhu, HM et al. Chinese Acad Med Sci, Inst Life, Beijing Proteome Res Ctr,State Key Lab Prote, Natl Ctr Prot Sci Beijing,Res Unit Prote & Res &, Beijing 102206, Peoples R China

ABSTRACT: Accurate genome annotation, the foundation of life science research in the genome era, is hampered by limited known gene models, nonstandard start codons, and the limited homology of annotated genes in other organisms. LysargiNase mirrors trypsin at the cleavage sites, providing the opportunity to identify peptides other than tryptic peptides. In this study, we used an in-house developed acetylated LysargiNase (Ac-LysargiNase) with higher activity and stability in non-pathogenic Mycolicibacterium smegmatis MC2 155 to supplement the widely used trypsin in proteomic studies.

We identified 27,582 peptides from 3844 annotated proteins and 332 novel genome search-specific peptides (GSSPs). Among these GSSPs, 88 peptides were annotated in another M.smeg-matis genome database, and 41 were verified as novel peptides by predicted theoretical spectra and their cor-responding 15N-labeling spectra. Further analysis revealed that 17 verified GSSPs corrected the N-terminus of the 13 annotated genes. The other 24 verified GSSPs helped identify 17 novel open reading frames (ORFs) missed in previously annotated M. smegmatis genomes. Among these novel ORFs, four relatively small proteins with amino acid residues less than 100 and three were precisely identified with C-terminal peptides. Ac-LysargiNase helps with genome reannotation by identifying new genes and events in proteogenomic studies. Significance: Correct genomic annotation is vital in the field of life sciences. The nonstandard start codons seriously affect the confirmation of the translation initiation sites (TISs) of an open reading frame (ORF), and unknown structural genes are easily missed in automated gene prediction. Although proteogenomics presents new avenues for validating gene expression and gene structure refinement based on conventional tryptic pep-tides, determining the TISs and potential encoding genes is complicated. Thus, validation of TISs and encoding ORFs is crucial and urgent. Therefore, we recommend Ac-LysargiNase, a mirror enzyme of trypsin that can identify additional novel peptides for N-terminal correction and ORF identification.

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A novel proteogenomic integration strategy expands the breadth of neo-epitope sources

Cancers. 2022. Xiang, Haitao et al. BGI Shenzhen, Shenzhen 518103, Peoples R China; Guangdong Prov Key Lab Human Dis Genom, Shenzhen Key Lab Genom, Shenzhen 518083, Peoples R China; BGI, Shenzhen 518083, Peoples R China

ABSTRACT: Simple Summary Tumor-specific antigens are ideal targets for cancer immunotherapy. Mass spectrometry, which is the main method that directly identifies neo-epitopes presented on tumor cells, focuses mainly on peptides derived from annotated protein-coding exomes. However, non-canonical peptides arising from alterations at genomic, transcriptional, and posttranslational levels have been identified in several pioneering studies, making it necessary to develop an integrated proteogenomic approach that can comprehensively identify neoantigens derived from all genomic regions.

Our novel strategy combining database searches with a de novo peptide sequencing method accurately identified multiple types of non-canonical peptides in the colorectal cancer cell line, HCT116. This practical proteogenomic strategy can be applied to neoantigen discovery in clinical tumor samples, improving cancer immunotherapy. Tumor-specific antigens can activate T cell-based antitumor immune responses and are ideal targets for cancer immunotherapy. However, their identification is still challenging. Although mass spectrometry can directly identify human leukocyte antigen (HLA) binding peptides in tumor cells, it focuses on tumor-specific antigens derived from annotated protein-coding regions constituting only 1.5% of the genome. We developed a novel proteogenomic integration strategy to expand the breadth of tumor-specific epitopes derived from all genomic regions. Using the colorectal cancer cell line HCT116 as a model, we accurately identified 10,737 HLA-presented peptides, 1293 of which were non-canonical peptides that traditional database searches could not identify. Moreover, we found eight tumor neo-epitopes derived from somatic mutations, four of which were not previously reported. Our findings suggest that this new proteogenomic approach holds great promise for increasing the number of tumor-specific antigen candidates, potentially enlarging the tumor target pool and improving cancer immunotherapy.

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De-sialylation of glycopeptides by acid treatment: enhancing sialic acid removal without reducing the identification

Analytical Methods. 2022. Dong, Wenbo et al. Northwest Univ, Coll Life Sci, Xian 710069, Shaanxi, Peoples R China

ABSTRACT: Sialic acid, a common terminal monosaccharide on many glycoconjugates, plays essential roles in many biological processes such as immune responses, pathogen recognition, and cancer development. For various purposes, sialic acids may need to be removed from glycopeptides or glycans, mainly using enzymatical or chemical approaches. In this study, we found that most commonly used chemical methods couldn't completely remove sialic acids from glycopeptides. Although the de-sialylation efficiency could be further enhanced by increasing the treatment time or acid concentration, the undesirable side reactions on the peptide portion would decrease glycopeptide identification.

By adding the deamidation on carbamidomethyl-cysteine (C), asparagine (N), and glutamine (Q) residues as a variable modification during database search, most of the unidentified spectra could be recovered. This optional acid-treatment and database search method for the complete removal of sialic acids without losing much spectral identification should be quite useful for many glycomic and glycoproteomic studies.

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Quantitative model suggests both intrinsic and contextual features contribute to the transcript coding ability determination in cells

Briefings in Bioinformatics. 2022. Kang, Yu-Jian et al. Peking Univ, Biomed Pioneering Innovat Ctr BIOPIC, Beijing Adv Innovat Ctr Genom ICG, Ctr Bioinformat CBI,Sch Life Sci, Beijing 100871, Peoples R China; Peking Univ, State Key Lab Pry Lein & Plant Gene Res, Sch Life Sci, Beijing 100871, Peoples R China

ABSTRACT: Gene transcription and protein translation are two key steps of the 'central dogma.' It is still a major challenge to quantitatively deconvolute factors contributing to the coding ability of transcripts in mammals. Here, we propose ribosome calculator (RiboCalc) for quantitatively modeling the coding ability of RNAs in human genome. In addition to effectively predicting the experimentally confirmed coding abundance via sequence and transcription features with high accuracy, RiboCalc provides interpretable parameters with biological information.

Large-scale analysis further revealed a number of transcripts with a variety of coding ability for distinct types of cells (i.e. context-dependent coding transcripts), suggesting that, contrary to conventional wisdom, a transcript's coding ability should be modeled as a continuous spectrum with a context-dependent nature.

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Angiotensin-converting enzyme genotype--specific immune response contributes to the susceptibility of COVID-19: a nested case--control study

Frontiers in pharmacology. 2022. Gong, Pengyun et al. Hubei Ctr Dis Control & Prevent, Wuhan, Peoples R China; Beihang Univ, Sch Biol Sci & Med Engn, Beijing, Peoples R China; Capital Med Univ, Beijing YouAn Hosp, Dept Radiol, Beijing, Peoples R China; Beihang Univ, Sch Engn Med, Beijing, Peoples R China

ABSTRACT: Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019 (COVID-19), which has resulted in a global pandemic.Methodology: We used a two-step polymerase chain reaction to detect the ACE genotype and ELISA kits to detect the cytokine factor. We also used proteomics to identify the immune pathway related to the ACE protein expression.Result: In this study, we found that the angiotensin-converting enzyme (ACE) deletion polymorphism was associated with the susceptibility to COVID-19 in a risk-dependent manner among the Chinese population.

D/D genotype distributions were higher in the COVID-19 disease group than in the control group (D/D odds ratio is 3.87 for mild (p value < 0.0001), 2.59 for moderate (p value = 0.0002), and 4.05 for severe symptoms (p value < 0.0001), logic regression analysis. Moreover, genotype-specific cytokine storms and immune responses were found enriched in patients with the ACE deletion polymorphism, suggesting the contribution to the susceptibility to COVID-19. Finally, we identified the immune pathway such as the complement system related to the ACE protein expression of patients by lung and plasma proteomics.Conclusion: Our results demonstrated that it is very important to consider gene polymorphisms in the population to discover a host-based COVID-19 vaccine and drug design for preventive and precision medicine.

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Discovery of 194 Unreported Conopeptides and Identification of a New Protein Disulfide Isomerase in Conus caracteristicus Using Integrated Transcriptomic and Proteomic Analysis

Frontiers in Marine Science. 2022. Zhang, Han et al. Southern Med Univ, Guangdong Prov Key Lab Single Cell Technol & Appl, Guangzhou, Peoples R China; Southern Med Univ, Guangdong Hong Kong Macao Greater Bay Area Ctr Br, Hong Kong, Guangdong, Peoples R China; Southern Med Univ, Sch Basic Med Sci, Dept Biochem & Mol Biol, Guangzhou, Peoples R China

ABSTRACT: Current ConoServer database accumulates 8,134 conopeptides from 122 species of cone snail, which are pharmaceutically attractive marine resource. However, many more conopeptides remain to be discovered, and the enzymes involved in their synthesis and processing are unclear. In this report, firstly we screened and analyzed the differentially expressed genes (DEGs) between venom duct (VD) and venom bulb (VB) of C. caracteristicus, and obtained 3,289 transcripts using a comprehensive assembly strategy.

Then using de novo deep transcriptome sequencing and analysis under a strict merit, we discovered 194 previously unreported conopeptide precursors in Conus caracteristicus. Meanwhile, 2 predicted conopeptides from Consort were verified using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Furthermore, we demonstrated that both VD and VB of C. caracteristicus secreted hundreds of different conotoxins, which showed a high diversity among individuals of the species. Finally, we identified a protein disulfide isomerase (PDI) gene, which, functioning for intramolecular disulfide-bond folding, was shared among C. caracteristicus, C. textile, and C. bartschi and was the first PDI identified with five thioredoxin domains. Our results provide novel insights and fuel further studies of the molecular evolution and function of the novel conotoxins.

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MetaLab-MAG: A Metaproteomic Data Analysis Platform for Genome-Level Characterization of Microbiomes from the Metagenome-Assembled Genomes Database

Journal of Proteome Research. 2022. Cheng, Kai et al. Univ Ottawa, Fac Med, Sch Pharmaceut Sci, Ottawa, ON K1H 8M5, Canada

ABSTRACT: The studies of microbial communities have drawn increased attention in various research fields such as agriculture, environment, and human health. Recently, metaproteomics has become a powerful tool to interpret the roles of the community members by investigating the expressed proteins of the microbes. However, analyzing the metaproteomic data sets at genome resolution is still challenging because of the lack of efficient bioinformatics tools. Here we develop MetaLab-MAG, a specially designed tool for the characterization of microbiomes from metagenome-assembled genomes databases.

MetaLab-MAG was evaluated by analyzing various human gut microbiota data sets and performed comparably or better than searching the gene catalog protein database directly. MetaLab-MAG can quantify the genome-level microbiota compositions and supports both label-free and isobaric labeling-based quantification strategies. MetaLab-MAG removes the obstacles of metaproteomic data analysis and provides the researchers with in-depth and comprehensive information from the microbiomes.

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Structure of a TOC-TIC supercomplex spanning two chloroplast envelope membranes

CELL. 2022. Jin, Zeyu et al. Westlake Univ, Sch Life Sci, Key Lab Struct Biol Zhejiang Prov, Hangzhou 310024, Zhejiang, Peoples R China; Westlake Inst Adv Study, Inst Biol, Hangzhou 310024, Zhejiang, Peoples R China; Westlake Lab Life Sci & Biomed, Hangzhou 310024, Zhejiang, Peoples R China

ABSTRACT: The TOC and TIC complexes are essential translocons that facilitate the import of the nuclear genome-en-coded preproteins across the two envelope membranes of chloroplast, but their exact molecular identities and assembly remain unclear. Here, we report a cryoelectron microscopy structure of TOC-TIC supercom-plex from Chlamydomonas, containing a total of 14 identified components. The preprotein-conducting pore of TOC is a hybrid b-barrel co-assembled by Toc120 and Toc75, while the potential translocation path of TIC is formed by transmembrane helices from Tic20 and YlmG, rather than a classic model of Tic110.

A rigid intermembrane space (IMS) scaffold bridges two chloroplast membranes, and a large hydro-philic cleft on the IMS scaffold connects TOC and TIC, forming a pathway for preprotein translocation. Our study provides structural insights into the TOC-TIC supercomplex composition, assembly, and preprotein translocation mechanism, and lays a foundation to interpret the evolutionary conservation and diversity of this fundamental translocon machinery.

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Assessing the dark field of metaproteome

Analytical Chemistry. 2022. Duan, Haonan et al. Univ Ottawa, Fac Med, Daniel Figeys Sch Pharmaceut Sci, Ottawa, ON K1H 8L1, Canada; Univ Ottawa, Ottawa Inst Syst Biol, Ottawa, ON K1H 8L1, Canada

ABSTRACT: The human gut microbiome is a complex system composed of hundreds of species, and metaproteomics can be used to explore their expressed functions. However, many lower abundance species are not detected by current metaproteomic techniques and represent the dark field of metaproteomics. We do not know the minimal abundance of a bacterium in a microbiome-(depth) that can be detected by shotgun metaproteomics. In this study, we spiked 15N-labeled E. coli peptides at different percentages into peptides mixture derived from the human gut microbiome to evaluate the depth that can be achieved by shotgun metaproteomics.

We observed that the number of identified peptides and peptide intensity from 15N-labeled E. coli were linearly correlated with the spike-in levels even when 15N-labeled E. coli was down to 0.5% of the biomass. Below that level, it was not detected. Interestingly, the match-between-run strategy significantly increased the number of quantified peptides even when 15N-labeled E. coli peptides were at low abundance. This is indicative that in metaproteomics of complex gut microbiomes many peptides from low abundant species are likely observable in MS1 but are not selected for MS2 by standard shotgun strategies.

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Changes to Urinary Proteome in High-Fat-Diet ApoE-/- Mice

Biomolecules. 2022. Hua, Yuanrui et al. Beijing Normal Univ, Coll Life Sci, Gene Engn Drug & Biotechnol Beijing Key Lab, Beijing 100875, Peoples R China

ABSTRACT: Cardiovascular disease is currently the leading cause of death worldwide. Atherosclerosis is an important pathological basis of cardiovascular disease, and its early diagnosis is of great significance. Urine bears no need nor mechanism to be stable, so it accumulates many small changes and is therefore a good source of biomarkers in the early stages of disease. In this study, ApoE-/- mice were fed a high-fat diet for 5 months. Urine samples from the experimental group and control group (C57BL/6 mice fed a normal diet) were collected at seven time points.

Proteomic analysis was used for comparison within the experimental group and for comparison between the experimental group and the control group. The results of the comparison within the experimental group showed a significant difference in the urinary proteome before and after a one-week high-fat diet, and several of the differential proteins have been reported to be associated with atherosclerosis and/or as biomarker candidates. The results of the comparison between the experimental group and the control group indicated that the biological processes enriched by the GO analysis of the differential proteins correspond to the progression of atherosclerosis. The differences in chemical modifications of urinary proteins have also been reported to be associated with the disease. This study demonstrates that urinary proteomics has the potential to sensitively monitor changes in the body and provides the possibility of identifying early biomarkers of atherosclerosis.

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The cytosolic thiol peroxidase PRXIIB is an intracellular sensor for H2O2 that regulates plant immunity through a redox relay

NATURE PLANTS. 2022. Bi, Guozhi et al. Chinese Acad Sci, Inst Genet & Dev Biol, State Key Lab Plant Genom, Beijing, Peoples R China; Hainan Yazhou Bay Seed Lab, Sanya, Peoples R China; Univ Chinese Acad Sci, Coll Adv Agr Sci, Beijing, Peoples R China; Univ Chinese Acad Sci, CAS Ctr Excellence Biot Interact, Beijing, Peoples R China; Beijing Inst Life, Natl Ctr Prot Sci, Beijing Proteome Res Ctr, State Key Lab Prote, Beijing, Peoples R China

ABSTRACT: Rapid production of H2O2 is a hallmark of plant responses to diverse pathogens and plays a crucial role in signalling downstream of various receptors that perceive immunogenic patterns. However, mechanisms by which plants sense H2O2 to regulate immunity remain poorly understood. We show that endogenous H2O2 generated upon immune activation is sensed by the thiol peroxidase PRXIIB via oxidation at Cys51, and this is essential for stomatal immunity against Pseudomonas syringae. We further show that in immune-stimulated cells, PRXIIB conjugates via Cys51 with the type 2C protein phosphatase ABA insensitive 2 (ABI2), subsequently transducing H2O2 signal to ABI2.

This oxidation dramatically sensitizes H2O2-mediated inhibition of the ABI2 phosphatase activity in vitro and is required for stomatal immunity in plants. Together, our results illustrate a redox relay, with PRXIIB as a sensor for H2O2 and ABI2 as a target protein, that mediates reactive oxygen species signalling during plant immunity.

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PBC, an easy and efficient strategy for high-throughput protein C-terminome profiling

Frontiers in Cell and Developmental Biology. 2022. Zhai, Linhui et al. Chinese Acad Sci, Shanghai Inst Mat Med, State Key Lab Drug Res, Shanghai, Peoples R China; Nanjing Univ Chinese Med, Sch Pharm, Jiangsu Key Lab Funct Subst Chinese Med, Nanjing, Jiangsu, Peoples R China; Nanjing Univ Chinese Med, Sch Chinese Mat Med, Sch Pharm, Nanjing, Jiangsu, Peoples R China

ABSTRACT: High-throughput profiling of protein C-termini is still a challenging task. Proteomics provides a powerful technology for systematic and high-throughput study of protein C-termini. Various C-terminal peptide enrichment strategies based on chemical derivatization and chromatography separation have been reported. However, they are still costly and time-consuming, with low enrichment efficiency for C-terminal peptides. In this study, by taking advantage of the high reaction selectivity of 2-pyridinecarboxaldehyde (2-PCA) with an alpha-amino group on peptide N-terminus and high affinity between biotin and streptavidin, we developed a 2-PCA- and biotin labeling-based C-terminomic (PBC) strategy for a high-efficiency and high-throughput analysis of protein C-terminome.

Triplicates of PBC experiments identified a total of 1,975 C-terminal peptides corresponding to 1,190 proteins from 293 T cell line, which is 180% higher than the highest reported number of C-terminal peptides identified from mammalian cells by chemical derivatization-based C-terminomics study. The enrichment efficiency (68%) is the highest among the C-terminomics methods currently reported. In addition, we not only uncovered 50 proteins with truncated C-termini which were significantly enriched in extracellular exosome, vesicle, and ribosome by a bioinformatic analysis but also systematically characterized the whole PTMs on C-terminal in 293 T cells, suggesting PBC as a powerful tool for protein C-terminal degradomics and PTMs investigation. In conclusion, the PBC strategy would benefit high-efficiency and high-throughput profiling of protein C-terminome.

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Many kinds of oxidized proteins are present more in the urine of the elderly

Clinical proteomics. 2022. Liu, YT et al. Beijing Normal Univ, Dept Biochem & Mol Biol, Beijing Key Lab Gene Engn Drug & Biotechnol, Beijing 100875, Peoples R China

ABSTRACT: Background Many studies have shown an association between aging and oxidation. To our knowledge, there have been no studies exploring aging-related urine proteome modifications. The purpose of this study was to explore differences in global chemical modifications of urinary protein at different ages. Methods Discovery (n=38) cohort MS data including children, young and old groups were downloaded from three published studies, and this data was analyzed using open-pFind for identifying modifications.

Verification cohort human samples (n=28) including young, middle-aged, and old groups, rat samples (n=7) at three-time points after birth, adulthood, and old age were collected and processed in the laboratory simultaneously based on label-free quantification combined with pFind. Results Discovery cohort: there were 28 kinds of differential oxidations in the old group that were higher than those in the young or children group in. Verification cohort: there were 17 kinds of differential oxidations of 49 oxidized proteins in the middle and old groups, which were significantly higher than those in the young group. Both oxidations and oxidized proteins distinguished different age groups well. There were also 15 kinds of differential oxidations in old age higher than others in the rat cohort. The results showed that the validation experiment was basically consistent with the results of the discovery experiment, showing that the level of oxidized proteins in urine increased significantly with age. Conclusions Our study is the first to show that oxidative proteins occur in urine and that oxidations are higher in older than younger ages. Perhaps improving the degree of excretion of oxidative protein in vivo through the kidney is helpful for maintaining the homeostasis of the body's internal environment, delaying aging and the occurrence of senile diseases.

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PML Body Component Sp100A Is a Cytosolic Responder to IFN and Activator of Antiviral ISGs

Mbio. 2022. Dong, HC et al. Sun Yat Sen Univ, Key Lab Trop Dis Control, Minist Educ, Guangzhou, Peoples R China; Sun Yat Sen Univ, Ctr Infect & Immun Studies, Sch Med, Shenzhen, Peoples R China

ABSTRACT: PML bodies sit at the center stage of various important biological processes; however, the signal transduction networks of these macromolecular protein complexes remain enigmatic. The present study illustrates, in detail and for the first time, the course of signal receiving, processing, and implementation by PML bodies in response to IFN and virus infection.Promyelocytic leukemia protein (PML) bodies are implicated in one of the key pathways in the establishment of antiviral status in response to interferon (IFN), yet the molecular mechanisms bridging the cross talk remain elusive.

Herein, we report that a major constitutive component of the PML body, Sp100A, is ubiquitously located in the cytosol of various cell types and is an immediate responder to multiple extracellular stimuli, including virus infection, IFN, epidermal growth factor (EGF), glial cell-derived nerve factor (GDNF), etc., signaling through the phosphatidylinositol 3-kinase (PI3K) pathway. IFN-beta induces phosphorylation of Sp100A on Ser(188), which fortifies the binding of Sp100A to pyruvate kinase 2 (PKM2) and facilitates its nuclear importation through the extracellular signal-regulated kinase 1/2 (ERK1/2)-PKM2-PIN1-importin axes. Blocking PI3K pathway signaling or interference with the ERK1/2-PKM2-PIN1-importin axes independently hampers nuclear translocation of Sp100A in response to IFN, reflecting a dual-regulation mechanism governing this event. In the nucleus, Sp100A is enriched in the promoter regions of essential antiviral interferon-stimulated genes (ISGs), such as those coding for IFI16, OAS2, and RIG-I, and activates their transcription. Importantly, nuclear importation of Sp100A, but not accumulation of a mutant Sp100A that failed to respond to IFN, during infection potently enhanced transcription of these antiviral ISGs and restricted virus propagation. These findings depict a novel IFN response mechanism by PML bodies in the cytosol and shed light on the complex sensing-regulatory network of PML bodies.IMPORTANCE PML bodies sit at the center stage of various important biological processes; however, the signal transduction networks of these macromolecular protein complexes remain enigmatic. The present study illustrates, in detail and for the first time, the course of signal receiving, processing, and implementation by PML bodies in response to IFN and virus infection. It shows that PML body constitutive component Sp100A was phosphorylated on Ser(188) by IFN signaling through the PI3K pathway in the cytosol, cotranslocated into the nucleus with PKM2, enriched on the promoter regions of essential antiviral ISGs such as those coding for IFI16, RIG-I, OAS2, etc., and mediating their transcriptional activation.

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The effect of lactulose thermal degradation products on $\beta$-lactoglobulin: Linear-, loop-, and cross-link structural modifications and reduced digestibility

FOOD CHEMISTRY. 2022. Dong, L et al. Nankai Univ, Sch Med, Tianjin Key Lab Food Sci & Hlth, Tianjin 300071, Peoples R China

ABSTRACT: The thermal degradation products of lactulose and the interaction between lactulose and beta-lactoglobulin (beta Lg) were investigated in a thermal model system. Lactulose was thermally degraded into fructose and galactose, which were further degraded into methylglyoxal, glyoxal, 3-deoxyglucosone, and 2, 3-butanedione via heating. After incubating with lactulose, the structure of beta Lg was changed, which manifested by the formation of new band with doubled the molecular weight of beta Lg in the mobility spectrum and the changes in the internal fluo-rescence spectrum.

Furthermore, the lysine and arginine residues of beta Lg were confirmed to be the modification sites of the thermal degradation products of lactulose, and the modification types of linear-, loop-, and cross -linked were detected. The digestibility of beta Lg incubated with lactulose was significantly decreased due to the modification of trypsin digestion sites and the formation of cross-linked conjunctions. Therefore, the adverse effects of lactulose application in thermally processed foods should be concerned.

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FBB18 participates in preassembly of almost all axonemal dyneins independent of R2TP complex

PLoS genetics. 2022. Wang, LM et al. Qingdao Natl Lab Marine Sci & Technol, Lab Marine Biol & Biotechnol, Qingdao, Shandong, Peoples R China; Tsinghua Univ, Sch Life Sci, MOE Key Lab Prot Sci, Beijing, Peoples R China

ABSTRACT: Assembly of dynein arms requires cytoplasmic processes which are mediated by dynein preassembly factors (DNAAFs). CFAP298, which is conserved in organisms with motile cilia, is required for assembly of dynein arms but with obscure mechanisms. Here, we show that FBB18, a Chlamydomonas homologue of CFAP298, localizes to the cytoplasm and functions in folding/stabilization of almost all axonemal dyneins at the early steps of dynein preassembly. Mutation of FBB18 causes no or short cilia accompanied with partial loss of both outer and inner dynein arms.

Comparative proteomics using N-15 labeling suggests partial degradation of almost all axonemal dynein heavy chains (DHCs). A mutant mimicking a patient variant induces particular loss of DHC alpha. FBB18 associates with 9 DNAAFs and 14 out of 15 dynein HCs but not with IC1/IC2. FBB18 interacts with RuvBL1/2, components of the HSP90 co-chaperone R2TP complex but not the holo-R2TP complex. Further analysis suggests simultaneous formation of multiple DNAAF complexes involves dynein folding/stability and thus provides new insights into axonemal dynein preassembly.

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MStoCIRC: A powerful tool for downstream analysis of MS/MS data to predict translatable circRNAs

Frontiers in Molecular Biosciences. 2022. Cao, Zhou et al. Shaanxi Normal Univ, Coll Life Sci, Key Lab Minist Educ Med Plant Resource & Nat Pharm, Natl Engn Lab Resource Dev Endangered Crude Drugs, Xian, Peoples R China

ABSTRACT: CircRNAs are formed by a non-canonical splicing method and appear circular in nature. CircRNAs are widely distributed in organisms and have the features of time- and tissue-specific expressions. CircRNAs have attracted increasing interest from scientists because of their non-negligible effects on the growth and development of organisms. The translation capability of circRNAs is a novel and valuable direction in the functional research of circRNAs. To explore the translation potential of circRNAs, some progress has been made in both experimental identification and computational prediction.

For computational prediction, both CircCode and CircPro are ribosome profiling-based software applications for predicting translatable circRNAs, and the online databases riboCIRC and TransCirc analyze as many pieces of evidence as possible and list the predicted translatable circRNAs of high confidence. Simultaneously, mass spectrometry in proteomics is often recognized as an efficient method to support the identification of protein and peptide sequences from diverse complex templates. However, few applications fully utilize mass spectrometry to predict translatable circRNAs. Therefore, this research aims to build up a scientific analysis pipeline with two salient features: 1) it starts with the data analysis of raw tandem mass spectrometry data; and 2) it also incorporates other translation evidence such as IRES. The pipeline has been packaged into an analysis tool called mass spectrometry to translatable circRNAs (MStoCIRC). MStoCIRC is mainly implemented by Python3 language programming and could be downloaded from GitHub (). The tool contains a main program and several small, independent function modules, making it more multifunctional. MStoCIRC can process data efficiently and has obtained hundreds of translatable circRNAs in humans and Arabidopsis thaliana.

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Efficient detection of the alternative spliced human proteome using translatome sequencing

Frontiers in Molecular Biosciences. 2022. Wu, Chun et al. Jinan Univ, Inst Life & Hlth Engn, MOE Key Lab Tumor Mol Biol, Guangzhou, Peoples R China; Jinan Univ, Key Lab Funct Prot Res Guangdong Higher Educ Inst, Inst Life & Hlth Engn, Guangzhou, Peoples R China

ABSTRACT: Alternative splicing (AS) isoforms create numerous proteoforms, expanding the complexity of the genome. Highly similar sequences, incomplete reference databases and the insufficient sequence coverage of mass spectrometry limit the identification of AS proteoforms. Here, we demonstrated full-length translating mRNAs (ribosome nascent-chain complex-bound mRNAs, RNC-mRNAs) sequencing (RNC-seq) strategy to sequence the entire translating mRNA using next-generation sequencing, including short-read and long-read technologies, to construct a protein database containing all translating AS isoforms.

Taking the advantage of read length, short-read RNC-seq identified up to 15,289 genes and 15,906 AS isoforms in a single human cell line, much more than the Ribo-seq. The single-molecule long-read RNC-seq supplemented 4,429 annotated AS isoforms that were not identified by short-read datasets, and 4,525 novel AS isoforms that were not included in the public databases. Using such RNC-seq-guided database, we identified 6,766 annotated protein isoforms and 50 novel protein isoforms in mass spectrometry datasets. These results demonstrated the potential of full-length RNC-seq in investigating the proteome of AS isoforms.

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AlphaPeptDeep: a modular deep learning framework to predict peptide properties for proteomics

Nature Communications. 2022. Zeng, Wen-Feng et al. Univ Copenhagen, Fac Hlth Sci, NNF Ctr Prot Res, Prote Program, Copenhagen, Denmark; Max Planck Inst Biochem, Dept Prote & Signal Transduct, Martinsried, Germany

ABSTRACT: Machine learning and in particular deep learning (DL) are increasingly important in mass spectrometry (MS)-based proteomics. Recent DL models can predict the retention time, ion mobility and fragment intensities of a peptide just from the amino acid sequence with good accuracy. However, DL is a very rapidly developing field with new neural network architectures frequently appearing, which are challenging to incorporate for proteomics researchers. Here we introduce AlphaPeptDeep, a modular Python framework built on the PyTorch DL library that learns and predicts the properties of peptides (https://github.com/MannLabs/alphapeptdeep).

It features a model shop that enables non-specialists to create models in just a few lines of code. AlphaPeptDeep represents post-translational modifications in a generic manner, even if only the chemical composition is known. Extensive use of transfer learning obviates the need for large data sets to refine models for particular experimental conditions. The AlphaPeptDeep models for predicting retention time, collisional cross sections and fragment intensities are at least on par with existing tools. Additional sequence-based properties can also be predicted by AlphaPeptDeep, as demonstrated with a HLA peptide prediction model to improve HLA peptide identification for data-independent acquisition (https://github.com/MannLabs/PeptDeep-HLA).

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Cryo-EM structure of the human CST--Pol$\alpha$/primase complex in a recruitment state

Nature Structural & Molecular Biology. 2022. Cai, Sarah W. et al. Rockefeller Univ, Lab Mol Electron Microscopy, New York, NY 10065 USA; Rockefeller Univ, Lab Cell Biol & Genet, New York, NY 10065 USA

ABSTRACT: The CST-Pol alpha/primase complex is essential for telomere maintenance and functions to counteract resection at double-strand breaks. We report a 4.6-angstrom resolution cryo-EM structure of human CST-Pol alpha/primase, captured prior to catalysis in a recruitment state stabilized by chemical cross-linking. Our structure reveals an evolutionarily conserved interaction between the C-terminal domain of the catalytic POLA1 subunit and an N-terminal expansion in metazoan CTC1. Cross-linking mass spectrometry and negative-stain EM analysis provide insight into CST binding by the flexible POLA1 N-terminus.

Finally, Coats plus syndrome disease mutations previously characterized to disrupt formation of the CST-Pol alpha/primase complex map to protein-protein interfaces observed in the recruitment state. Together, our results shed light on the architecture and stoichiometry of the metazoan fill-in machinery.

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Protein posttranslational signatures identified in COVID-19 patient plasma

Frontiers in Cell and Developmental Biology. 2022. Vedula, Pavan et al. Univ Penn, Sch Vet Med, Dept Biomed Sci, Philadelphia, PA 19104 USA

ABSTRACT: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a highly contagious virus of the coronavirus family that causes coronavirus disease-19 (COVID-19) in humans and a number of animal species. COVID-19 has rapidly propagated in the world in the past 2 years, causing a global pandemic. Here, we performed proteomic analysis of plasma samples from COVID-19 patients compared to healthy control donors in an exploratory study to gain insights into protein-level changes in the patients caused by SARS-CoV-2 infection and to identify potential proteomic and posttranslational signatures of this disease.

Our results suggest a global change in protein processing and regulation that occurs in response to SARS-CoV-2, and the existence of a posttranslational COVID-19 signature that includes an elevation in threonine phosphorylation, a change in glycosylation, and a decrease in arginylation, an emerging posttranslational modification not previously implicated in infectious disease. This study provides a resource for COVID-19 researchers and, longer term, and will inform our understanding of this disease and its treatment.

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De-sialylation of glycopeptides by acid treatment: enhancing sialic acid removal without reducing the identification

Analytical Methods. 2022. Dong, Wenbo et al. Northwest Univ, Coll Life Sci, Xian 710069, Shaanxi, Peoples R China

ABSTRACT: Sialic acid, a common terminal monosaccharide on many glycoconjugates, plays essential roles in many biological processes such as immune responses, pathogen recognition, and cancer development. For various purposes, sialic acids may need to be removed from glycopeptides or glycans, mainly using enzymatical or chemical approaches. In this study, we found that most commonly used chemical methods couldn't completely remove sialic acids from glycopeptides. Although the de-sialylation efficiency could be further enhanced by increasing the treatment time or acid concentration, the undesirable side reactions on the peptide portion would decrease glycopeptide identification.

By adding the deamidation on carbamidomethyl-cysteine (C), asparagine (N), and glutamine (Q) residues as a variable modification during database search, most of the unidentified spectra could be recovered. This optional acid-treatment and database search method for the complete removal of sialic acids without losing much spectral identification should be quite useful for many glycomic and glycoproteomic studies.

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Quantitative model suggests both intrinsic and contextual features contribute to the transcript coding ability determination in cells

Briefings in Bioinformatics. 2022. Kang, Yu-Jian et al. Peking Univ, Biomed Pioneering Innovat Ctr BIOPIC, Beijing Adv Innovat Ctr Genom ICG, Ctr Bioinformat CBI,Sch Life Sci, Beijing 100871, Peoples R China; Peking Univ, State Key Lab Pry Lein & Plant Gene Res, Sch Life Sci, Beijing 100871, Peoples R China

ABSTRACT: Gene transcription and protein translation are two key steps of the 'central dogma.' It is still a major challenge to quantitatively deconvolute factors contributing to the coding ability of transcripts in mammals. Here, we propose ribosome calculator (RiboCalc) for quantitatively modeling the coding ability of RNAs in human genome. In addition to effectively predicting the experimentally confirmed coding abundance via sequence and transcription features with high accuracy, RiboCalc provides interpretable parameters with biological information.

Large-scale analysis further revealed a number of transcripts with a variety of coding ability for distinct types of cells (i.e. context-dependent coding transcripts), suggesting that, contrary to conventional wisdom, a transcript's coding ability should be modeled as a continuous spectrum with a context-dependent nature.

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Discovery of 194 Unreported Conopeptides and Identification of a New Protein Disulfide Isomerase in Conus caracteristicus Using Integrated Transcriptomic and Proteomic Analysis

Frontiers in Marine Science. 2022. Zhang, Han et al. Southern Med Univ, Guangdong Prov Key Lab Single Cell Technol & Appl, Guangzhou, Peoples R China; Southern Med Univ, Guangdong Hong Kong Macao Greater Bay Area Ctr Br, Hong Kong, Guangdong, Peoples R China; Southern Med Univ, Sch Basic Med Sci, Dept Biochem & Mol Biol, Guangzhou, Peoples R China

ABSTRACT: Current ConoServer database accumulates 8,134 conopeptides from 122 species of cone snail, which are pharmaceutically attractive marine resource. However, many more conopeptides remain to be discovered, and the enzymes involved in their synthesis and processing are unclear. In this report, firstly we screened and analyzed the differentially expressed genes (DEGs) between venom duct (VD) and venom bulb (VB) of C. caracteristicus, and obtained 3,289 transcripts using a comprehensive assembly strategy.

Then using de novo deep transcriptome sequencing and analysis under a strict merit, we discovered 194 previously unreported conopeptide precursors in Conus caracteristicus. Meanwhile, 2 predicted conopeptides from Consort were verified using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Furthermore, we demonstrated that both VD and VB of C. caracteristicus secreted hundreds of different conotoxins, which showed a high diversity among individuals of the species. Finally, we identified a protein disulfide isomerase (PDI) gene, which, functioning for intramolecular disulfide-bond folding, was shared among C. caracteristicus, C. textile, and C. bartschi and was the first PDI identified with five thioredoxin domains. Our results provide novel insights and fuel further studies of the molecular evolution and function of the novel conotoxins.

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Angiotensin-converting enzyme genotype--specific immune response contributes to the susceptibility of COVID-19: a nested case--control study

Frontiers in pharmacology. 2022. Gong, Pengyun et al. Hubei Ctr Dis Control & Prevent, Wuhan, Peoples R China; Beihang Univ, Sch Biol Sci & Med Engn, Beijing, Peoples R China; Capital Med Univ, Beijing YouAn Hosp, Dept Radiol, Beijing, Peoples R China; Beihang Univ, Sch Engn Med, Beijing, Peoples R China

ABSTRACT: Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019 (COVID-19), which has resulted in a global pandemic.Methodology: We used a two-step polymerase chain reaction to detect the ACE genotype and ELISA kits to detect the cytokine factor. We also used proteomics to identify the immune pathway related to the ACE protein expression.Result: In this study, we found that the angiotensin-converting enzyme (ACE) deletion polymorphism was associated with the susceptibility to COVID-19 in a risk-dependent manner among the Chinese population.

D/D genotype distributions were higher in the COVID-19 disease group than in the control group (D/D odds ratio is 3.87 for mild (p value < 0.0001), 2.59 for moderate (p value = 0.0002), and 4.05 for severe symptoms (p value < 0.0001), logic regression analysis. Moreover, genotype-specific cytokine storms and immune responses were found enriched in patients with the ACE deletion polymorphism, suggesting the contribution to the susceptibility to COVID-19. Finally, we identified the immune pathway such as the complement system related to the ACE protein expression of patients by lung and plasma proteomics.Conclusion: Our results demonstrated that it is very important to consider gene polymorphisms in the population to discover a host-based COVID-19 vaccine and drug design for preventive and precision medicine.

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Mirror proteases of Ac-Trypsin and Ac-LysargiNase precisely improve novel event identifications in Mycolicibacterium smegmatis MC2 155 by proteogenomic analysis

Frontiers in Microbiology. 2022. Jiang, Songhao et al. Hebei Univ, Sch Life Sci, Key Lab Microbial Divers Res & Applicat Hebei, Baoding, Peoples R China; Guangzhou Univ Chinese Med, Clin Med Coll 2, Guangzhou Higher Educ Mega Ctr, Guangzhou, Peoples R China; Chinese Acad Med Sci, Inst Life, Beijing Proteome Res Ctr, Natl Ctr Prot Sci Beijing,State Key Lab Prote,Res, Beijing, Peoples R China; Chinese Acad Med Sci & Peking Union Med Coll, Inst Med Biotechnol, Res Unit Prote & Res & Dev New Drug, Beijing, Peoples R China

ABSTRACT: Accurate identification of novel peptides remains challenging because of the lack of evaluation criteria in large-scale proteogenomic studies. Mirror proteases of trypsin and lysargiNase can generate complementary b/y ion series, providing the opportunity to efficiently assess authentic novel peptides in experiments other than filter potential targets by different false discovery rates (FDRs) ranking. In this study, a pair of in-house developed acetylated mirror proteases, Ac-Trypsin and Ac-LysargiNase, were used in Mycolicibacterium smegmatis MC2 155 for proteogenomic analysis.

The mirror proteases accurately identified 368 novel peptides, exhibiting 75-80% b and y ion coverages against 65-68% y or b ion coverages of Ac-Trypsin (38.9% b and 68.3% y) or Ac-LysargiNase (65.5% b and 39.6% y) as annotated peptides from M. smegmatis MC2 155. The complementary b and y ion series largely increased the reliability of overlapped sequences derived from novel peptides. Among these novel peptides, 311 peptides were annotated in other public M. smegmatis strains, and 57 novel peptides with more continuous b and y pairs were obtained for further analysis after spectral quality assessment. This enabled mirror proteases to successfully correct six annotated proteins' N-termini and detect 17 new coding open reading frames (ORFs). We believe that mirror proteases will be an effective strategy for novel peptide detection in both prokaryotic and eukaryotic proteogenomics.

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PBC, an easy and efficient strategy for high-throughput protein C-terminome profiling

Frontiers in Cell and Developmental Biology. 2022. Zhai, Linhui et al. Chinese Acad Sci, Shanghai Inst Mat Med, State Key Lab Drug Res, Shanghai, Peoples R China; Nanjing Univ Chinese Med, Sch Pharm, Jiangsu Key Lab Funct Subst Chinese Med, Nanjing, Jiangsu, Peoples R China; Nanjing Univ Chinese Med, Sch Chinese Mat Med, Sch Pharm, Nanjing, Jiangsu, Peoples R China

ABSTRACT: High-throughput profiling of protein C-termini is still a challenging task. Proteomics provides a powerful technology for systematic and high-throughput study of protein C-termini. Various C-terminal peptide enrichment strategies based on chemical derivatization and chromatography separation have been reported. However, they are still costly and time-consuming, with low enrichment efficiency for C-terminal peptides. In this study, by taking advantage of the high reaction selectivity of 2-pyridinecarboxaldehyde (2-PCA) with an alpha-amino group on peptide N-terminus and high affinity between biotin and streptavidin, we developed a 2-PCA- and biotin labeling-based C-terminomic (PBC) strategy for a high-efficiency and high-throughput analysis of protein C-terminome.

Triplicates of PBC experiments identified a total of 1,975 C-terminal peptides corresponding to 1,190 proteins from 293 T cell line, which is 180% higher than the highest reported number of C-terminal peptides identified from mammalian cells by chemical derivatization-based C-terminomics study. The enrichment efficiency (68%) is the highest among the C-terminomics methods currently reported. In addition, we not only uncovered 50 proteins with truncated C-termini which were significantly enriched in extracellular exosome, vesicle, and ribosome by a bioinformatic analysis but also systematically characterized the whole PTMs on C-terminal in 293 T cells, suggesting PBC as a powerful tool for protein C-terminal degradomics and PTMs investigation. In conclusion, the PBC strategy would benefit high-efficiency and high-throughput profiling of protein C-terminome.

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MetaLab-MAG: A Metaproteomic Data Analysis Platform for Genome-Level Characterization of Microbiomes from the Metagenome-Assembled Genomes Database

Journal of Proteome Research. 2022. Cheng, Kai et al. Univ Ottawa, Fac Med, Sch Pharmaceut Sci, Ottawa, ON K1H 8M5, Canada

ABSTRACT: The studies of microbial communities have drawn increased attention in various research fields such as agriculture, environment, and human health. Recently, metaproteomics has become a powerful tool to interpret the roles of the community members by investigating the expressed proteins of the microbes. However, analyzing the metaproteomic data sets at genome resolution is still challenging because of the lack of efficient bioinformatics tools. Here we develop MetaLab-MAG, a specially designed tool for the characterization of microbiomes from metagenome-assembled genomes databases.

MetaLab-MAG was evaluated by analyzing various human gut microbiota data sets and performed comparably or better than searching the gene catalog protein database directly. MetaLab-MAG can quantify the genome-level microbiota compositions and supports both label-free and isobaric labeling-based quantification strategies. MetaLab-MAG removes the obstacles of metaproteomic data analysis and provides the researchers with in-depth and comprehensive information from the microbiomes.

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Structure of a TOC-TIC supercomplex spanning two chloroplast envelope membranes

CELL. 2022. Jin, Zeyu et al. Westlake Univ, Sch Life Sci, Key Lab Struct Biol Zhejiang Prov, Hangzhou 310024, Zhejiang, Peoples R China; Westlake Inst Adv Study, Inst Biol, Hangzhou 310024, Zhejiang, Peoples R China; Westlake Lab Life Sci & Biomed, Hangzhou 310024, Zhejiang, Peoples R China

ABSTRACT: The TOC and TIC complexes are essential translocons that facilitate the import of the nuclear genome-en-coded preproteins across the two envelope membranes of chloroplast, but their exact molecular identities and assembly remain unclear. Here, we report a cryoelectron microscopy structure of TOC-TIC supercom-plex from Chlamydomonas, containing a total of 14 identified components. The preprotein-conducting pore of TOC is a hybrid b-barrel co-assembled by Toc120 and Toc75, while the potential translocation path of TIC is formed by transmembrane helices from Tic20 and YlmG, rather than a classic model of Tic110.

A rigid intermembrane space (IMS) scaffold bridges two chloroplast membranes, and a large hydro-philic cleft on the IMS scaffold connects TOC and TIC, forming a pathway for preprotein translocation. Our study provides structural insights into the TOC-TIC supercomplex composition, assembly, and preprotein translocation mechanism, and lays a foundation to interpret the evolutionary conservation and diversity of this fundamental translocon machinery.

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Assessing the Dark Field of Metaproteome

Analytical Chemistry. 2022. Duan, Haonan et al. Univ Ottawa, Fac Med, Daniel Figeys Sch Pharmaceut Sci, Ottawa, ON K1H 8L1, Canada; Univ Ottawa, Ottawa Inst Syst Biol, Ottawa, ON K1H 8L1, Canada

ABSTRACT: The human gut microbiome is a complex system composed of hundreds of species, and metaproteomics can be used to explore their expressed functions. However, many lower abundance species are not detected by current metaproteomic techniques and represent the dark field of metaproteomics. We do not know the minimal abundance of a bacterium in a microbiome-(depth) that can be detected by shotgun metaproteomics. In this study, we spiked 15N-labeled E. coli peptides at different percentages into peptides mixture derived from the human gut microbiome to evaluate the depth that can be achieved by shotgun metaproteomics.

We observed that the number of identified peptides and peptide intensity from 15N-labeled E. coli were linearly correlated with the spike-in levels even when 15N-labeled E. coli was down to 0.5% of the biomass. Below that level, it was not detected. Interestingly, the match-between-run strategy significantly increased the number of quantified peptides even when 15N-labeled E. coli peptides were at low abundance. This is indicative that in metaproteomics of complex gut microbiomes many peptides from low abundant species are likely observable in MS1 but are not selected for MS2 by standard shotgun strategies.

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Changes to Urinary Proteome in High-Fat-Diet ApoE-/- Mice

Biomolecules. 2022. Hua, Yuanrui et al. Beijing Normal Univ, Coll Life Sci, Gene Engn Drug & Biotechnol Beijing Key Lab, Beijing 100875, Peoples R China

ABSTRACT: Cardiovascular disease is currently the leading cause of death worldwide. Atherosclerosis is an important pathological basis of cardiovascular disease, and its early diagnosis is of great significance. Urine bears no need nor mechanism to be stable, so it accumulates many small changes and is therefore a good source of biomarkers in the early stages of disease. In this study, ApoE-/- mice were fed a high-fat diet for 5 months. Urine samples from the experimental group and control group (C57BL/6 mice fed a normal diet) were collected at seven time points.

Proteomic analysis was used for comparison within the experimental group and for comparison between the experimental group and the control group. The results of the comparison within the experimental group showed a significant difference in the urinary proteome before and after a one-week high-fat diet, and several of the differential proteins have been reported to be associated with atherosclerosis and/or as biomarker candidates. The results of the comparison between the experimental group and the control group indicated that the biological processes enriched by the GO analysis of the differential proteins correspond to the progression of atherosclerosis. The differences in chemical modifications of urinary proteins have also been reported to be associated with the disease. This study demonstrates that urinary proteomics has the potential to sensitively monitor changes in the body and provides the possibility of identifying early biomarkers of atherosclerosis.

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The cytosolic thiol peroxidase PRXIIB is an intracellular sensor for H2O2 that regulates plant immunity through a redox relay

NATURE PLANTS. 2022. Bi, Guozhi et al. Chinese Acad Sci, Inst Genet & Dev Biol, State Key Lab Plant Genom, Beijing, Peoples R China; Hainan Yazhou Bay Seed Lab, Sanya, Peoples R China; Univ Chinese Acad Sci, Coll Adv Agr Sci, Beijing, Peoples R China; Univ Chinese Acad Sci, CAS Ctr Excellence Biot Interact, Beijing, Peoples R China; Beijing Inst Life, Natl Ctr Prot Sci, Beijing Proteome Res Ctr, State Key Lab Prote, Beijing, Peoples R China

ABSTRACT: Rapid production of H2O2 is a hallmark of plant responses to diverse pathogens and plays a crucial role in signalling downstream of various receptors that perceive immunogenic patterns. However, mechanisms by which plants sense H2O2 to regulate immunity remain poorly understood. We show that endogenous H2O2 generated upon immune activation is sensed by the thiol peroxidase PRXIIB via oxidation at Cys51, and this is essential for stomatal immunity against Pseudomonas syringae. We further show that in immune-stimulated cells, PRXIIB conjugates via Cys51 with the type 2C protein phosphatase ABA insensitive 2 (ABI2), subsequently transducing H2O2 signal to ABI2.

This oxidation dramatically sensitizes H2O2-mediated inhibition of the ABI2 phosphatase activity in vitro and is required for stomatal immunity in plants. Together, our results illustrate a redox relay, with PRXIIB as a sensor for H2O2 and ABI2 as a target protein, that mediates reactive oxygen species signalling during plant immunity.

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A Novel Proteogenomic Integration Strategy Expands the Breadth of Neo-Epitope Sources

Cancers. 2022. Xiang, Haitao et al. BGI Shenzhen, Shenzhen 518103, Peoples R China; Guangdong Prov Key Lab Human Dis Genom, Shenzhen Key Lab Genom, Shenzhen 518083, Peoples R China; BGI, Shenzhen 518083, Peoples R China

ABSTRACT: Simple Summary Tumor-specific antigens are ideal targets for cancer immunotherapy. Mass spectrometry, which is the main method that directly identifies neo-epitopes presented on tumor cells, focuses mainly on peptides derived from annotated protein-coding exomes. However, non-canonical peptides arising from alterations at genomic, transcriptional, and posttranslational levels have been identified in several pioneering studies, making it necessary to develop an integrated proteogenomic approach that can comprehensively identify neoantigens derived from all genomic regions.

Our novel strategy combining database searches with a de novo peptide sequencing method accurately identified multiple types of non-canonical peptides in the colorectal cancer cell line, HCT116. This practical proteogenomic strategy can be applied to neoantigen discovery in clinical tumor samples, improving cancer immunotherapy. Tumor-specific antigens can activate T cell-based antitumor immune responses and are ideal targets for cancer immunotherapy. However, their identification is still challenging. Although mass spectrometry can directly identify human leukocyte antigen (HLA) binding peptides in tumor cells, it focuses on tumor-specific antigens derived from annotated protein-coding regions constituting only 1.5% of the genome. We developed a novel proteogenomic integration strategy to expand the breadth of tumor-specific epitopes derived from all genomic regions. Using the colorectal cancer cell line HCT116 as a model, we accurately identified 10,737 HLA-presented peptides, 1293 of which were non-canonical peptides that traditional database searches could not identify. Moreover, we found eight tumor neo-epitopes derived from somatic mutations, four of which were not previously reported. Our findings suggest that this new proteogenomic approach holds great promise for increasing the number of tumor-specific antigen candidates, potentially enlarging the tumor target pool and improving cancer immunotherapy.

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Accurate discrimination of leucine and isoleucine residues by combining continuous digestion with multiple MS3 spectra integration in protein sequence

Talanta. 2022. Zhang, Weijie et al. Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog R&A Ctr, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Liaoning, Peoples R China

ABSTRACT: Protein de novo sequencing based on tandem mass spectrometry is a crucial technology that enables the identification of peptides without searching databases and assembling unknown sequence proteins, especially for monoclonal antibodies (mAbs). However, the discrimination of leucine (Leu) and isoleucine (Ile) residues in the target protein sequence is still challenging. Herein, we developed an accurate method by continuous digestion with MS3-based fragmentation and multiple spectra integration (evaluated by combined verification score, CVS) to distinguish Leu and Ile residues.

Continuous digestion promotes the diversity of peptides in order to expose more Leu and Ile at the N-terminal. CVS integrates multiple MS3 spectra to reduce the interference from noise and co-fragmented ions and improve accuracy. This method successfully resolved all 75 Leu/Ile in bovine serum albumin, especially 3 consecutive Leu/Ile. We further applied the method to analyze trastuzumab and 67 out of the 68 Leu/Ile from the light chain and heavy chain were accurately discriminated, demonstrating the great potential in mAbs sequencing.

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AlphaFold and structural mass spectrometry enable interrogations on the intrinsically disordered regions in cyanobacterial light-harvesting complex phycobilisome

JOURNAL OF MOLECULAR BIOLOGY. 2022. Liu, Haijun et al. Washington Univ St Louis, Dept Biol, One Brookings Dr,POB 1137, St Louis, MO 63130 USA

ABSTRACT: Intrinsically disordered proteins/regions (IDPRs) are a very large and functionally important class of pro-teins that participate in weak multivalent interactions in protein complexes. They are recalcitrant for inter-rogations using X-ray crystallography and cryo-EM. The IDPRs observed at the interface of the photosynthetic pigment protein complexes (PPCs) remain much less clear, e.g., the major cyanobacterial light-harvesting complex (PBS) contains an unstructured PB-loop insertion in the phycocyanobilin domain (PB domain) of ApcE (the largest polypeptide in PBS).

Here, a joint platform is built to probe such struc-tural domains. This platform is characterized by two-round progressive justifications of in silico models by using the structural mass spectrometry data. First, the AlphaFold-generated 3D structure of the PB domain (containing PB-loop) was justified in the context of PBS. Second, docking the AlphaFold-generated ApcG (a ligand) into the first-step justified structure (a receptor). The final ligand-receptor com-plex was then subjected to a second-round justification, again, by using unequivocal isotopically-encoded cross-links identified in LC-MS/MS. This work reveals a full-length PB-loop structure modelled in the PBS basal cylinder, free from any spatial conflicts against the other subunits in PBS. The structure of PB domain highlights the close associations of the intrinsically disordered PB-loop with its binding partners in PBS, including ApcG, another IDPR. The PB-loop region involved in the binding of photosystem II (PSII) is also discussed in the context of excitation energy transfer regulation. This work calls attention to the highly disordered, yet interrogatable interface between the light-harvesting antenna complexes and the reaction centers.(c) 2022 Elsevier Ltd. All rights reserved.

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A Hybrid Spectral Library and Protein Sequence Database Search Strategy for Bottom-Up and Top-Down Proteomic Data Analysis

Journal of Proteome Research. 2022. Dai, YL et al. Univ Wisconsin, Dept Chem, Madison, WI 53706 USA

ABSTRACT: Tandem mass spectrometry (MS/MS) is widely employed for the analysis of complex proteomic samples. While protein sequence database searching and spectral library searching are both well-established peptide identification methods, each has shortcomings. Protein sequence databases lack fragment peak intensity information, which can result in poor discrimination between correct and incorrect spectrum assignments. Spectral libraries usually contain fewer peptides than protein sequence databases, which limits the number of peptides that can be identified.

Notably, few post-translationally modified peptides are represented in spectral libraries. This is because few search engines can both identify a broad spectrum of PTMs and create corresponding spectral libraries. Also, programs that generate spectral libraries using deep learning approaches are not yet able to accurately predict spectra for the vast majority of PTMs. Here, we address these limitations through use of a hybrid search strategy that combines protein sequence database and spectral library searches to improve identification success rates and sensitivity. This software uses Global PTM Discovery (G-PTM-D) to produce spectral libraries for a wide variety of different PTMs. These features, along with a new spectrum annotation and visualization tool, have been integrated into the freely available and open-source search engine MetaMorpheus.

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High-throughput proteomic sample preparation using pressure cycling technology

Nature protocols. 2022. Cai, X et al. Westlake Univ, Sch Life Sci, Westlake Lab Life Sci & Biomed, Key Lab Struct Biol Zhejiang Prov, Hangzhou, Peoples R China; Westlake Inst Adv Study, Inst Basic Med Sci, Hangzhou, Peoples R China

ABSTRACT: High-throughput lysis and proteolytic digestion of biopsy-level tissue specimens is a major bottleneck for clinical proteomics. Here we describe a detailed protocol of pressure cycling technology (PCT)-assisted sample preparation for proteomic analysis of biopsy tissues. A piece of fresh frozen or formalin-fixed paraffin-embedded tissue weighing similar to 0.1-2 mg is placed in a 150 mu L pressure-resistant tube called a PCT-MicroTube with proper lysis buffer. After closing with a PCT-MicroPestle, a batch of 16 PCT-MicroTubes are placed in a Barocycler, which imposes oscillating pressure to the samples from one atmosphere to up to similar to 3,000 times atmospheric pressure.

The pressure cycling schemes are optimized for tissue lysis and protein digestion, and can be programmed in the Barocycler to allow reproducible, robust and efficient protein extraction and proteolysis digestion for mass spectrometry-based proteomics. This method allows effective preparation of not only fresh frozen and formalin-fixed paraffin-embedded tissue, but also cells, feces and tear strips. It takes similar to 3 h to process 16 samples in one batch. The resulting peptides can be analyzed by various mass spectrometry-based proteomics methods. We demonstrate the applications of this protocol with mouse kidney tissue and eight types of human tumors.

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Identification and mechanism of G protein-biased ligands for chemokine receptor CCR1

Nature chemical biology. 2022. Shao, ZH et al. Zhejiang Univ, MOE Frontier Sci Ctr Brain Res & Brain Machine In, Sch Med, Hangzhou, Peoples R China; Zhejiang Univ, Affiliated Hosp 2, Dept Pharmacol, Key Lab Resp Dis Zhejiang Prov,Sch Med, Hangzhou, Peoples R China; Zhejiang Univ, Dept Pathol, Sir Run Run Shaw Hosp, Sch Med, Hangzhou, Peoples R China; Zhejiang Univ, Affiliated Hosp 2, Dept Resp & Crit Care Med, Key Lab Resp Dis Zhejiang Prov,Sch Med, Hangzhou, Peoples R China; Zhejiang Prov Key Lab Immun & Inflammatory Dis, Hangzhou, Peoples R China; Zhejiang Univ, Key Lab Resp Dis Zhejiang Prov, Dept Resp & Crit Care Med, Affiliated Hosp 2,Sch Med, Hangzhou, Peoples R China; State Key Lab Resp Dis, Guangzhou, Peoples R China; Zhejiang Univ, Liangzhu Lab, Med Ctr, Hangzhou, Peoples R China; Zhejiang Univ, Int Inst Med, Sch Med, Affiliated Hosp 4, Yiwu, Peoples R China; Zhejiang Univ, Dept Biophys, Sir Run Run Shaw Hosp, Sch Med, Hangzhou, Peoples R China

ABSTRACT: Biased signaling of G protein-coupled receptors describes an ability of different ligands that preferentially activate an alternative downstream signaling pathway. In this work, we identified and characterized different N-terminal truncations of endogenous chemokine CCL15 as balanced or biased agonists targeting CCR1, and presented three cryogenic-electron microscopy structures of the CCR1-G(i) complex in the ligand-free form or bound to different CCL15 truncations with a resolution of 2.6-2.9 angstrom, illustrating the structural basis of natural biased signaling that initiates an inflammation response.

Complemented with pharmacological and computational studies, these structures revealed it was the conformational change of Tyr291 (Y291(7.)(43)) in CCR1 that triggered its polar network rearrangement in the orthosteric binding pocket and allosterically regulated the activation of beta-arrestin signaling. Our structure of CCL15-bound CCR1 also exhibited a critical site for ligand binding distinct from many other chemokine-receptor complexes, providing new insights into the mode of chemokine recognition.

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A mechanism for oxidative damage repair at gene regulatory elements

Nature. 2022. Ray, S et al. Univ Sheffield, Hlth Lifespan & Neurosci Inst, Sheffield, S Yorkshire, England; Univ Bradford, Sch Pharm & Med Sci, Inst Canc Therapeut, Bradford, W Yorkshire, England; Univ Sheffield, Sch Biosci, Sheffield, S Yorkshire, England

ABSTRACT: Oxidative genome damage is an unavoidable consequence of cellular metabolism. It arises at gene regulatory elements by epigenetic demethylation during transcriptional activation(1,2). Here we show that promoters are protected from oxidative damage via a process mediated by the nuclear mitotic apparatus protein NuMA (also known as NUMA1). NuMA exhibits genomic occupancy approximately 100 bp around transcription start sites. It binds the initiating form of RNA polymerase II, pause-release factors and single-strand break repair (SSBR) components such as TDP1.

The binding is increased on chromatin following oxidative damage, and TDP1 enrichment at damaged chromatin is facilitated by NuMA. Depletion of NuMA increases oxidative damage at promoters. NuMA promotes transcription by limiting the polyADP-ribosylation of RNA polymerase II, increasing its availability and release from pausing at promoters. Metabolic labelling of nascent RNA identifies genes that depend on NuMA for transcription including immediate-early response genes. Complementation of NuMA-deficient cells with a mutant that mediates binding to SSBR, or a mitotic separation-of-function mutant, restores SSBR defects. These findings underscore the importance of oxidative DNA damage repair at gene regulatory elements and describe a process that fulfils this function.

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Mechanistic Insights into the Functioning of a Two-Subunit GMP Synthetase, an Allosterically Regulated, Ammonia Channeling Enzyme

BIOCHEMISTRY. 2022. Shivakumaraswamy, S et al. Jawaharlal Nehru Ctr Adv Sci Res, Mol Biol & Genet Unit, Bengaluru 560064, India

ABSTRACT: Guanosine 5 & PRIME;-monophosphate (GMP) synthetases, enzymes that catalyze the conversion of xanthosine 5 & PRIME;-monophosphate (XMP) to GMP, are composed of two different catalytic units, which are either two domains of a polypeptide chain or two subunits that associate to form a complex. The glutamine amidotransferase (GATase) unit hydrolyzes glutamine generating ammonia, and the ATP pyrophosphatase (ATPPase) unit catalyzes the formation of an AMP-XMP intermediate. The substrate-bound ATPPase allosterically activates GATase, and the ammonia thus generated is tunneled to the ATPPase active site where it reacts with AMP-XMP generating GMP.

In ammonia channeling enzymes reported thus far, a tight complex of the two subunits is observed, while the interaction of the two subunits of Methanocaldococcus jannaschii GMP synthetase (MjGMPS) is transient with the underlying mechanism of allostery and substrate channeling largely unclear. Here, we present a mechanistic model encompassing the various steps in the catalytic cycle of MjGMPS based on biochemical experiments, crystal structure, and cross-linking mass spectrometry guided integrative modeling. pH dependence of enzyme kinetics establishes that ammonia is tunneled across the subunits with the lifetime of the complex being & LE;0.5 s. The crystal structure of the XMP-bound ATPPase subunit reported herein highlights the role of conformationally dynamic loops in enabling catalysis. The structure of MjGMPS derived using restraints obtained from cross-linking mass spectrometry has enabled the visualization of subunit interactions that enable allostery under catalytic conditions. We integrate the results and propose a functional mechanism for MjGMPS detailing the various steps involved in catalysis.

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Probing strigolactone perception mechanisms with rationally designed small-molecule agonists stimulating germination of root parasitic weeds

Nature communications. 2022. Wang, DW et al. Hunan Univ, State Key Lab Chemo Biosensing & Chemometr, Hunan Prov Key Lab Plant Funct Genom & Dev Regula, Coll Biol, Changsha 410082, Peoples R China; Nankai Univ, Collaborat Innovat Ctr Chem Sci & Engn, Natl Pesticide Engn Res Ctr, Coll Chem,Dept Chem Biol, Tianjin 300071, Peoples R China; Univ Amsterdam, Swammerdam Inst Life Sci SILS, Sci Pk 904, NL-1098 XH Amsterdam, Netherlands; Nankai Univ, State Key Lab Elementoorgan Chem, Collaborat Innovat Ctr Chem Sci & Engn, Coll Chem,Natl Pesticide Engn Res Ctr, Tianjin 300071, Peoples R China

ABSTRACT: The development of potent strigolactone (SL) agonists as suicidal germination inducers could be a useful strategy for controlling root parasitic weeds, but uncertainty about the SL perception mechanism impedes real progress. Here we describe small-molecule agonists that efficiently stimulate Phelipanchce aegyptiaca, and Striga hermonthica, germination in concentrations as low as 10(-8) to 10(-17) M. We show that full efficiency of synthetic SL agonists in triggering signaling through the Striga SL receptor, ShHTL7, depends on the receptor-catalyzed hydrolytic reaction of the agonists.

Additionally, we reveal that the stereochemistry of synthetic SL analogs affects the hydrolytic ability of ShHTL7 by influencing the probability of the privileged conformations of ShHTL7. Importantly, an alternative ShHTL7-mediated hydrolysis mechanism, proceeding via nucleophilic attack of the NE2 atom of H246 to the 2 ' C of the D-ring, is reported. Together, our findings provide insight into SL hydrolysis and structure-perception mechanisms, and potent suicide germination stimulants, which would contribute to the elimination of the noxious parasitic weeds.Strigolactone agonists could potentially help control noxious weeds by promoting suicidal germination. Here the authors describe a series of small molecule agonists that stimulate germination via the Striga ShHTL7 receptor and show that stereochemistry and hydrolysis-independent signalling mediate potency.

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Deep coverage proteome analysis of hair shaft for forensic individual identification

Forensic Science International: Genetics. 2022. Wu, JL et al. Chinese Acad Sci, Natl Chromatog Res & Anal Ctr, Dalian Inst Chem Phys, CAS Key Lab Separat Sci Analyt Chem, 457 Zhongshan Rd, Dalian 116023, Peoples R China; Peoples Publ Secur Univ China, Grad Sch, 1 Muxidi Nanli, Beijing 100038, Peoples R China; Inst Forens Sci, Natl Engn Lab Forens Sci, Key Lab Forens Genet, Minist Publ Secur, 17 Muxidi Nanli, Beijing 100038, Peoples R China

ABSTRACT: Hair shaft is one of the most common biological evidence found at crime scenes. However, due to the biogenic degradation of nuclear DNA in hair shaft, it is difficult to achieve individual identification through routine DNA analysis. In contrast, the proteins in hair shaft are stable and contain genetic polymorphisms in the form of single amino acid polymorphisms (SAPs), translated from non-synonymous single nucleotide polymorphisms (nsSNPs) in the genome. However, the number of SAPs detected still cannot meet the requirements of practical applications.

This paper developed a deep coverage proteome analysis method by combining a three-step sequential ionic liquid-based protein extraction and 2D-RPLC-MS/MS with high and low pH to identify both variant and reference SAPs from 2-cm-long hair shafts. We identified 632 +/- 243 protein groups from 10 individuals, with the average number of SAPs reaching 167 +/- 21/person. These were further used to calculate random match probabilities (RMPs), a widely accepted forensic statistical term for human identification. The RMPs ranged from 6.53 x 10(-4) to 3.10 x 10(-14) (median = 2.62 x 10(-8)) when calculated with frequency of matching nsSNP genotype data from exomes, and ranged from 2.62 x 10(-3) to 2.07 x 10(-10) (median = 4.88 x 10(-6)) with SAP genotype frequency. All these results indicate that the deep coverage proteomics method is beneficial for improving SAP-based forensic individual identification in hair shaft, with great potential in crime investigation.

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Characterization of protein unfolding by fast cross-linking mass spectrometry using di-ortho-phthalaldehyde cross-linkers

Nature communications. 2022. Wang, JH et al. Natl Inst Biol Sci NIBS, Beijing 102206, Peoples R China; Tsinghua Univ, Tsinghua Inst Multidisciplinary Biomed Res, Beijing 102206, Peoples R China; Peking Univ, Coll Chem & Mol Engn, Peking Tsinghua Ctr Life Sci,Minist Educ, Beijing Natl Lab Mol Sci,Key Lab Bioorgan Chem &, Beijing 100871, Peoples R China; Chinese Acad Sci, Innovat Acad Precis Measurement Sci & Technol, Wuhan 430071, Peoples R China

ABSTRACT: Conformations sampled by a protein while it unfolds are difficult to visualize. Here, the authors develop di-ortho-phthalaldehyde cross-linkers for rapid chemical cross-linking mass spectrometry analysis and demonstrate that this method captures the conformations of protein unfolding intermediates.Chemical cross-linking of proteins coupled with mass spectrometry is widely used in protein structural analysis. In this study we develop a class of non-hydrolyzable amine-selective di-ortho-phthalaldehyde (DOPA) cross-linkers, one of which is called DOPA2.

Cross-linking of proteins with DOPA2 is 60-120 times faster than that with the N-hydroxysuccinimide ester cross-linker DSS. Compared with DSS cross-links, DOPA2 cross-links show better agreement with the crystal structures of tested proteins. More importantly, DOPA2 has unique advantages when working at low pH, low temperature, or in the presence of denaturants. Using staphylococcal nuclease, bovine serum albumin, and bovine pancreatic ribonuclease A, we demonstrate that DOPA2 cross-linking provides abundant spatial information about the conformations of progressively denatured forms of these proteins. Furthermore, DOPA2 cross-linking allows time-course analysis of protein conformational changes during denaturant-induced unfolding.

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Comprehensive identification of protein orthologs in the family Ascoviridae facilitates an understanding of phylogenomics, protein conservation, and phosphorylation

Archives of Virology. 2022. Shi, YH et al. Weifang Med Univ, Sch Life Sci & Technol, Weifang 261053, Peoples R China; Hunan Agr Univ, Hunan Prov Key Lab Biol & Control Plant Dis & Ins, Changsha 410128, Hunan, Peoples R China

ABSTRACT: Analysis of orthology is important for understanding protein conservation, function, and phylogenomics. In this study, we performed a comprehensive analysis of gene orthology in the family Ascoviridae based on identification of 366 protein homologue groups and phylogenetic analysis of 34 non-single-copy proteins. Our findings revealed 90 newly annotated proteins, five newly identified core proteins for the family Ascoviridae, and 14 core proteins for the genus Ascovirus. A phylogenomic tree of 11 Ascoviridae members was constructed based on a concatenation of 35 of the 45 ortholog groups.

In combination with phosphoproteomic results and conservation estimations, 30 conserved phosphorylation sites on 17 phosphoproteins were identified from a total of 176 phosphosites on 57 phosphoproteins from Heliothis virescens ascovirus 3h (HvAV-3h), providing potential research targets for investigating the role of these protein in the regulation of viral infection. This study will facilitate genome annotation and comparison of further Ascoviridae members as well as functional genomic investigations.

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PML Body Component Sp100A Is a Cytosolic Responder to IFN and Activator of Antiviral ISGs

Mbio. 2022. Dong, HC et al. Sun Yat Sen Univ, Key Lab Trop Dis Control, Minist Educ, Guangzhou, Peoples R China; Sun Yat Sen Univ, Ctr Infect & Immun Studies, Sch Med, Shenzhen, Peoples R China

ABSTRACT: PML bodies sit at the center stage of various important biological processes; however, the signal transduction networks of these macromolecular protein complexes remain enigmatic. The present study illustrates, in detail and for the first time, the course of signal receiving, processing, and implementation by PML bodies in response to IFN and virus infection.Promyelocytic leukemia protein (PML) bodies are implicated in one of the key pathways in the establishment of antiviral status in response to interferon (IFN), yet the molecular mechanisms bridging the cross talk remain elusive.

Herein, we report that a major constitutive component of the PML body, Sp100A, is ubiquitously located in the cytosol of various cell types and is an immediate responder to multiple extracellular stimuli, including virus infection, IFN, epidermal growth factor (EGF), glial cell-derived nerve factor (GDNF), etc., signaling through the phosphatidylinositol 3-kinase (PI3K) pathway. IFN-beta induces phosphorylation of Sp100A on Ser(188), which fortifies the binding of Sp100A to pyruvate kinase 2 (PKM2) and facilitates its nuclear importation through the extracellular signal-regulated kinase 1/2 (ERK1/2)-PKM2-PIN1-importin axes. Blocking PI3K pathway signaling or interference with the ERK1/2-PKM2-PIN1-importin axes independently hampers nuclear translocation of Sp100A in response to IFN, reflecting a dual-regulation mechanism governing this event. In the nucleus, Sp100A is enriched in the promoter regions of essential antiviral interferon-stimulated genes (ISGs), such as those coding for IFI16, OAS2, and RIG-I, and activates their transcription. Importantly, nuclear importation of Sp100A, but not accumulation of a mutant Sp100A that failed to respond to IFN, during infection potently enhanced transcription of these antiviral ISGs and restricted virus propagation. These findings depict a novel IFN response mechanism by PML bodies in the cytosol and shed light on the complex sensing-regulatory network of PML bodies.IMPORTANCE PML bodies sit at the center stage of various important biological processes; however, the signal transduction networks of these macromolecular protein complexes remain enigmatic. The present study illustrates, in detail and for the first time, the course of signal receiving, processing, and implementation by PML bodies in response to IFN and virus infection. It shows that PML body constitutive component Sp100A was phosphorylated on Ser(188) by IFN signaling through the PI3K pathway in the cytosol, cotranslocated into the nucleus with PKM2, enriched on the promoter regions of essential antiviral ISGs such as those coding for IFI16, RIG-I, OAS2, etc., and mediating their transcriptional activation.

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Many kinds of oxidized proteins are present more in the urine of the elderly

Clinical proteomics. 2022. Liu, YT et al. Beijing Normal Univ, Dept Biochem & Mol Biol, Beijing Key Lab Gene Engn Drug & Biotechnol, Beijing 100875, Peoples R China

ABSTRACT: Background Many studies have shown an association between aging and oxidation. To our knowledge, there have been no studies exploring aging-related urine proteome modifications. The purpose of this study was to explore differences in global chemical modifications of urinary protein at different ages. Methods Discovery (n=38) cohort MS data including children, young and old groups were downloaded from three published studies, and this data was analyzed using open-pFind for identifying modifications.

Verification cohort human samples (n=28) including young, middle-aged, and old groups, rat samples (n=7) at three-time points after birth, adulthood, and old age were collected and processed in the laboratory simultaneously based on label-free quantification combined with pFind. Results Discovery cohort: there were 28 kinds of differential oxidations in the old group that were higher than those in the young or children group in. Verification cohort: there were 17 kinds of differential oxidations of 49 oxidized proteins in the middle and old groups, which were significantly higher than those in the young group. Both oxidations and oxidized proteins distinguished different age groups well. There were also 15 kinds of differential oxidations in old age higher than others in the rat cohort. The results showed that the validation experiment was basically consistent with the results of the discovery experiment, showing that the level of oxidized proteins in urine increased significantly with age. Conclusions Our study is the first to show that oxidative proteins occur in urine and that oxidations are higher in older than younger ages. Perhaps improving the degree of excretion of oxidative protein in vivo through the kidney is helpful for maintaining the homeostasis of the body's internal environment, delaying aging and the occurrence of senile diseases.

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The effect of lactulose thermal degradation products on β-lactoglobulin: linear-, loop-, and cross-link structural modifications and reduced digestibility

FOOD CHEMISTRY. 2022. Dong, L et al. Nankai Univ, Sch Med, Tianjin Key Lab Food Sci & Hlth, Tianjin 300071, Peoples R China

ABSTRACT: The thermal degradation products of lactulose and the interaction between lactulose and beta-lactoglobulin (beta Lg) were investigated in a thermal model system. Lactulose was thermally degraded into fructose and galactose, which were further degraded into methylglyoxal, glyoxal, 3-deoxyglucosone, and 2, 3-butanedione via heating. After incubating with lactulose, the structure of beta Lg was changed, which manifested by the formation of new band with doubled the molecular weight of beta Lg in the mobility spectrum and the changes in the internal fluo-rescence spectrum.

Furthermore, the lysine and arginine residues of beta Lg were confirmed to be the modification sites of the thermal degradation products of lactulose, and the modification types of linear-, loop-, and cross -linked were detected. The digestibility of beta Lg incubated with lactulose was significantly decreased due to the modification of trypsin digestion sites and the formation of cross-linked conjunctions. Therefore, the adverse effects of lactulose application in thermally processed foods should be concerned.

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FBB18 participates in preassembly of almost all axonemal dyneins independent of R2TP complex

PLoS genetics. 2022. Wang, LM et al. Qingdao Natl Lab Marine Sci & Technol, Lab Marine Biol & Biotechnol, Qingdao, Shandong, Peoples R China; Tsinghua Univ, Sch Life Sci, MOE Key Lab Prot Sci, Beijing, Peoples R China

ABSTRACT: Assembly of dynein arms requires cytoplasmic processes which are mediated by dynein preassembly factors (DNAAFs). CFAP298, which is conserved in organisms with motile cilia, is required for assembly of dynein arms but with obscure mechanisms. Here, we show that FBB18, a Chlamydomonas homologue of CFAP298, localizes to the cytoplasm and functions in folding/stabilization of almost all axonemal dyneins at the early steps of dynein preassembly. Mutation of FBB18 causes no or short cilia accompanied with partial loss of both outer and inner dynein arms.

Comparative proteomics using N-15 labeling suggests partial degradation of almost all axonemal dynein heavy chains (DHCs). A mutant mimicking a patient variant induces particular loss of DHC alpha. FBB18 associates with 9 DNAAFs and 14 out of 15 dynein HCs but not with IC1/IC2. FBB18 interacts with RuvBL1/2, components of the HSP90 co-chaperone R2TP complex but not the holo-R2TP complex. Further analysis suggests simultaneous formation of multiple DNAAF complexes involves dynein folding/stability and thus provides new insights into axonemal dynein preassembly.

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HRS phosphorylation drives immunosuppressive exosome secretion and restricts CD8+ T-cell infiltration into tumors

Nature communications. 2022. Guan, L et al. Univ Penn, Sch Arts & Sci, Dept Biol, Philadelphia, PA 19104 USA

ABSTRACT: The lack of tumor infiltration by CD8(+) T cells is associated with poor patient response to anti-PD-1 therapy. Understanding how tumor infiltration is regulated is key to improving treatment efficacy. Here, we report that phosphorylation of HRS, a pivotal component of the ESCRT complex involved in exosome biogenesis, restricts tumor infiltration of cytolytic CD8(+) T cells. Following ERK-mediated phosphorylation, HRS interacts with and mediates the selective loading of PD-L1 to exosomes, which inhibits the migration of CD8(+) T cells into tumors.

In tissue samples from patients with melanoma, CD8(+) T cells are excluded from the regions where tumor cells contain high levels of phosphorylated HRS. In murine tumor models, overexpression of phosphorylated HRS increases resistance to anti-PD-1 treatment, whereas inhibition of HRS phosphorylation enhances treatment efficacy. Our study reveals a mechanism by which phosphorylation of HRS in tumor cells regulates anti-tumor immunity by inducing PD-L1(+) immunosuppressive exosomes, and suggests HRS phosphorylation blockade as a potential strategy to improve the efficacy of cancer immunotherapy.Lack of CD8(+) T-cell infiltration into solid tumors is associated with poor responsiveness to immune checkpoint therapy (ICT). Here, the authors show that blocking the phosphorylation of HRS to reduce the induction of immunosuppressive exosomes promotes CD8(+) T-cell infiltration into tumors and enhances the efficacy of ICT in mouse melanoma models.

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Orally efficacious lead of the AVG inhibitor series targeting a dynamic interface in the respiratory syncytial virus polymerase

Science Advances. 2022. Sourimant, J et al. Georgia State Univ, Ctr Translat Antiviral Res, Inst Biomed Sci, Atlanta, GA 30303 USA

ABSTRACT: Respiratory syncytial virus (RSV) is a leading cause of lower respiratory infections in infants and the immuno-compromised, yet no efficient therapeutic exists. We have identified the AVG class of allosteric inhibitors of RSV RNA synthesis. Here, we demonstrate through biolayer interferometry and in vitro RNA-dependent RNA polymerase (RdRP) assays that AVG compounds bind to the viral polymerase, stalling the polymerase in initiation conformation. Resistance profiling revealed a unique escape pattern, suggesting a discrete docking pose.

Affinity mapping using photoreactive AVG analogs identified the interface of polymerase core, capping, and connector domains as a molecular target site. A first-generation lead showed nanomolar potency against RSV in human airway epithelium organoids but lacked in vivo efficacy. Docking pose-informed synthetic optimization generated orally efficacious AVG-388, which showed potent efficacy in the RSV mouse model when administered therapeutically. This study maps a druggable target in the RSV RdRP and establishes clinical potential of the AVG chemotype against RSV disease.

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Ac-LysargiNase efficiently helps genome reannotation of Mycolicibacterium smegmatis MC2 155

Journal of Proteomics. 2022. Zhu, HM et al. Chinese Acad Med Sci, Inst Life, Beijing Proteome Res Ctr,State Key Lab Prote, Natl Ctr Prot Sci Beijing,Res Unit Prote & Res &, Beijing 102206, Peoples R China

ABSTRACT: Accurate genome annotation, the foundation of life science research in the genome era, is hampered by limited known gene models, nonstandard start codons, and the limited homology of annotated genes in other organisms. LysargiNase mirrors trypsin at the cleavage sites, providing the opportunity to identify peptides other than tryptic peptides. In this study, we used an in-house developed acetylated LysargiNase (Ac-LysargiNase) with higher activity and stability in non-pathogenic Mycolicibacterium smegmatis MC2 155 to supplement the widely used trypsin in proteomic studies.

We identified 27,582 peptides from 3844 annotated proteins and 332 novel genome search-specific peptides (GSSPs). Among these GSSPs, 88 peptides were annotated in another M.smeg-matis genome database, and 41 were verified as novel peptides by predicted theoretical spectra and their cor-responding 15N-labeling spectra. Further analysis revealed that 17 verified GSSPs corrected the N-terminus of the 13 annotated genes. The other 24 verified GSSPs helped identify 17 novel open reading frames (ORFs) missed in previously annotated M. smegmatis genomes. Among these novel ORFs, four relatively small proteins with amino acid residues less than 100 and three were precisely identified with C-terminal peptides. Ac-LysargiNase helps with genome reannotation by identifying new genes and events in proteogenomic studies. Significance: Correct genomic annotation is vital in the field of life sciences. The nonstandard start codons seriously affect the confirmation of the translation initiation sites (TISs) of an open reading frame (ORF), and unknown structural genes are easily missed in automated gene prediction. Although proteogenomics presents new avenues for validating gene expression and gene structure refinement based on conventional tryptic pep-tides, determining the TISs and potential encoding genes is complicated. Thus, validation of TISs and encoding ORFs is crucial and urgent. Therefore, we recommend Ac-LysargiNase, a mirror enzyme of trypsin that can identify additional novel peptides for N-terminal correction and ORF identification.

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Deephos: predicted spectral database search for TMT-labeled phosphopeptides and its false discovery rate estimation

Bioinformatics. 2022. Na, S et al. Hanyang Univ, Inst Artificial Intelligence Res, Seoul 04763, South Korea; Hanyang Univ, Dept Comp Sci, Seoul 04763, South Korea

ABSTRACT: Motivation: Tandem mass tag (TMT)-based tandem mass spectrometry (MS/MS) has become the method of choice for the quantification of post-translational modifications in complex mixtures. Many cancer proteogenomic studies have highlighted the importance of large-scale phosphopeptide quantification coupled with TMT labeling. Herein, we propose a predicted Spectral DataBase (pSDB) search strategy called Deephos that can improve both sensitivity and specificity in identifying MS/MS spectra of TMT-labeled phosphopeptides.Results: With deep learning-based fragment ion prediction, we compiled a pSDB of TMT-labeled phosphopeptides generated from similar to 8000 human phosphoproteins annotated in UniProt.

Deep learning could successfully recognize the fragmentation patterns altered by both TMT labeling and phosphorylation. In addition, we discuss the decoy spectra for false discovery rate (FDR) estimation in the pSDB search. We show that FDR could be inaccurately estimated by the existing decoy spectra generation methods and propose an innovative method to generate decoy spectra for more accurate FDR estimation. The utilities of Deephos were demonstrated in multi-stage analyses (coupled with database searches) of glioblastoma, acute myeloid leukemia and breast cancer phosphoproteomes.

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EGLN1 prolyl hydroxylation of hypoxia-induced transcription factor HIF1$\alpha$ is repressed by SET7-catalyzed lysine methylation

Journal of Biological Chemistry. 2022. Tang, JH et al. Univ Chinese Acad Sci, Beijing, Peoples R China; Chinese Acad Sci, Innovat Seed Design, Wuhan, Peoples R China; Hubei Hongshan Lab, Wuhan, Peoples R China; Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan, Peoples R China

ABSTRACT: Egg-laying defective nine 1 (EGLN1) functions as an oxygen sensor to catalyze prolyl hydroxylation of the transcription factor hypoxia-inducible factor-1 alpha under normoxia conditions, leading to its proteasomal degradation. Thus, EGLN1 plays a central role in the hypoxia-inducible factor-mediated hypoxia signaling pathway; however, the posttranslational modifications that control EGLN1 function remain largely unknown. Here, we identified that a lysine monomethylase, SET7, catalyzes EGLN1 methylation on lysine 297, resulting in the repression of EGLN1 activity in catalyzing prolyl hydroxylation of hypoxia-inducible factor-1 alpha.

Notably, we demonstrate that the methylation mimic mutant of EGLN1 loses the capability to suppress the hypoxia signaling pathway, leading to the enhancement of cell proliferation and the oxygen consumption rate. Collectively, our data identify a novel modification of EGLN1 that is critical for inhibiting its enzymatic activity and which may benefit cellular adaptation to conditions of hypoxia.

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Glyco-Decipher enables glycan database-independent peptide matching and in-depth characterization of site-specific N-glycosylation

Nature Communications. 2022. Fang, Z et al. Chinese Acad Sci, Dalian Inst Chem Phys, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Peoples R China; Univ Chinese Acad Sci, Beijing 100049, Peoples R China; Dalian Univ Technol, Sch Bioengn, Dalian 116024, Peoples R China

ABSTRACT: Glycopeptides with unusual glycans or poor peptide backbone fragmentation in tandem mass spectrometry are unaccounted for in typical site-specific glycoproteomics analysis and thus remain unidentified. Here, we develop a glycoproteomics tool, Glyco-Decipher, to address these issues. Glyco-Decipher conducts glycan database-independent peptide matching and exploits the fragmentation pattern of shared peptide backbones in glycopeptides to improve the spectrum interpretation. We benchmark Glyco-Decipher on several large-scale datasets, demonstrating that it identifies more peptide-spectrum matches than Byonic, MSFragger-Glyco, StrucGP and pGlyco 3.0, with a 33.5%-178.5% increase in the number of identified glycopeptide spectra.

The database-independent and unbiased profiling of attached glycans enables the discovery of 164 modified glycans in mouse tissues, including glycans with chemical or biological modifications. By enabling in-depth characterization of site-specific protein glycosylation, Glyco-Decipher is a promising tool for advancing glycoproteomics analysis in biological research.Poor peptide fragmentation and unusual glycan structures limit mass spectrometry-based analysis of intact N-glycopeptides. Here, the authors develop Glyco-Decipher, a glycan-independent peptide search tool, to tackle these issues and improve the coverage of site-specific glycan analysis.

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Crucial role and mechanism of transcription-coupled DNA repair in bacteria

Nature. 2022. Bharati, BK et al. NYU, Sch Med, Dept Biochem & Mol Pharmacol, New York, NY 10012 USA; NYU, Sch Med, Howard Hughes Med Inst, New York, NY 10012 USA; Chinese Acad Sci, Inst Plant Physiol & Ecol, Ctr Excellence Mol Plant Sci, Key Lab Synthet Biol, Shanghai, Peoples R China

ABSTRACT: Transcription-coupled DNA repair (TCR) is presumed to be a minor sub-pathway of nucleotide excision repair (NER) in bacteria. Global genomic repair is thought to perform the bulk of repair independently of transcription. TCR is also believed to be mediated exclusively by Mfd-a DNA translocase of a marginal NER phenotype(1-3). Here we combined in cellulo cross-linking mass spectrometry with structural, biochemical and genetic approaches to map the interactions within the TCR complex (TCRC) and to determine the actual sequence of events that leads to NER in vivo.

We show that RNA polymerase (RNAP) serves as the primary sensor of DNA damage and acts as a platform for the recruitment of NER enzymes. UvrA and UvrD associate with RNAP continuously, forming a surveillance pre-TCRC. In response to DNA damage, pre-TCRC recruits a second UvrD monomer to form a helicase-competent UvrD dimer that promotes backtracking of the TCRC. The weakening of UvrD-RNAP interactions renders cells sensitive to genotoxic stress. TCRC then recruits a second UvrA molecule and UvrB to initiate the repair process. Contrary to the conventional view, we show that TCR accounts for the vast majority of chromosomal repair events; that is, TCR thoroughly dominates over global genomic repair. We also show that TCR is largely independent of Mfd. We propose that Mfd has an indirect role in this process: it participates in removing obstructive RNAPs in front of TCRCs and also in recovering TCRCs from backtracking after repair has been completed.

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Mucus sialylation determines intestinal host-commensal homeostasis

Cell. 2022. Yao, YK et al. NIAID, Mol Dev Immune Syst Sect, Lab Immune Syst Biol & Clin Genom Program, NIH, Bethesda, MD 20892 USA; NCI, Expt Immunol Branch, NIH, Bethesda, MD 20892 USA

ABSTRACT: Intestinal mucus forms the first line of defense against bacterial invasion while providing nutrition to support microbial symbiosis. How the host controls mucus barrier integrity and commensalism is unclear. We show that terminal sialylation of glycans on intestinal mucus by ST6GALNAC1 (ST6), the dominant sialyltransferase specifically expressed in goblet cells and induced by microbial pathogen-associated molecular patterns, is essential for mucus integrity and protecting against excessive bacterial proteolytic degradation.

Glycoproteomic profiling and biochemical analysis of ST6 mutations identified in patients show that decreased sialylation causes defective mucus proteins and congenital inflammatory bowel disease (IBD). Mice harboring a patient ST6 mutation have compromised mucus barriers, dysbiosis, and susceptibility to intestinal inflammation. Based on our understanding of the ST6 regulatory network, we show that treatment with sialylated mucin or a Foxo3 inhibitor can ameliorate IBD.

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Itaconate and itaconate derivatives target JAK1 to suppress alternative activation of macrophages

Cell Metabolism. 2022. Runtsch, MC et al. Trinity Coll Dublin, Sch Biochem & Immunol, Trinity Biomed Sci Inst, 152-160 Pearse St, Dublin D02 R590, Ireland

ABSTRACT: The Krebs cycle-derived metabolite itaconate and its derivatives suppress the inflammatory response in pro-inflammatory "M1" macrophages. However, alternatively activated "M2" macrophages can take up itaconate. We therefore examined the effect of itaconate and 4-octyl itaconate (OI) on M2 macrophage activation. We demonstrate that itaconate and OI inhibit M2 polarization and metabolic remodeling. Examination of IL-4 signaling revealed inhibition of JAK1 and STAT6 phosphorylation by both itaconate and OI.

JAK1 activation was also inhibited by OI in response to IL-13, interferon-beta, and interferon-gamma in macrophages and in T helper 2 (Th2) cells. Importantly, JAK1 was directly modified by itaconate derivatives at multiple residues, including cysteines 715, 816, 943, and 1130. Itaconate and OI also inhibited JAK1 kinase activity. Finally, OI treatment suppressed M2 macrophage polarization and JAK1 phosphorylation in vivo. We therefore identify itaconate and OI as JAK1 inhibitors, suggesting a new strategy to inhibit JAK1 in M2 macrophage-driven diseases.

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Identification of Microproteins in Hep3B Cells at Different Cell Cycle Stages

Journal of Proteome Research. 2022. Li, B et al. Cent China Normal Univ, Sch Life Sci, Wuhan 430079, Hubei, Peoples R China; Cent China Normal Univ, Hubei Key Lab Genet Regulat & Integrat Biol, Wuhan 430079, Hubei, Peoples R China

ABSTRACT: Microproteins are generated from small open reading frames andturn out to play various vital biological functions. As an essential biological event ofeukaryotic cells, the cell cycle is involved in cell replication and division. For such ahighly regulated event, microproteins associated with cell cycle regulation remainedunclarified. Utilizing a combination of bottom-up and top-down proteomics, weanalyzed microproteins at specific cell cycle stages of Hep3B cells. A total of 657microproteins were identified under three cell cycle stages, including 151 in the G0/G1 stage, 163 in the S stage, and 132 in the G2/M stage.

The annotation of these microproteins showed their cell cycle-specific functions, such as translation, nuclear assembly,chromatin organization, and the G2/M transition of the mitotic cell cycle. Meanwhile, more than 50% of identified microproteinswere ncRNA-encoded. These nonannotated novel microproteins contain several function domains, such as the nucleosidediphosphate kinase domain, the high mobility group domain, and the DNA-binding domain. This suggested the potential functionsof these novel microproteins in specific cell cycle stages. This study presented a large-scale profile of microproteins at different cellcycle stages from Hep3B and may provide new perspectives on the regulation mechanism of the cell cycle. Liquid chromatography-mass spectrometry data were deposited to ProteomeXchange using the identifier PXD030286.

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Targeting tumor endothelial hyperglycolysis enhances immunotherapy through remodeling tumor microenvironment

Acta Pharmaceutica Sinica B. 2022. Shan, YL et al. China Pharmaceut Univ, Key Lab Drug Metab & Pharmacokinet, State Key Lab Nat Med, Nanjing 210009, Peoples R China; Nanjing Med Univ, Dept Hematol & Oncol, Affiliated Huaian 1 Peoples Hosp, Huaian 223300, Peoples R China

ABSTRACT: Vascular abnormality isa hallmark of most solid tumors and facilitates immune evasion. Targeting the abnormal metabolism of tumor endothelial cells (TECs) may provide an opportunity to improve the outcome of immunotherapy. Here, in comparison to vascular endothelial cells from adjacent peritumoral tissues in patients with colorectal cancer (CRC), TECs presented enhanced glycolysis with higher glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. Then an unbiased screening identified that osimertinib could modify the GAPDH and thus inhibit its activity in TECs.

Low-dose osimertinib treatment caused tumor regression with vascular normalization and increased infiltration of immune effector cells in tumor, which was due to the reduced secretion of lactate from TECs by osimertinib through the inhibition of GAPDH. Moreover, osimertinib and anti-PD-1 blockade synergistically retarded tumor growth. This study provides a potential strategy to enhance immunotherapy by targeting the abnormal metabolism of TECs.

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Histone deacetylase 3 contributes to the antiviral innate immunity of macrophages by interacting with FOXK1 to regulate STAT1/2 transcription

Cell Reports. 2022. Yang, LP et al. Zhejiang Univ, Affiliated Hosp 2, Dept Infect Dis, Sch Med, Hangzhou 310009, Peoples R China; Chinese Acad Med Sci & Peking Union Med Coll, Inst Syst Med, Beijing 100005, Peoples R China; Suzhou Inst Syst Med, Suzhou 215123, Peoples R China; Univ Calif Los Angeles, Dept Microbiol Immunol & Mol Genet, Los Angeles, CA 90095 USA

ABSTRACT: It is well known that interferon (IFN)-alpha/-beta activates the JAK/STAT signaling pathway and suppresses viral replication through the induction of IFN stimulated genes (ISGs). Here, we report that knockout of HDAC3 from macrophages results in the decreased expression of STAT1 and STAT2, leading to defective antiviral immunity in cells and mice. Further studies show that HDAC3 interacts with a conserved transcription factor Forkhead Box K1 (FOXK1), co-localizes with FOXK1 at the promoter of STAT1 and STAT2, and is required for protecting FOXK1 from lysosomal system-mediated degradation, FOXK1 -deficient macrophages also show low STAT1 and STAT2 expression with defective responses to viruses.

Thus, our studies uncover the biological importance of HDAC3 in regulating the antiviral immunity of macrophages through interacting with FOXK1 to regulate the expression of STAT1 and STAT2.

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Shelterin is a Dimeric Complex with Extensive Structural Heterogeneity

PNAS. 2022. Zinder, John C. et al. Rockefeller Univ, Lab Cell Biol & Genet, New York, NY 10065 USA; Rockefeller Univ, Lab Mol Elect Microscopy, New York, NY 10065 USA

ABSTRACT: Human shelterin is a six-subunit complex-composed of TRF1, TRF2, Rap1, TIN2, TPP1, and POT1-that binds telomeres, protects them from the DNA-damage response, and regulates the maintenance of telomeric DNA. Although high-resolution structures have been generated of the individual structured domains within shelterin, the architecture and stoichiometry of the full complex are currently unknown. Here, we report the purification of shelterin subcomplexes and reconstitution of the entire complex using full-length, recombinant subunits.

By combining negative-stain electron microscopy (EM), cross-linking mass spectrometry (XLMS), AlphaFold modeling, mass photometry, and native mass spectrometry (MS), we obtain stoichiometries as well as domain-scale architectures of shelterin subcomplexes and determine that they feature extensive conformational heterogeneity. For POT1/TPP1 and POT1/TPP1/TIN2, we observe high variability in the positioning of the POT1 DNA-binding domain, the TPP1 oligonucleotide/oligosaccharide-binding (OB) fold, and the TIN2 TRFH domain with respect to the C-terminal domains of POT1. Truncation of unstructured linker regions in TIN2, TPP1, and POT1 did not reduce the conformational variability of the heterotrimer. Shelterin and TRF1-containing subcomplexes form fully dimeric stoichiometries, even in the absence of DNA substrates. Shelterin and its subcomplexes showed extensive conformational variability, regardless of the presence of DNA substrates. We conclude that shelterin adopts a multitude of conformations and argue that its unusual architectural variability is beneficial for its many functions at telomeres.

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Iso-seco-tanapartholide activates Nrf2 signaling pathway through Keap1 modification and oligomerization to exert anti-inflammatory effects

Free Radical Biology and Medicine. 2022. Zhu, DR et al. China Pharmaceut Univ, Nanjing 210009, Peoples R China

ABSTRACT: Covalent modification of Keap1 results in reducing ubiquitination and the accumulation of Nrf2, which subsequently initiates the transcription of cellular anti-oxidant and anti-inflammatory genes. Iso-seco-tanapartholide (IST), a sesquiterpene isolated from the traditional Chinese medicine Artemisia argyi, had been reported to possess NF-Kappa B inhibitory activity. However, its deep anti-inflammatory effects and direct target have never been reported. Here we show that IST activated Nrf2 and increased its target gene expression.

In particular, LPScaused inflammation in vitro and in vivo was mitigated by IST-induced Nrf2 activation but aggravated by Nrf2 inhibition. Mechanically, IST targeted Keap1 proteins via alkylating its cysteine residues 151, 273, 288, and so on. Subsequently, the modifying agent IST was displaced by intermolecular sulfhydryl disulfide interchange to lead to a disulfide dimer of Keap1. The resulting conformational change of Keap1 liberated Nrf2 from sequestration and allowed it translocation to the nucleus to activate the transcriptional program. Further studies demonstrated that Keap1 dimer formation contributed to the anti-inflammatory effects of IST. Taken together, our findings reveal a new mechanism for Nrf2 activation and provide a potential lead compound to treat inflammatory diseases through targeting Keap1.

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Deep N-terminomics of Mycobacterium tuberculosis H37Rv extensively correct annotated encoding genes

Genomics. 2022. Shi, JH et al. Chinese Acad Med Sci, Beijing Proteome Res Ctr, Natl Ctr Prot Sci Beijing,State Key Lab Proteom, Res Unit Prote & Res & Dev New Drug,Inst Lifeom, Beijing 102206, Peoples R China

ABSTRACT: Mycobacterium tuberculosis (MTB) is a severe causing agent of tuberculosis (TB). Although H37Rv, the type strain of M. tuberculosis was sequenced in 1998, annotation errors of encoding genes have been frequently reported in hundreds of papers. This phenomenon is particularly severe at the 5 ' end of the genes. Here, we applied a TMPP [(N-Succinimidyloxycarbonylmethyl) tris (2,4,6-trimethoxyphenyl) phosphonium bromide] labeling combined with StageTip separating strategy on M. tuberculosis H37Rv to characterize the N-terminal start sites of its annotated encoding genes.

Totally, 1047 proteins were identified with 2058 TMPP labeled N-terminal peptides from all the 2625 mass spectrometer (MS) sequenced proteins. Comparative genomics analysis allowed the reannotation of 43 proteins' N-termini in H37Rv and 762 proteins in Mycobacteriaceae. All revised N-termini start sites were distributed in 5'-UTR of annotated genes due to over-annotation of previous N-terminal initiation codon, especially the ATG. In addition, we identified and verified a novel gene Rv1078A in +3 frame different from the annotated gene Rv1078 in +2 frame. Altogether, our findings contribute to the better understanding of N-terminal of H37Rv and other species from Mycobacteriaceae that can assist future studies on biological study.

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Structure of human chromatin-remodelling PBAF complex bound to a nucleosome

Nature. 2022. Yuan, JJ et al. Tsinghua Univ, MOE Key Lab Prot Sci, Beijing, Peoples R China; Tsinghua Univ, Sch Life Sci, Beijing, Peoples R China; Beijing Adv Innovat Ctr Struct Biol, Tsinghua Peking Joint Ctr Life Sci, Beijing, Peoples R China

ABSTRACT: DNA wraps around the histone octamer to form nucleosomes(1), the repeating unit of chromatin, which create barriers for accessing genetic information. Snf2-like chromatin remodellers couple the energy of ATP binding and hydrolysis to reposition and recompose the nucleosome, and have vital roles in various chromatin-based transactions(2,3). Here we report the cryo-electron microscopy structure of the 12-subunit human chromatin-remodelling polybromo-associated BRG1-associated factor (PBAF) complex bound to the nucleosome.

The motor subunit SMARCA4 engages the nucleosome in the active conformation, which reveals clustering of multiple disease-associated mutations at the interfaces that are essential for chromatin-remodelling activity. SMARCA4 recognizes the H2A-H2B acidic pocket of the nucleosome through three arginine anchors of the Snf2 ATP coupling (SnAc) domain. PBAF shows notable functional modularity, and most of the auxiliary subunits are interwoven into three lobe-like submodules for nucleosome recognition. The PBAF-specific auxiliary subunit ARID2 acts as the structural core for assembly of the DNA-binding lobe, whereas PBRM1, PHF10 and BRD7 are collectively incorporated into the lobe for histone tail binding. Together, our findings provide mechanistic insights into nucleosome recognition by PBAF and a structural basis for understanding SMARCA4-related human diseases.

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Structural basis of SNAPc-dependent snRNA transcription initiation by RNA polymerase II

Nature Structural & Molecular Biology. 2022. Rengachari, S et al. Inst Canc Res, Div Struct Biol, London, England; Human Technopole, Milan, Italy; Max Planck Inst Multidisciplinary Sci, Dept Mol Biol, Gottingen, Germany

ABSTRACT: Rengachari et al. provide a structural investigation of Pol II initiation at snRNA gene promoters and find that the snRNA-activating protein complex enables DNA opening and transcription initiation independent of TFIIE and TFIIH in vitro.RNA polymerase II (Pol II) carries out transcription of both protein-coding and non-coding genes. Whereas Pol II initiation at protein-coding genes has been studied in detail, Pol II initiation at non-coding genes, such as small nuclear RNA (snRNA) genes, is less well understood at the structural level.

Here, we study Pol II initiation at snRNA gene promoters and show that the snRNA-activating protein complex (SNAPc) enables DNA opening and transcription initiation independent of TFIIE and TFIIH in vitro. We then resolve cryo-EM structures of the SNAPc-containing Pol IIpre-initiation complex (PIC) assembled on U1 and U5 snRNA promoters. The core of SNAPc binds two turns of DNA and recognizes the snRNA promoter-specific proximal sequence element (PSE), located upstream of the TATA box-binding protein TBP. Two extensions of SNAPc, called wing-1 and wing-2, bind TFIIA and TFIIB, respectively, explaining how SNAPc directs Pol II to snRNA promoters. Comparison of structures of closed and open promoter complexes elucidates TFIIH-independent DNA opening. These results provide the structural basis of Pol II initiation at non-coding RNA gene promoters.

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Structural basis of Tom20 and Tom22 cytosolic domains as the human TOM complex receptors

PNAS. 2022. Su, JY et al. Southern Univ Sci & Technol, Dept Biol, Shenzhen 518055, Guangdong, Peoples R China; Tsinghua Univ, Beijing Adv Innovat Ctr Struct Biol, Beijing Frontier Res Ctr Biol Struct, State Key Lab Membrane Biol,Sch Life Sci, Beijing 100084, Peoples R China

ABSTRACT: Mitochondrial preproteins synthesized in cytosol are imported into mitochondria by a multisubunit translocase of the outer membrane (TOM) complex. Functioned as the receptor, the TOM complex components, Tom 20, Tom22, and Tom70, recognize the presequence and further guide the protein translocation. Their deficiency has been linked with neurodegenerative diseases and cardiac pathology. Although several structures of the TOM complex have been reported by cryoelectron microscopy (cryo-EM), how Tom22 and Tom20 function as TOM receptors remains elusive.

Here we determined the structure of TOM core complex at 2.53 angstrom and captured the structure of the TOM complex containing Tom22 and Tom20 cytosolic domains at 3.74 angstrom. Structural analysis indicates that Tom20 and Tom22 share a similar three-helix bundle structural feature in the cytosolic domain Further structure-guided biochemical analysis reveals that the Tom22 cytosolic domain is responsible for binding to the presequence, and the helix H1 is critical for this binding. Altogether, our results provide insights into the functional mechanism of the TOM complex recognizing and transferring preproteins across the mitochondrial membrane.

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Juxtaposition of Bub1 and Cdc20 on phosphorylated Mad1 during catalytic mitotic checkpoint complex assembly

Nature communications. 2022. Fischer, ES et al. MRC Lab Mol Biol, Cambridge Biomed Campus,Francis Crick Ave, Cambridge CB2 0QH, England

ABSTRACT: Formation of the mitotic checkpoint complex (MCC) is catalysed by a phosphorylation-dependent scaffold. This work provides structural details of how a tripartite Mad1:Bub1:Cdc20 complex presents Cdc20 to Mad2, triggering open-to-closed conversion of Mad2 to assemble the MCC.In response to improper kinetochore-microtubule attachments in mitosis, the spindle assembly checkpoint (SAC) assembles the mitotic checkpoint complex (MCC) to inhibit the anaphase-promoting complex/cyclosome, thereby delaying entry into anaphase.

The MCC comprises Mad2:Cdc20:BubR1:Bub3. Its assembly is catalysed by unattached kinetochores on a Mad1:Mad2 platform. Mad1-bound closed-Mad2 (C-Mad2) recruits open-Mad2 (O-Mad2) through self-dimerization. This interaction, combined with Mps1 kinase-mediated phosphorylation of Bub1 and Mad1, accelerates MCC assembly, in a process that requires O-Mad2 to C-Mad2 conversion and concomitant binding of Cdc20. How Mad1 phosphorylation catalyses MCC assembly is poorly understood. Here, we characterized Mps1 phosphorylation of Mad1 and obtained structural insights into a phosphorylation-specific Mad1:Cdc20 interaction. This interaction, together with the Mps1-phosphorylation dependent association of Bub1 and Mad1, generates a tripartite assembly of Bub1 and Cdc20 onto the C-terminal domain of Mad1 (Mad1(CTD)). We additionally identify flexibility of Mad1:Mad2 that suggests how the Cdc20:Mad1(CTD) interaction brings the Mad2-interacting motif (MIM) of Cdc20 near O-Mad2. Thus, Mps1-dependent formation of the MCC-assembly scaffold functions to position and orient Cdc20 MIM near O-Mad2, thereby catalysing formation of C-Mad2:Cdc20.

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Structure of Arabidopsis SOQ1 lumenal region unveils C-terminal domain essential for negative regulation of photoprotective qH

Nature Plants. 2022. Yu, GM et al. Umea Univ, Dept Plant Physiol, Umea Plant Sci Ctr UPSC, Umea, Sweden; Chinese Acad Sci, Inst Biophys, CAS Ctr Excellence Biomacromol, Natl Lab Biomacromol, Beijing, Peoples R China

ABSTRACT: Genetic, biochemical and high-resolution structural studies of chloroplast protein SOQ1 reveal the existence of a C-terminal lumenal domain with potential redox function and its essential role for suppressing photoprotection in plants.Non-photochemical quenching (NPQ) plays an important role for phototrophs in decreasing photo-oxidative damage. qH is a sustained form of NPQ and depends on the plastid lipocalin (LCNP). A thylakoid membrane-anchored protein SUPPRESSOR OF QUENCHING1 (SOQ1) prevents qH formation by inhibiting LCNP.

SOQ1 suppresses qH with its lumen-located thioredoxin (Trx)-like and NHL domains. Here we report structural data, genetic modification and biochemical characterization of Arabidopsis SOQ1 lumenal domains. Our results show that the Trx-like and NHL domains are associated together, with the cysteine motif located at their interface. Residue E859, required for SOQ1 function, is pivotal for maintaining the Trx-NHL association. Importantly, the C-terminal region of SOQ1 forms an independent beta-stranded domain that has structural homology to the N-terminal domain of bacterial disulfide bond protein D and is essential for negative regulation of qH. Furthermore, SOQ1 is susceptible to cleavage at the loops connecting the neighbouring lumenal domains both in vitro and in vivo, which could be a regulatory process for its suppression function of qH.

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Identification of an autoinhibitory, mitophagy-inducing peptide derived from the transmembrane domain of USP30

Autophagy. 2022. Qin, X et al. Pingshan Translat Med Ctr, Shenzhen Bay Lab, Shenzhen, Peoples R China; Peking Univ, Sch Chem Biol & Biotechnol, State Key Lab Chem Oncogen, Shenzhen Grad Sch, Shenzhen, Peoples R China; Tsinghua Univ, Tsinghua Shenzhen Int Grad Sch, Inst Biopharmaceut & Hlth Engn, Shenzhen, Peoples R China

ABSTRACT: The mitochondrial-anchored deubiquitinating enzyme USP30 (ubiquitin specific peptidase 30) antagonizes PRKN/parkin-mediated mitophagy, making it a potential target for treating Parkinson disease. However, few inhibitors targeting USP30 have been reported. Here, we report a novel peptide (Q14) derived from the transmembrane (TM) domain of USP30 that can target mitochondrial-anchored USP30 directly and increase mitophagy through two intriguing and distinct mechanisms: a novel autoinhibition mechanism in USP30 and accelerated autophagosome formation via the LC3-interacting region (LIR) of the Q14 peptide.

We identified the potential binding sites between the Q14 peptide and USP30 and postulated that an allosteric autoinhibition mechanism regulates USP30 activity. Furthermore, the LIR motif in the Q14 peptide offers additional binding with LC3 and accelerated autophagosome formation. The two mechanisms synergistically enhance mitophagy. Our work provides novel insight and direction to the design of inhibitors for USP30 or other deubiquitinating enzymes (DUBs).

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Activation of the essential kinase PDK1 by phosphoinositide-driven trans-autophosphorylation

Nature communications. 2022. Levina, A et al. Max Perutz Labs, Dept Struct & Computat Biol, Campus Vienna Bioctr 5, A-1030 Vienna, Austria; Med Univ Vienna, Dept Med Biochem, A-1090 Vienna, Austria

ABSTRACT: 3-phosphoinositide-dependent kinase 1 (PDK1) is an essential serine/threonine protein kinase, which plays a crucial role in cell growth and proliferation. It is often referred to as a 'master' kinase due to its ability to activate at least 23 downstream protein kinases implicated in various signaling pathways. In this study, we have elucidated the mechanism of phosphoinositide-driven PDK1 auto-activation. We show that PDK1 trans-autophosphorylation is mediated by a PIP3-mediated face-to-face dimer.

We report regulatory motifs in the kinase-PH interdomain linker that allosterically activate PDK1 autophosphorylation via a linker-swapped dimer mechanism. Finally, we show that PDK1 is autoinhibited by its PH domain and that positive cooperativity of PIP3 binding drives switch-like activation of PDK1. These results imply that the PDK1-mediated activation of effector kinases, including Akt, PKC, Sgk, S6K and RSK, many of whom are not directly regulated by phosphoinositides, is also likely to be dependent on PIP3 or PI(3,4)P-2.The essential protein kinase PDK1 is activated by phospoinositide-mediated dimerization and trans-autophosphorylation. Here, the authors show that in the absence of PIP3 or PI(3,4)P-2 phosphoinositides, PDK1 is maintained in an inactive, autoinhibited conformation in the cytosol.

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Conformational rearrangements upon start codon recognition in human 48S translation initiation complex

Nucleic Acids Research. 2022. Yi, Sung-Hui et al. Max Planck Inst Multidisciplinary Sci, Dept Struct Dynam, D-37077 Gottingen, Germany; Georg August Univ Gottingen, Inst Microbiol & Genet, Dept Mol Struct Biol, D-37077 Gottingen, Germany

ABSTRACT: Selection of the translation start codon is a key step during protein synthesis in human cells. We obtained cryo-EM structures of human 48S initiation complexes and characterized the intermediates of codon recognition by kinetic methods using eIF1A as a reporter. Both approaches capture two distinct ribosome populations formed on an mRNA with a cognate AUG codon in the presence of eIF1, eIF1A, eIF2-GTP-Met-tRNA(i)(Met) and eIF3. The 'open' 40S subunit conformation differs from the human 48S scanning complex and represents an intermediate preceding the codon recognition step.

The 'closed' form is similar to reported structures of complexes from yeast and mammals formed upon codon recognition, except for the orientation of eIF1A, which is unique in our structure. Kinetic experiments show how various initiation factors mediate the population distribution of open and closed conformations until 60S subunit docking. Our results provide insights into the timing and structure of human translation initiation intermediates and suggest the differences in the mechanisms of start codon selection between mammals and yeast.

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In-Depth In Vivo Crosslinking in Minutes by a Compact, Membrane-Permeable, and Alkynyl-Enrichable Crosslinker

Analytical Chemistry. 2022. Gao, H et al. Chinese Acad Sci, CAS Key Lab Separat Sci Analyt Chem, Natl Chromatog R&A Ctr, Dalian Inst Chem Phys, Dalian 116023, Liaoning, Peoples R China

ABSTRACT: Chemical crosslinking coupled with mass spectrometry (CXMS) has emerged as a powerful technique to obtain the dynamic conformations and interaction interfaces of protein complexes. Limited by the poor cell membrane permeability, chemical reactivity, and biocompatibility of crosslinkers, in vivo crosslinking to capture the dynamics of protein complexes with finer temporal resolution and higher coverage is attractive but challenging. In this work, a trifunctional crosslinker bis(succinimidyl) with propargyl tag (BSP), involving compact size, proper amphipathy, and enrichment capacity, was developed to enable better cell membrane permeability and efficient crosslinking in 5 min without obvious cellular interference.

Followed by a two-step enrichment method based on click chemistry at the peptide level, 13,098 crosslinked peptides (5068 inter-crosslinked peptides and 8030 intra-crosslinked peptides) were identified under the data threshold of peptide-spectrum matches (PSMs) >= 2 on the basic of the FDR control of 1%, which was the most comprehensive dataset for homo species cells by a non-cleavable crosslinker. Besides, the interactome network comprising 1519 proteins connected by 2913 interaction edges in various intracellular compartments, as well as 80S ribosome structural dynamics, were characterized, showing the great potential of our in vivo crosslinking approach in minutes. All these results demonstrated that our developed BSP could provide a valuable toolkit for the in-depth in vivo analysis of protein-protein interactions (PPIs) and protein architectures with finer temporal resolution.

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Mimicked synthetic ribosomal protein complex for benchmarking crosslinking mass spectrometry workflows

Nature Communications. 2022. Matzinger, Manuel et al. Austrian Acad Sci, Vienna BioCtr VBC, Inst Mol Biotechnol, Vienna, Austria; Vienna BioCtr VBC, Inst Mol Pathol IMP, Vienna, Austria

ABSTRACT: Cross-linking mass spectrometry has matured to a frequently used tool for the investigation of protein structures as well as interactome studies up to a system-wide level. The growing community generated a broad spectrum of applications, linker types, acquisition strategies and specialized data analysis tools, which makes it challenging to decide for an appropriate analysis workflow. Here, we report a large and flexible synthetic peptide library as reliable instrument to benchmark crosslink workflows.

Additionally, we provide a tool, IMP-X-FDR, that calculates the real, experimentally validated, FDR, compares results across search engine platforms and analyses crosslink properties in an automated manner. We apply the library with 6 commonly used linker reagents and analyse the data with 6 established search engines. We thereby show that the correct algorithm and search setting choice is highly important to improve identification rate and reliability. We reach identification rates of up to similar to 70 % of the theoretical maximum (i.e. 700 unique lysine-lysine cross-links) while maintaining a real false-discovery-rate of <3 % at cross-link level with high reproducibility, representatively showing that our test system delivers valuable and statistically solid results.

[more...]

Multistate structures of the MLL1-WRAD complex bound to H2B-ubiquitinated nucleosome

PNAS. 2022. Rahman, S et al. Johns Hopkins Univ, Dept Biophys & Biophys Chem, Sch Med, Baltimore, MD 21205 USA

ABSTRACT: The human Mixed Lineage Leukemia-1 (MLL1) complex methylates histone H3K4 to promote transcription and is stimulated by monoubiquitination of histone H2B. Recent structures of the MLL1-WRAD core complex, which comprises the MLL1 methyltransferase, WDR5, RbBp5, Ash2L, and DPY-30, have revealed variability in the docking of MLL1-WRAD on nucleosomes. In addition, portions of the Ash2L structure and the position of DPY30 remain ambiguous. We used an integrated approach combining cryoelectron microscopy (cryo-EM) and mass spectrometry cross-linking to determine a structure of the MLL1-WRAD complex bound to ubiquitinated nucleosomes.

The resulting model contains the Ash2L intrinsically disordered region (IDR), SPRY insertion region, Sdc1-DPY30 interacting region (SDI-motif), and the DPY30 dimer. We also resolved three additional states of MLL1-WRAD lacking one or more subunits, which may reflect different steps in the assembly of MLL1-WRAD. The docking of subunits in all four states differs from structures of MLL1- WRAD bound to unmodified nucleosomes, suggesting that H2B-ubiquitin favors assembly of the active complex. Our results provide a more complete picture of MLL1-WRAD and the role of ubiquitin in promoting formation of the active methyltransferase complex.

[more...]

Hsp multichaperone complex buffers pathologically modified Tau

Nature communications. 2022. Moll, A et al. Max Planck Inst Multidisciplinary Sci, Dept NMR Based Struct Biol, Fassberg 11, D-37077 Gottingen, Germany; German Ctr Neurodegenerat Dis DZNE, Von Siebold Str 3a, D-37075 Gottingen, Germany

ABSTRACT: Alzheimer's disease is a neurodegenerative disorder in which misfolding and aggregation of pathologically modified Tau is critical for neuronal dysfunction and degeneration. The two central chaperones Hsp70 and Hsp90 coordinate protein homeostasis, but the nature of the interaction of Tau with the Hsp70/Hsp90 machinery has remained enigmatic. Here we show that Tau is a high-affinity substrate of the human Hsp70/Hsp90 machinery. Complex formation involves extensive intermolecular contacts, blocks Tau aggregation and depends on Tau's aggregation-prone repeat region.

The Hsp90 co-chaperone p23 directly binds Tau and stabilizes the multichaperone/substrate complex, whereas the E3 ubiquitin-protein ligase CHIP efficiently disassembles the machinery targeting Tau to proteasomal degradation. Because phosphorylated Tau binds the Hsp70/Hsp90 machinery but is not recognized by Hsp90 alone, the data establish the Hsp70/Hsp90 multichaperone complex as a critical regulator of Tau in neurodegenerative diseases.Alzheimer's disease is characterized by the accumulation of aggregated tau protein. Here the authors find that Hsp chaperones, which normally protect cell homeostasis, can assemble with co-chaperones in a "multichaperone machinery" to target tau aggregation.

[more...]

Architecture of the human NALCN channelosome

CELL DISCOVERY. 2022. Zhou, LN et al. Westlake Univ, Sch Life Sci, Key Lab Struct Biol Zhejiang Prov, Hangzhou, Zhejiang, Peoples R China; Westlake Lab Life Sci & Biomed, Hangzhou, Zhejiang, Peoples R China; Westlake Inst Adv Study, Inst Biol, Hangzhou, Zhejiang, Peoples R China

ABSTRACT: NALCN regulates the resting membrane potential by mediating the Na+ leak current in neurons, and it functions as a channelosome in complex with FAM155A, UNC79, and UNC80. Dysfunction of the NALCN channelosome causes a broad range of neurological and developmental diseases called NALCN channelopathies in humans. How the auxiliary subunits, especially the two large components UNC79 and UNC80, assemble with NALCN and regulate its function remains unclear. Here we report an overall architecture of the human NALCN channelosome.

UNC79 and UNC80 each adopt an S-shape super-helical structure consisting of HEAT and armadillo repeats, forming a super-coiled heterodimeric assembly in the cytoplasmic side, which may provide a scaffold for the binding of other potential modulators of the channelosome. The UNC79-UNC80 assembly specifically associates with the NALCN-FAM155A subcomplex through the intracellular II-III linker of NALCN. Disruptions of the interaction interfaces between UNC79 and UNC80, and between the II-III linker of NALCN and the UNC79-UNC80 assembly, significantly reduce the NALCN-mediated currents in HEK293T system, suggesting the importance of the UNC79-UNC80 assembly in regulating channelosome function. Cross-linking mass spectrometry analysis identified an additional calmodulin (CaM) bound in the carboxyl-terminal domain of NALCN. Our study thus provides a structural basis for understanding the unique assembly mechanism and functional regulation of the NALCN channelosome, and also provides an opportunity for the interpretation of many disease-related mutations in UNC80.

[more...]

Optimized TMT-based quantitative cross-linking mass spectrometry strategy for large-scale interactomic studies

Analytical Chemistry. 2022. Ruwolt, M et al. Leibniz Forschungsinst Mol Pharmakol FMP, Dept Struct Biol, D-13125 Berlin, Germany

ABSTRACT: Cross-linking mass spectrometry (XL-MS) is a powerful method for theinvestigation of protein-protein interactions (PPI) from highly complex samples. XL-MScombined with tandem mass tag (TMT) labeling holds the promise of large-scale PPIquantification. However, a robust and efficient TMT-based XL-MS quantificationmethod has not yet been established due to the lack of a benchmarking dataset andthorough evaluation of various MS parameters. To tackle these limitations, we generate atwo-interactome dataset by spiking in TMT-labeled cross-linkedEscherichia colilysateinto TMT-labeled cross-linked HEK293T lysate using a defined mixing scheme.

Usingthis benchmarking dataset, we assess the efficacy of cross-link identification and accuracyof cross-link quantification using different MS acquisition strategies. For identification,we compare various MS2- and MS3-based XL-MS methods, and optimize stepped higherenergy collisional dissociation (HCD) energies for TMT-labeled cross-links. Weobserved a need for notably higher fragmentation energies compared to unlabeledcross-links. For quantification, we assess the quantification accuracy and dispersion of MS2-, MS3-, and synchronous precursorselection-MS3-based methods. We show that a stepped HCD-MS2 method with stepped collision energies 36-42-48 provides a vastnumber of quantifiable cross-links with high quantification accuracy. This widely applicable method paves the way for multiplexedquantitative PPI characterization from complex biological systems.

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Structural basis for c-di-AMP–dependent regulation of the bacterial stringent response by receptor protein DarB

Journal of Biological Chemistry. 2022. Heidemann, JL et al. Georg August Univ Gottingen, Dept Mol Struct Biol, Gottingen, Germany

ABSTRACT: The bacterial second messenger c-di-AMP controls essential cellular processes, including potassium and osmolyte homeo-stasis. This makes synthesizing enzymes and components involved in c-di-AMP signal transduction intriguing as poten-tial targets for drug development. The c-di-AMP receptor protein DarB of Bacillus subtilis binds the Rel protein and triggers the Rel-dependent stringent response to stress condi-tions; however, the structural basis for this trigger is unclear. Here, we report crystal structures of DarB in the ligand-free state and of DarB complexed with c-di-AMP, 3'3'-cGAMP, and AMP.

We show that DarB forms a homodimer with a parallel, head-to-head assembly of the monomers. We also confirm the DarB dimer binds two cyclic dinucleotide mole-cules or two AMP molecules; only one adenine of bound c-di -AMP is specifically recognized by DarB, while the second protrudes out of the donut-shaped protein. This enables DarB to bind also 3'3'-cGAMP, as only the adenine fits in the active site. In absence of c-di-AMP, DarB binds to Rel and stimulates (p)ppGpp synthesis, whereas the presence of c-di-AMP abol-ishes this interaction. Furthermore, the DarB crystal structures reveal no conformational changes upon c-di-AMP binding, leading us to conclude the regulatory function of DarB on Rel must be controlled directly by the bound c-di-AMP. We thus derived a structural model of the DarB-Rel complex via in silico docking, which was validated with mass spectrometric analysis of the chemically crosslinked DarB-Rel complex and mutagenesis studies. We suggest, based on the predicted complex structure, a mechanism of stringent response regula-tion by c-di-AMP.

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Characterization of protein complexes in extracellular vesicles by intact extracellular vesicle crosslinking mass spectrometry (iEVXL)

Journal of Extracellular Vesicles. 2022. Bauza-Martinez, J et al. ASTAR, Singapore Immunol Network SIgN, Singapore, Singapore

ABSTRACT: Extracellular vesicles (EVs) are blood-borne messengers that coordinate signalling between different tissues and organs in the body. The specificity of such crosstalk is determined by preferential EV docking to target sites, as mediated through protein-protein interactions. As such, the need to structurally characterize the EV surface precedes further understanding of docking selectivity and recipient-cell uptake mechanisms. Here, we describe an intact extracellular vesicle crosslinking mass spectrometry (iEVXL) method that can be applied for structural characterization of protein complexes in EVs.

By using a partially membrane-permeable disuccinimidyl suberate crosslinker, proteins on the EV outer-surface and inside EVs can be immobilized together with their interacting partners. This not only provides covalent stabilization of protein complexes before extraction from the membrane-enclosed environment, but also generates a set of crosslinking distance restraints that can be used for structural modelling and comparative screening of changes in EV protein assemblies. Here we demonstrate iEVXL as a powerful approach to reveal high-resolution information, about protein determinants that govern EV docking and signalling, and as a crucial aid in modelling docking interactions.

[more...]

Real-time library search increases cross-link identification depth across all levels of sample complexity

Analytical chemistry. 2022. Ruwolt, Max et al. Leibniz Forschungsinst Mol Pharmakol FMP, Dept Struct Biol, D-13125 Berlin, Germany; Charite Univ Med Berlin, D-10117 Berlin, Germany

ABSTRACT: Cross-linking mass spectrometry (XL-MS) is a universal tool for probing structural dynamics and protein-protein interactions in vitro and in vivo. Although cross-linked peptides are naturally less abundant than their unlinked counterparts, recent experimental advances improved cross-link identification by enriching the cross-linker-modified peptides chemically with the use of enrichable cross-linkers. However, mono-links (i.e., peptides modified with a hydrolyzed cross-linker) still hinder efficient cross-link identification since a large proportion of measurement time is spent on their MS2 acquisition.

Currently, cross-links and mono-links cannot be separated by sample preparation techniques or chromatography because they are chemically almost identical. Here, we found that based on the intensity ratios of four diagnostic peaks when using PhoX/tBu-PhoX cross-linkers, cross-links and mono-links can be partially distinguished. Harnessing their characteristic intensity ratios for real-time library search (RTLS)-based triggering of high-resolution MS2 scans increased the number of cross-link identifications from both single protein samples and intact E. coli cells. Specifically, RTLS improves cross-link identification from unenriched samples and short gradients, emphasizing its advantages in high-throughput approaches and when instrument time or sample amount is limited.

[more...]

Nitrogen Mustard Alkylates and Cross-Links p53 in Human Keratinocytes

Chemical Research in Toxicology. 2022. Jan, YH et al. Rutgers State Univ, Sch Publ Hlth, Dept Environm & Occupat Hlth & Justice, Piscataway, NJ 08854 USA

ABSTRACT: Cytotoxic blistering agents such as sulfur mustard and nitrogen mustard (HN2) were synthesized for chemical warfare. Toxicity is due to reactive chloroethyl side chains that modify and damage cellular macromolecules including DNA and proteins. In response to DNA damage, cells initiate a DNA damage response directed at the recruitment and activation of repair-related proteins. A central mediator of the DNA damage response is p53, a protein that plays a critical role in regulating DNA repair. We found that HN2 causes cytosolic and nuclear accumulation of p53 in HaCaT keratinocytes; HN2 also induced post-translational modifications on p53 including S15 phosphorylation and K382 acetylation, which enhance p53 stability, promote DNA repair, and mediate cellular metabolic responses to stress.

HN2 also cross-linked p53, forming dimers and high-molecular-weight protein complexes in the cells. Cross-linked multimers were also modified by K48-linked ubiquitination indicating that they are targets for proteasome degradation. HN2-induced modifications transiently suppressed the transcriptional activity of p53. Using recombinant human p53, HN2 alkylation was found to be concentration- and redox status-dependent. Dithiothreitol-reduced protein was more efficiently cross-linked indicating that p53 cysteine residues play a key role in protein modification. LC-MS/MS analysis revealed that HN2 directly alkylated p53 at C124, C135, C141, C176, C182, C275, C277, H115, H178, K132, and K139, forming both monoadducts and cross-links. The formation of intermolecular complexes was a consequence of HN2 cross-linked cysteine residues between two molecules of p53. Together, these data demonstrate that p53 is a molecular target for mustard vesicants. Modification of p53 likely mediates cellular responses to HN2 including DNA repair and cell survival contributing to vesicant-induced cytotoxicity.

[more...]

Enhanced protein--protein interaction network construction promoted by in vivo cross-linking with acid-cleavable click-chemistry enrichment

Frontiers in Chemistry. 2022. Zhao, Lili et al. Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog R&A Ctr, CAS Key Lab Separat Sci Analyt Chem, Dalian, Liaoning, Peoples R China

ABSTRACT: Chemical cross-linking coupled with mass spectrometry has emerged as a powerful strategy which enables global profiling of protein interactome with direct interaction interfaces in complex biological systems. The alkyne-tagged enrichable cross-linkers are preferred to improve the coverage of low-abundance cross-linked peptides, combined with click chemistry for biotin conjugation to allow the cross-linked peptide enrichment. However, a systematic evaluation on the efficiency of click approaches (protein-based or peptide-based) and diverse cleavable click-chemistry ligands (acid, reduction, and photo) for cross-linked peptide enrichment and release is lacking.

Herein, together with in vivo chemical cross-linking by alkyne-tagged cross-linkers, we explored the click-chemistry-based enrichment approaches on protein and peptide levels with three cleavable click-chemistry ligands, respectively. By comparison, the approach of protein-based click-chemistry conjugation with acid-cleavable tags was demonstrated to permit the most cross-linked peptide identification. The advancement of this strategy enhanced the proteome-wide cross-linking analysis, constructing a 5,518-protein-protein-interaction network among 1,871 proteins with widely abundant distribution in cells. Therefore, all these results demonstrated the guideline value of our work for efficient cross-linked peptide enrichment, thus facilitating the in-depth profiling of protein interactome for functional analysis.

[more...]

Architecture of the human NALCN channelosome

CELL DISCOVERY. 2022. Zhou, LN et al. Westlake Univ, Sch Life Sci, Key Lab Struct Biol Zhejiang Prov, Hangzhou, Zhejiang, Peoples R China; Westlake Lab Life Sci & Biomed, Hangzhou, Zhejiang, Peoples R China; Westlake Inst Adv Study, Inst Biol, Hangzhou, Zhejiang, Peoples R China

ABSTRACT: NALCN regulates the resting membrane potential by mediating the Na+ leak current in neurons, and it functions as a channelosome in complex with FAM155A, UNC79, and UNC80. Dysfunction of the NALCN channelosome causes a broad range of neurological and developmental diseases called NALCN channelopathies in humans. How the auxiliary subunits, especially the two large components UNC79 and UNC80, assemble with NALCN and regulate its function remains unclear. Here we report an overall architecture of the human NALCN channelosome.

UNC79 and UNC80 each adopt an S-shape super-helical structure consisting of HEAT and armadillo repeats, forming a super-coiled heterodimeric assembly in the cytoplasmic side, which may provide a scaffold for the binding of other potential modulators of the channelosome. The UNC79-UNC80 assembly specifically associates with the NALCN-FAM155A subcomplex through the intracellular II-III linker of NALCN. Disruptions of the interaction interfaces between UNC79 and UNC80, and between the II-III linker of NALCN and the UNC79-UNC80 assembly, significantly reduce the NALCN-mediated currents in HEK293T system, suggesting the importance of the UNC79-UNC80 assembly in regulating channelosome function. Cross-linking mass spectrometry analysis identified an additional calmodulin (CaM) bound in the carboxyl-terminal domain of NALCN. Our study thus provides a structural basis for understanding the unique assembly mechanism and functional regulation of the NALCN channelosome, and also provides an opportunity for the interpretation of many disease-related mutations in UNC80.

[more...]

Multistate structures of the MLL1-WRAD complex bound to H2B-ubiquitinated nucleosome

PNAS. 2022. Rahman, S et al. Johns Hopkins Univ, Dept Biophys & Biophys Chem, Sch Med, Baltimore, MD 21205 USA

ABSTRACT: The human Mixed Lineage Leukemia-1 (MLL1) complex methylates histone H3K4 to promote transcription and is stimulated by monoubiquitination of histone H2B. Recent structures of the MLL1-WRAD core complex, which comprises the MLL1 methyltransferase, WDR5, RbBp5, Ash2L, and DPY-30, have revealed variability in the docking of MLL1-WRAD on nucleosomes. In addition, portions of the Ash2L structure and the position of DPY30 remain ambiguous. We used an integrated approach combining cryoelectron microscopy (cryo-EM) and mass spectrometry cross-linking to determine a structure of the MLL1-WRAD complex bound to ubiquitinated nucleosomes.

The resulting model contains the Ash2L intrinsically disordered region (IDR), SPRY insertion region, Sdc1-DPY30 interacting region (SDI-motif), and the DPY30 dimer. We also resolved three additional states of MLL1-WRAD lacking one or more subunits, which may reflect different steps in the assembly of MLL1-WRAD. The docking of subunits in all four states differs from structures of MLL1- WRAD bound to unmodified nucleosomes, suggesting that H2B-ubiquitin favors assembly of the active complex. Our results provide a more complete picture of MLL1-WRAD and the role of ubiquitin in promoting formation of the active methyltransferase complex.

[more...]

Structures of transcription preinitiation complex engaged with the+ 1 nucleosome

Nature Structural & Molecular Biology. 2022. Wang, HB et al. Max Planck Inst Multidisciplinary Sci, Dept Mol Biol, Gottingen, Germany

ABSTRACT: The preinitiation complex (PIC) assembles on promoters of protein-coding genes to position RNA polymerase II (Pol II) for transcription initiation. Previous structural studies revealed the PIC on different promoters, but did not address how the PIC assembles within chromatin. In the yeast Saccharomyces cerevisiae, PIC assembly occurs adjacent to the +1 nucleosome that is located downstream of the core promoter. Here we present cryo-EM structures of the yeast PIC bound to promoter DNA and the +1 nucleosome located at three different positions.

The general transcription factor TFIIH engages with the incoming downstream nucleosome and its translocase subunit Ssl2 (XPB in human TFIIH) drives the rotation of the +1 nucleosome leading to partial detachment of nucleosomal DNA and intimate interactions between TFIIH and the nucleosome. The structures provide insights into how transcription initiation can be influenced by the +1 nucleosome and may explain why the transcription start site is often located roughly 60 base pairs upstream of the dyad of the +1 nucleosome in yeast.Cryo-EM structures of the transcription preinitiation complex in the presence of the +1 nucleosome show how the general transcription factor TFIIH can interact with the nucleosome at several positions.

[more...]

A toxin-deformation dependent inhibition mechanism in the T7SS toxin-antitoxin system of Gram-positive bacteria

Nature Communications. 2022. Wang, YJ et al. Jinan Univ, Coll Pharm, Int Cooperat Lab Tradit Chinese Med Modernizat &, Chinese Minist Educ MOE, Guangzhou 510632, Peoples R China; Weill Cornell Med, Dept Physiol & Biophys, New York, NY 10065 USA; Guangdong Youmei Inst Intelligent Biomfg, Foshan 528200, Guangdong, Peoples R China

ABSTRACT: Toxin EsaD secreted by some S. aureus strains through the type VII secretion system (T7SS) specifically kills those strains lacking the antitoxin EsaG. Here we report the structures of EsaG, the nuclease domain of EsaD and their complex, which together reveal an inhibition mechanism that relies on significant conformational change of the toxin. To inhibit EsaD, EsaG breaks the nuclease domain of EsaD protein into two independent fragments that, in turn, sandwich EsaG. The originally well-folded beta beta alpha-metal finger connecting the two fragments is stretched to become a disordered loop, leading to disruption of the catalytic site of EsaD and loss of nuclease activity.

This mechanism is distinct from that of the other Type II toxin-antitoxin systems, which utilize an intrinsically disordered region on the antitoxins to cover the active site of the toxins. This study paves the way for developing therapeutic approaches targeting this antagonism.Antimicrobial toxins are secreted by bacteria to kill rival species. Here the authors report the mechanism of inhibition of EsaD, a toxin secreted by some S. aureus strains to kill competitors that lack the antitoxin EsaG, showing marked mechanistic differences to other Type II toxin-antitoxin systems.

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Mapping of the plant SnRK1 kinase signalling network reveals a key regulatory role for the class II T6P synthase-like proteins

Nature Plants. 2022. Van Leene, J et al. Univ Ghent, Dept Plant Biotechnol & Bioinformat, Ghent, Belgium; VIB Ctr Plant Syst Biol, Ghent, Belgium

ABSTRACT: The central metabolic regulator SnRK1 controls plant growth and survival upon activation by energy depletion, but detailed molecular insight into its regulation and downstream targets is limited. Here we used phosphoproteomics to infer the sucrose-dependent processes targeted upon starvation by kinases as SnRK1, corroborating the relation of SnRK1 with metabolic enzymes and transcriptional regulators, while also pointing to SnRK1 control of intracellular trafficking. Next, we integrated affinity purification, proximity labelling and crosslinking mass spectrometry to map the protein interaction landscape, composition and structure of the SnRK1 heterotrimer, providing insight in its plant-specific regulation.

At the intersection of this multi-dimensional interactome, we discovered a strong association of SnRK1 with class II T6P synthase (TPS)-like proteins. Biochemical and cellular assays show that TPS-like proteins function as negative regulators of SnRK1. Next to stable interactions with the TPS-like proteins, similar intricate connections were found with known regulators, suggesting that plants utilize an extended kinase complex to fine-tune SnRK1 activity for optimal responses to metabolic stress.SnRK1 is a key metabolic sensor that controls plant development and stress responses. This study integrates phosphoproteomics, affinity purification coupled to mass spectrometry, proximity labelling and crosslinking mass spectrometry to obtain more insight into its upstream regulation and downstream target processes.

[more...]

Subcellular Interactomes Revealed by Merging APEX with Cross-Linking Mass Spectrometry

Analytical Chemistry. 2022. Sun, MZ et al. Chinese Inst Brain Res CIBR, Beijing 102206, Peoples R China; Peking Univ, Synthet & Funct Biomol Ctr, Coll Chem & Mol Engn, Dept Chem Biol,Beijing Natl Lab Mol Sci,Key Lab Bi, Beijing 100871, Peoples R China; Inst Canc Res, Shenzhen Bay Lab, Shenzhen 518107, Peoples R China; Peking Univ, Peking Tsinghua Ctr Life Sci, Beijing 100871, Peoples R China; Peking Univ, PKU IDG McGovern Inst Brain Res, Beijing 100871, Peoples R China

ABSTRACT: Subcellular protein-protein interactions (PPIs) are essential to understanding the mechanism of diverse cellular signaling events and the pathogenesis of diseases. Herein, we report an integrated APEX proximity labeling and chemical cross-linking coupled with mass spectrometry (CXMS) platform named APEX-CXMS for spatially resolved subcellular interactome profiling in a high-throughput manner. APEX proximity labeling rapidly captures subcellular proteomes, and the highly reactive chemical cross-linkers can capture weak and dynamic interactions globally without extra genetic manipulation.

APEX-CXMS was first applied to mitochondria and identified 653 pairs of interprotein cross-links. Six pairs of new interactions were selected and verified by coimmunoprecipitation, the mammalian two-hybrid system, and surface plasmon resonance method. Besides, our approach was further applied to the nucleus, capturing 336 pairs of interprotein cross-links with approximately 94% nuclear specificity. APEX-CXMS thus provides a simple, fast, and general alternative to map diverse subcellular PPIs.

[more...]

Genetically encoded chemical crosslinking of carbohydrate

Nature Chemistry. 2022. Li, SS et al. Univ Calif San Francisco, Dept Pharmaceut Chem, San Francisco, CA 94143 USA; Univ Calif San Francisco, Cardiovasc Res Inst, Box 0544, San Francisco, CA 94143 USA

ABSTRACT: Protein-carbohydrate interactions play important roles in various biological processes, such as organism development, cancer metastasis, pathogen infection and immune response, but they remain challenging to study and exploit due to their low binding affinity and non-covalent nature. Here we site-specifically engineered covalent linkages between proteins and carbohydrates under biocompatible conditions. We show that sulfonyl fluoride reacts with glycans via a proximity-enabled reactivity, and to harness this a bioreactive unnatural amino acid (SFY) that contains sulfonyl fluoride was genetically encoded into proteins.

SFY-incorporated Siglec-7 crosslinked with its sialoglycan ligand specifically in vitro and on the surface of cancer cells. Through irreversible cloaking of sialoglycan at the cancer cell surface, SFY-incorporated Siglec-7 enhanced the killing of cancer cells by natural killer cells. Genetically encoding the chemical crosslinking of proteins to carbohydrates (GECX-sugar) offers a solution to address the low affinity and weak strength of protein-sugar interactions.

[more...]

Divergent polo boxes in KKT2 bind KKT1 to initiate the kinetochore assembly cascade in Trypanosoma brucei

Molecular Biology of the Cell. 2022. Ishii, Midori et al. Univ Oxford, Dept Biochem, Oxford OX1 3QU, England

ABSTRACT: Chromosome segregation requires assembly of the macromolecular kinetochore complex onto centromeric DNA. While most eukaryotes have canonical kinetochore proteins that are widely conserved among eukaryotes, evolutionarily divergent kinetoplastids have a unique set of kinetochore proteins. Little is known about the mechanism of kinetochore assembly in kinetoplastids. Here we characterize two homologous kinetoplastid kinetochore proteins, KKT2 and KKT3, that constitutively localize at centromeres. They have three domains that are highly conserved among kinetoplastids: an N-terminal kinase domain of unknown function, the centromere localization domain in the middle, and the C-terminal domain that has weak similarity to polo boxes of Polo-like kinases.

We show that the kinase activity of KKT2 is essential for accurate chromosome segregation, while that of KKT3 is dispensable for cell growth in Trypanosoma brucei. Crystal structures of their divergent polo boxes reveal differences between KKT2 and KKT3. We also show that the divergent polo boxes of KKT3 are sufficient to recruit KKT2 in trypanosomes. Furthermore, we demonstrate that the divergent polo boxes of KKT2 interact directly with KKT1 and that KKT1 interacts with KKT6. These results show that the divergent polo boxes of KKT2 and KKT3 are protein-protein interaction domains that initiate kinetochore assembly in T. brucei.

[more...]

Alkynyl-Enrichable Carboxyl-Selective Crosslinkers to Increase the Crosslinking Coverage for Deciphering Protein Structures

Analytical Chemistry. 2022. Gao, H et al. Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog R&A Ctr, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Liaoning, Peoples R China

ABSTRACT: The coverage of chemical crosslinking coupled with mass spectrometry (CXMS) is of great importance to determine its ability for deciphering protein structures. At present, N- hydroxysuccinimidyl (NHS) ester-based crosslinkers targeting lysines have been predominantly used in CXMS. However, they are not always effective for some proteins with few lysines. Other amino acid residues such as carboxyl could be crosslinked to complement lysines and improve the crosslinking coverage of CXMS, but the low intrinsic chemical reactivity of carboxyl compromises the application of carboxyl-selective crosslinkers for complex samples.

To enhance the crosslinking efficiency targeting acidic residues and realize in-depth crosslinking analysis of complex samples, we developed three new alkynyl-enrichable carboxyl-selective crosslinkers with different reactive groups such as hydrazide, amino, and aminooxy. The crosslinking efficiencies of the three crosslinkers were systematically evaluated, giving the best reactivity of the amino-functionalized crosslinker BAP. Furthermore, BAP was extended to the crosslinking analysis of Escherichia coli lysate in combination with efficient crosslink enrichment. A total of 1291 D/E-D/E crosslinks involved in 392 proteins were identified under a false discovery rate (FDR) of >= 1%. Obvious structural complementarity of BAP was exhibited to the lysine-targeting crosslinker, facilitating the capability of CXMS for protein structure elucidation. To the best of our knowledge, this was the first time for the carboxyl-selective crosslinker to achieve proteome-wide crosslinking analysis of the whole cell lysate. Collectively, we believe that this work not only expands on a promising toolkit of CXMS targeting acidic residues but also provides a valuable guideline to advance the performance of carboxyl-selective crosslinkers.

[more...]

DPY30 acts as an ASH2L-specific stabilizer to stimulate the enzyme activity of MLL family methyltransferases on different substrates

Iscience. 2022. Zhao, LJ et al. Chinese Acad Sci, Shanghai Inst Biochem & Cell Biol, Natl Ctr Prot Sci Shanghai, Ctr Excellence Mol Cell Sci, Shanghai 200031, Peoples R China; ShanghaiTech Univ, Sch Life Sci & Technol, 100 Haike Rd, Shanghai 201210, Peoples R China; Univ Chinese Acad Sci, Beijing 100049, Peoples R China

ABSTRACT: Dumpy-30 (DPY30) is a conserved component of the mixed lineage leukemia (MLL) family complex and is essential for robust methyltransferase activity of MLL complexes. However, the biochemical role of DPY30 in stimulating methyl-transferase activity of MLL complexes remains elusive. Here, we demonstrate that DPY30 plays a crucial role in regulating MLL1 activity through two com-plementary mechanisms: A nucleosome-independent mechanism and a nucleo-some-specific mechanism. DPY30 functions as an ASH2L-specific stabilizer to increase the stability of ASH2L and enhance ASH2L-mediated interactions.

As a result, DPY30 promotes the compaction and stabilization of the MLL1 complex, consequently increasing the HKMT activity of the MLL1 complex on diverse sub-strates. DPY30-stabilized ASH2L further acquires additional interfaces with H3 and nucleosomal DNA, thereby boosting the methyltransferase activity of the MLL1 complex on nucleosomes. These results collectively highlight the crucial and conserved roles of DPY30 in the complex assembly and activity regulation of MLL family complexes.

[more...]

Structural insights into dsRNA processing by Drosophila Dicer-2–Loqs-PD

Nature. 2022. Su, SC et al. Tsinghua Univ, Beijing Adv Innovat Ctr Struct Biol, Tsinghua Peking Joint Ctr Life Sci, Sch Life Sci,Minist Educ,Key Lab Prot Sci, Beijing, Peoples R China; Fudan Univ, Sch Life Sci, Dept Biochem & Biophys, Collaborat Innovat Ctr Genet & Dev, Shanghai, Peoples R China

ABSTRACT: Small interfering RNAs (siRNAs) are the key components for RNA interference (RNAi), a conserved RNA-silencing mechanism in many eukaryotes(1,2). In Drosophila, an RNase III enzyme Dicer-2 (Dcr-2), aided by its cofactor Loquacious-PD (Loqs-PD), has an important role in generating 21 bp siRNA duplexes from long double-stranded RNAs (dsRNAs)(3,4). ATP hydrolysis by the helicase domain of Dcr-2 is critical to the successful processing of a long dsRNA into consecutive siRNA duplexes(5,6). Here we report the cryo-electron microscopy structures of Dcr-2-Loqs-PD in the apo state and in multiple states in which it is processing a 50 bp dsRNA substrate.

The structures elucidated interactions between Dcr-2 and Loqs-PD, and substantial conformational changes of Dcr-2 during a dsRNA-processing cycle. The N-terminal helicase and domain of unknown function 283 (DUF283) domains undergo conformational changes after initial dsRNA binding, forming an ATP-binding pocket and a 5'-phosphate-binding pocket. The overall conformation of Dcr-2-Loqs-PD is relatively rigid during translocating along the dsRNA in the presence of ATP, whereas the interactions between the DUF283 and RIIIDb domains prevent non-specific cleavage during translocation by blocking the access of dsRNA to the RNase active centre. Additional ATP-dependent conformational changes are required to form an active dicing state and precisely cleave the dsRNA into a 21 bp siRNA duplex as confirmed by the structure in the post-dicing state. Collectively, this study revealed the molecular mechanism for the full cycle ofATP-dependent dsRNA processing by Dcr-2-Loqs-PD.

[more...]

High-resolution mass spectrometry unveils the molecular changes of ovalbumin induced by heating and their influence on IgE binding capacity

Food Chemistry. 2022. Cherkaoui, M et al. INRAE, UR1268 BIA, F-44316 Nantes, France

ABSTRACT: Ovalbumin (OVA) is a food allergen whose allergenicity is modulated by heating. We aimed to establish a molecular connection between heat-induced structural modifications and the modulation of the IgE binding capacity of OVA. For this, we used model samples of heat-modified OVA with increasing complexity; glycated, aggregated, or glycated and aggregated. Using sera from egg-allergic individuals, we show that both aggregation and glycation strongly impacted IgE binding capacity, despite limited structural changes for glycated OVA.

A molecular exploration at the amino acid level using high-resolution mass spectrometry revealed extensive cross -linking, mostly through disulfide and dehydroprotein bridges, and moderate but significant glycation. Structural modifications affected residues located within or at a few amino acids distance of known human linear IgE epitopes, such as C121, K123, S169, K190, K207, H332 and C368. We thus unveil key amino residues implicated in the changes in IgE binding of OVA induced by heating.

[more...]

Cryo-EM structures of human m6A writer complexes

Cell Research. 2022. Su, SC et al. Univ Sci & Technol China, MOE Key Lab Cellular Dynam, Hefei, Anhui, Peoples R China; Fudan Univ, Sch Life Sci, Dept Biochem & Biophys,Multiscale Res Inst Comple, State Key Lab Genet Engn,Collaborat Innovat Ctr G, Shanghai, Peoples R China; Univ Sci & Technol China, Div Life Sci & Med, Hefei, Anhui, Peoples R China

ABSTRACT: N-6-methyladenosine (m(6)A) is the most abundant ribonucleotide modification among eukaryotic messenger RNAs. The m(6)A "writer" consists of the catalytic subunit m(6)A-METTL complex (MAC) and the regulatory subunit m(6)A-METTL-associated complex (MACOM), the latter being essential for enzymatic activity. Here, we report the cryo-electron microscopy (cryo-EM) structures of MACOM at a 3.0-angstrom resolution, uncovering that WTAP and VIRMA form the core structure of MACOM and that ZC3H13 stretches the conformation by binding VIRMA.

Furthermore, the 4.4-angstrom resolution cryo-EM map of the MACOM-MAC complex, combined with crosslinking mass spectrometry and GST pull-down analysis, elucidates a plausible model of the m(6)A writer complex, in which MACOM binds to MAC mainly through WTAP and METTL3 interactions. In combination with in vitro RNA substrate binding and m(6)A methyltransferase activity assays, our results illustrate the molecular basis of how MACOM assembles and interacts with MAC to form an active m(6)A writer complex.

[more...]

Comprehensive structure and functional adaptations of the yeast nuclear pore complex

Cell. 2022. Akey, Christopher W. et al. Univ Calif San Diego, Div Biol Sci, Sect Mol Biol, La Jolla, CA 92093 USA; Univ Basque Country, Inst Biofis, UPV EHU, CSIC, Leioa 48940, Spain; Univ Calif San Diego, Howard Hughes Med Inst, La Jolla, CA 92093 USA; Baylor Coll Med, Verna & Marrs McLean Dept Biochem & Mol Biol, 1 Baylor Plaza, Houston, TX 77030 USA; Rockefeller Univ, Lab Cellular & Struct Biol, New York, NY 10065 USA; Basque Fdn Sci, Ikerbasque, Bilbao 48013, Spain

ABSTRACT: Nuclear pore complexes (NPCs) mediate the nucleocytoplasmic transport of macromolecules. Here we provide a structure of the isolated yeast NPC in which the inner ring is resolved by cryo-EM at sub-nanometer resolution to show how flexible connectors tie together different structural and functional layers. These connectors may be targets for phosphorylation and regulated disassembly in cells with an open mitosis. Moreover, some nucleoporin pairs and transport factors have similar interaction motifs, which suggests an evolutionary and mechanistic link between assembly and transport.

We provide evidence for three major NPC variants that may foreshadow functional specializations at the nuclear periphery. Cryo-electron tomography extended these studies, providing a model of the in situ NPC with a radially expanded inner ring. Our comprehensive model reveals features of the nuclear basket and central transporter, suggests a role for the lumenal Pom152 ring in restricting dilation, and highlights structural plasticity that may be required for transport.

[more...]

Structure of the metastatic factor P-Rex1 reveals a two-layered autoinhibitory mechanism

Nature structural & molecular biology. 2022. Chang, YG et al. Monash Univ, Biomed Discovery Inst, Clayton, Vic, Australia

ABSTRACT: P-Rex (PI(3,4,5)P-3-dependent Rac exchanger) guanine nucleotide exchange factors potently activate Rho GTPases. P-Rex guanine nucleotide exchange factors are autoinhibited, synergistically activated by G beta gamma and PI(3,4,5)P-3 binding and dysregulated in cancer. Here, we use X-ray crystallography, cryogenic electron microscopy and crosslinking mass spectrometry to determine the structural basis of human P-Rex1 autoinhibition. P-Rex1 has a bipartite structure of N- and C-terminal modules connected by a C-terminal four-helix bundle that binds the N-terminal Pleckstrin homology (PH) domain.

In the N-terminal module, the Dbl homology (DH) domain catalytic surface is occluded by the compact arrangement of the DH-PH-DEP1 domains. Structural analysis reveals a remarkable conformational transition to release autoinhibition, requiring a 126 degrees opening of the DH domain hinge helix. The off-axis position of G beta gamma and PI(3,4,5)P-3 binding sites further suggests a counter-rotation of the P-Rex1 halves by 90 degrees facilitates PH domain uncoupling from the four-helix bundle, releasing the autoinhibited DH domain to drive Rho GTPase signaling.Cryo-EM, X-ray crystallography and crosslinking mass spectrometry are harnessed to solve the structure of the full-length Rho-GEF P-Rex1, uncovering a two-layered mechanism of autoinhibition released upon G beta gamma and PI(3,4,5)P3 binding.

[more...]

Structural basis for assembly of TRAPPII complex and specific activation of GTPase Ypt31/32

Science Advances. 2022. Mi, Chenchen et al. Southern Univ Sci & Technol, Dept Biol, Shenzhen 518055, Guangdong, Peoples R China; Tsinghua Univ, Beijing Adv Innovat Ctr Struct Biol, Beijing Frontier Res Ctr Biol Struct, Sch Life Sci,State Key Lab Membrane Biol, Beijing 100084, Peoples R China

ABSTRACT: Transport protein particle (TRAPP) complexes belong to the multiprotein tethering complex and exist in three forms-core TRAPP/TRAPPI, TRAPPII, and TRAPPIII. TRAPPII activates GTPase Ypt31/Ypt32 as the guanine nucleotide exchange factor in the trans-Golgi network to determine the maturation of Golgi cisternae into post-Golgi carriers in yeast. Here, we present cryo-EM structures of yeast TRAPPII in apo and Ypt32-bound states. All the structures show a dimeric architecture assembled by two triangle-shaped monomers, while the monomer in the apo state exhibits both open and closed conformations, and the monomer in the Ypt32-bound form only captures the closed conformation.

Located in the interior of the monomer, Ypt32 binds with both core TRAPP/TRAPPI and Trs120 via its nucleotide-binding domain and binds with Trs31 via its hypervariable domain. Combined with functional analysis, the structures provide insights into the assembly of TRAPPII and the mechanism of the specific activation of Ypt31/Ypt32 by TRAPPII.

[more...]

Cryo-EM structures reveal the dynamic transformation of human alpha-2-macroglobulin working as a protease inhibitor

Science China Life Sciences. 2022. Huang, XX et al. Univ Chinese Acad Sci, Beijing 100049, Peoples R China; Chinese Acad Sci, CAS Ctr Excellence Biomacromol, Inst Biophys, Natl Lab Biomacromol, Beijing 100101, Peoples R China

ABSTRACT: Human alpha-2-macroglobulin is a well-known inhibitor of a broad spectrum of proteases and plays important roles in immunity, inflammation, and infections. Here, we report the cryo-EM structures of human alpha-2-macroglobulin in its native state, induced state transformed by its authentic substrate, human trypsin, and serial intermediate states between the native and fully induced states. These structures exhibit distinct conformations, which reveal the dynamic transformation of alpha-2-macro-globulin that acts as a protease inhibitor.

The results shed light on the molecular mechanism of alpha-2-macroglobulin in entrapping substrates.

[more...]

Mechanism of Bloom syndrome complex assembly required for double Holliday junction dissolution and genome stability

PNAS. 2022. Hodson, Charlotte et al. Univ Melbourne, Dept Med St Vincents, Fitzroy, Vic 3065, Australia; St Vincents Inst Med Res, Genome Stabil Unit, Fitzroy, Vic 3065, Australia

ABSTRACT: The RecQ-like helicase BLM cooperates with topoisomerase IIIa, RMI1, and RMI2 in a heterotetrameric complex (the "Bloom syndrome complex") for dissolution of double Holliday junctions, key intermediates in homologous recombination. Mutations in any component of the Bloom syndrome complex can cause genome instability and a highly cancer-prone disorder called Bloom syndrome. Some heterozygous carriers are also predisposed to breast cancer. To understand how the activities of BLM helicase and topoisomerase IIIa are coupled, we purified the active four-subunit complex.

Chemical cross-linking and mass spectrometry revealed a unique architecture that links the helicase and topoisomerase domains. Using biochemical experiments, we demonstrated dimerization mediated by the N terminus of BLM with a 2:2:2:2 stoichiometry within the Bloom syndrome complex. We identified mutations that independently abrogate dimerization or association of BLM with RMI1, and we show that both are dysfunctional for dissolution using in vitro assays and cause genome instability and synthetic lethal interactions with GEN1/MUS81 in cells. Truncated BLM can also inhibit the activity of full-length BLM in mixed dimers, suggesting a putative mechanism of dominant-negative action in carriers of BLM truncation alleles. Our results identify critical molecular determinants of Bloom syndrome complex assembly required for double Holliday junction dissolution and maintenance of genome stability.

[more...]

Specific binding of Hsp27 and phosphorylated Tau mitigates abnormal Tau aggregation-induced pathology

Elife. 2022. Zhang, SN et al. Shanghai Jiao Tong Univ, Bio X Inst, Key Lab Genet Dev & Neuropsychiat Disorders, Minist Educ, Shanghai, Peoples R China; Shanghai Jiao Tong Univ, Zhangjiang Inst Adv Study, Shanghai, Peoples R China; Univ Miami, Miller Sch Med, Dept Mol & Cellular Pharmacol, Miami, FL 33136 USA

ABSTRACT: Amyloid aggregation of phosphorylated Tau (pTau) into neurofibrillary tangles is closely associated with Alzheimer's disease (AD). Several molecular chaperones have been reported to bind Tau and impede its pathological aggregation. Recent findings of elevated levels of Hsp27 in the brains of patients with AD suggested its important role in pTau pathology. However, the molecular mechanism of Hsp27 in pTau aggregation remains poorly understood. Here, we show that Hsp27 partially co-localizes with pTau tangles in the brains of patients with AD.

Notably, phosphoryla-tion of Tau by microtubule affinity regulating kinase 2 (MARK2), dramatically enhances the binding affinity of Hsp27 to Tau. Moreover, Hsp27 efficiently prevents pTau fibrillation in vitro and mitigates neuropathology of pTau aggregation in a Drosophila tauopathy model. Further mechanistic study reveals that Hsp27 employs its N-terminal domain to directly interact with multiple phosphorylation sites of pTau for specific binding. Our work provides the structural basis for the specific recognition of Hsp27 to pathogenic pTau, and highlights the important role of Hsp27 in preventing abnormal aggregation and pathology of pTau in AD.

[more...]

SpotLink enables sensitive and precise identification of site nonspecific cross-links at the proteome scale

Briefings in Bioinformatics. 2022. Zhang, Weijie et al. Beihang Univ, Sch Biol Sci & Med Engn, Beijing 10010, Peoples R China; Beihang Univ, Sch Engn Med, Beijing 10010, Peoples R China; Chinese Acad Sci, Key Lab Separat Sci Analyt Chem, Dalian Inst Chem Phys, Dalian 116023, Peoples R China

ABSTRACT: Nonspecific cross-linker can provide distance restraints between surface residues of any type, which could be used to investigate protein structure construction and protein-protein interaction (PPI). However, the vast number of potential combinations of cross-linked residues or sites obtained with such a cross-linker makes the data challenging to analyze, especially for the proteome-wide applications. Here, we developed SpotLink software for identifying site nonspecific cross-links at the proteome scale.

Contributed by the dual pointer dynamic pruning algorithm and the quality control of cross-linking sites, SpotLink identified > 3000 cross-links from human cell samples within a short period of days. We demonstrated that SpotLink outperformed other approaches in terms of sensitivity and precision on the datasets of the simulated succinimidyl 4,4 '-azipentanoate dataset and the condensin complexes with known structures. In addition, some valuable PPI were discovered in the datasets of the condensin complexes and the HeLa dataset, indicating the unique identification advantages of site nonspecific cross-linking. These findings reinforce the importance of SpotLink as a fundamental characteristic of site nonspecific cross-linking technologies.

[more...]

Selective Removal of Unhydrolyzed Monolinked Peptides from Enriched Crosslinked Peptides To Improve the Coverage of Protein Complex Analysis

Analytical Chemistry. 2022. An, YX et al. Chinese Acad Sci, Dalian Inst Chem Phys, CAS Key Lab Separat Sci Analyt Chem, Natl Chromatog R&A Ctr, Dalian 116023, Liaoning, Peoples R China

ABSTRACT: Chemical crosslinking combined with mass spectrometry (CXMS) has allowed the global characterization of protein complexes with high throughput and accuracy. Although enrichable crosslinkers have been introduced to exclude the interference of regular peptides, the crosslinked peptide identification is still severely inhibited by a large amount of monolinked peptides. In this work, we proposed a strategy called MoTE (unhydrolyzed Monolinked peptide Targeting Elimination) to remove the unhydrolyzed monolinked peptides, while enriching crosslinked peptides for regular peptide removal.

In this strategy, followed by the crosslinking reaction, an amine biotin reagent was used to block the unreacted reactive groups on the crosslinker, and subsequently, the crosslinked proteins were tagged by a cleavable biotin-azide ligand based on click chemistry for enrichment. The following crosslinked protein digestion, purification by streptavidin beads, and release by chemical cleavage of the biotin-azide ligand were sequentially performed. In this case, the amine biotin-blocked unhydrolyzed monolinked peptides with the unbreakable arm remained on the streptavidin beads, which realized selective removal without any additional steps. By combining in vivo crosslinking with our proposed MoTE strategy for protein complex analysis of the HeLa cell, the number of high reliability (score

[more...]

The mouse nicotinamide mononucleotide adenylyltransferase chaperones diverse pathological amyloid client proteins

Journal of Biological Chemistry. 2022. Huang, CA et al. Chinese Acad Sci, Shanghai Inst Organ Chem, Interdisciplinary Res Ctr Biol & Chem, Shanghai, Peoples R China; Shanghai Jiao Tong Univ, Biox Renji Hosp Res Ctr, Renji Hosp, Sch Med, Shanghai, Peoples R China; Shanghai Jiao Tong Univ, Biox Inst, Minist Educ, Key Lab Genet Dev & Neuropsychiat Disorders, Shanghai, Peoples R China; Shanghai Jiao Tong Univ, Zhangjiang Inst Adv Study, Shanghai, Peoples R China

ABSTRACT: Molecular chaperones safeguard cellular protein homeostasis and obviate proteotoxicity. In the process of aging, as chaperone networks decline, aberrant protein amyloid aggregation accumulates in a mechanism that underpins neurodegeneration, leading to pathologies such as Alzheimer's disease and Parkinson's disease. Thus, it is important to identify and characterize chaperones for preventing such protein aggregation. In this work, we identified that the NAD(+) synthase-nicotinamide mononucleotide adenylyltransferase (NMNAT) 3 from mouse (mN3) exhibits potent chaperone activity to antagonize aggregation of a wide spectrum of pathological amyloid client proteins including alpha-synuclein, Tau (K19), amyloid beta, and islet amyloid polypeptide.

By combining NMR spectroscopy, cross-linking mass spectrometry, and computational modeling, we further reveal that mN3 uses different region of its amphiphilic surface near the active site to directly bind different amyloid client proteins. Our work demonstrates a client recognition mechanism of NMNAT via which it chaperones different amyloid client proteins against pathological aggregation and implies a potential protective role for NMNAT in different amyloid-associated diseases.

[more...]

D/E-rich peptides are less suitable than D/E-deficient peptides for identification by negative-ion HCD due to scarce production of sequencing ions from multiply …

International Journal of Mass Spectrometry. 2022. Zuo, Mei-Qing et al. Natl Inst Biol Sci, Beijing, Peoples R China

ABSTRACT: Highly acidic, D/E-rich peptides or proteins are difficult to identify by positive-ion-mode mass spec-trometry (MS), and negative-ion-mode MS is an attractive but insufficiently explored alternative. Based on high-resolution and accurate-mass MS analysis of 115 synthetic peptides of 5-28 amino acids, we confirmed that higher-energy collisional dissociation (HCD) of deprotonated peptides induced abundant backbone or side-chain neutral losses (NL), and updated the ranking list of NLs by abundance. The most abundant fragment ion types are y-> x-, z-> c-if the NL ions are included, or c-> y-> z-> 6-if not.

The most frequent side-chain NLs involve amino acids C, S, T, D, E, N, Q, and R. Although NL of CO2 is common for all peptides, it is markedly enhanced in D/E-containing peptides. Long peptides and D/E-rich peptides are prone to carrying multiple negative charges. HCD spectra produced from multiply deprotonated peptides have a lower fraction of sequencing ions (i.e., a-, 6-, c-, x-, y-, z -ions) than those produced from 1- ([M -H]-) and 2- ([M -2H]2-) precursors. Based on the above findings, we predict that under negative-ion HCD, D/E-rich peptides should be difficult to identify and that choosing a protease to generate peptides containing fewer D/E residues will improve identification of highly acidic proteins.(c) 2022 Elsevier B.V. All rights reserved.

[more...]

Autologous K63 deubiquitylation within the BRCA1-A complex licenses DNA damage recognition

Journal of Cell Biology. 2022. Jiang, QQ et al. Univ Penn, Penn Ctr Genome Integr, Basser Ctr BRCA, Perelman Sch Med,Dept Canc Biol, Philadelphia, PA 19104 USA; Univ Leeds, Fac Biol Sci, Astbury Ctr Struct Mol Biol, Sch Mol & Cellular Biol, Leeds, W Yorkshire, England

ABSTRACT: The BRCA1-A complex contains matching lysine-63 ubiquitin (K63-Ub) binding and deubiquitylating activities. How these functionalities are coordinated to effectively respond to DNA damage remains unknown. We generated Brcc36 deubiquitylating enzyme (DUB) inactive mice to address this gap in knowledge in a physiologic system. DUB inactivation impaired BRCA1-A complex damage localization and repair activities while causing early lethality when combined with Brca2 mutation. Damage response dysfunction in DUB-inactive cells corresponded to increased K63-Ub on RAP80 and BRCC36.

Chemical cross-linking coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and cryogenic-electron microscopy (cryo-EM) analyses of isolated BRCA1-A complexes demonstrated the RAP80 ubiquitin interaction motifs are occupied by ubiquitin exclusively in the DUB-inactive complex, linking auto-inhibition by internal K63-Ub chains to loss of damage site ubiquitin recognition. These findings identify RAP80 and BRCC36 as autologous DUB substrates in the BRCA1-A complex, thus explaining the evolution of matching ubiquitin-binding and hydrolysis activities within a single macromolecular assembly.

[more...]

Mimicked synthetic ribosomal protein complex for benchmarking crosslinking mass spectrometry workflows

Nature Communications. 2022. Matzinger, Manuel et al. Austrian Acad Sci, Vienna BioCtr VBC, Inst Mol Biotechnol, Vienna, Austria; Vienna BioCtr VBC, Inst Mol Pathol IMP, Vienna, Austria

ABSTRACT: Cross-linking mass spectrometry has matured to a frequently used tool for the investigation of protein structures as well as interactome studies up to a system-wide level. The growing community generated a broad spectrum of applications, linker types, acquisition strategies and specialized data analysis tools, which makes it challenging to decide for an appropriate analysis workflow. Here, we report a large and flexible synthetic peptide library as reliable instrument to benchmark crosslink workflows.

Additionally, we provide a tool, IMP-X-FDR, that calculates the real, experimentally validated, FDR, compares results across search engine platforms and analyses crosslink properties in an automated manner. We apply the library with 6 commonly used linker reagents and analyse the data with 6 established search engines. We thereby show that the correct algorithm and search setting choice is highly important to improve identification rate and reliability. We reach identification rates of up to similar to 70 % of the theoretical maximum (i.e. 700 unique lysine-lysine cross-links) while maintaining a real false-discovery-rate of <3 % at cross-link level with high reproducibility, representatively showing that our test system delivers valuable and statistically solid results.

[more...]

Conformational rearrangements upon start codon recognition in human 48S translation initiation complex

Nucleic Acids Research. 2022. Yi, Sung-Hui et al. Max Planck Inst Multidisciplinary Sci, Dept Struct Dynam, D-37077 Gottingen, Germany; Georg August Univ Gottingen, Inst Microbiol & Genet, Dept Mol Struct Biol, D-37077 Gottingen, Germany

ABSTRACT: Selection of the translation start codon is a key step during protein synthesis in human cells. We obtained cryo-EM structures of human 48S initiation complexes and characterized the intermediates of codon recognition by kinetic methods using eIF1A as a reporter. Both approaches capture two distinct ribosome populations formed on an mRNA with a cognate AUG codon in the presence of eIF1, eIF1A, eIF2-GTP-Met-tRNA(i)(Met) and eIF3. The 'open' 40S subunit conformation differs from the human 48S scanning complex and represents an intermediate preceding the codon recognition step.

The 'closed' form is similar to reported structures of complexes from yeast and mammals formed upon codon recognition, except for the orientation of eIF1A, which is unique in our structure. Kinetic experiments show how various initiation factors mediate the population distribution of open and closed conformations until 60S subunit docking. Our results provide insights into the timing and structure of human translation initiation intermediates and suggest the differences in the mechanisms of start codon selection between mammals and yeast.

[more...]

Enhanced protein--protein interaction network construction promoted by in vivo cross-linking with acid-cleavable click-chemistry enrichment

Frontiers in Chemistry. 2022. Zhao, Lili et al. Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog R&A Ctr, CAS Key Lab Separat Sci Analyt Chem, Dalian, Liaoning, Peoples R China

ABSTRACT: Chemical cross-linking coupled with mass spectrometry has emerged as a powerful strategy which enables global profiling of protein interactome with direct interaction interfaces in complex biological systems. The alkyne-tagged enrichable cross-linkers are preferred to improve the coverage of low-abundance cross-linked peptides, combined with click chemistry for biotin conjugation to allow the cross-linked peptide enrichment. However, a systematic evaluation on the efficiency of click approaches (protein-based or peptide-based) and diverse cleavable click-chemistry ligands (acid, reduction, and photo) for cross-linked peptide enrichment and release is lacking.

Herein, together with in vivo chemical cross-linking by alkyne-tagged cross-linkers, we explored the click-chemistry-based enrichment approaches on protein and peptide levels with three cleavable click-chemistry ligands, respectively. By comparison, the approach of protein-based click-chemistry conjugation with acid-cleavable tags was demonstrated to permit the most cross-linked peptide identification. The advancement of this strategy enhanced the proteome-wide cross-linking analysis, constructing a 5,518-protein-protein-interaction network among 1,871 proteins with widely abundant distribution in cells. Therefore, all these results demonstrated the guideline value of our work for efficient cross-linked peptide enrichment, thus facilitating the in-depth profiling of protein interactome for functional analysis.

[more...]

Comprehensive structure and functional adaptations of the yeast nuclear pore complex

Cell. 2022. Akey, Christopher W. et al. Univ Calif San Diego, Div Biol Sci, Sect Mol Biol, La Jolla, CA 92093 USA; Univ Basque Country, Inst Biofis, UPV EHU, CSIC, Leioa 48940, Spain; Univ Calif San Diego, Howard Hughes Med Inst, La Jolla, CA 92093 USA; Baylor Coll Med, Verna & Marrs McLean Dept Biochem & Mol Biol, 1 Baylor Plaza, Houston, TX 77030 USA; Rockefeller Univ, Lab Cellular & Struct Biol, New York, NY 10065 USA; Basque Fdn Sci, Ikerbasque, Bilbao 48013, Spain

ABSTRACT: Nuclear pore complexes (NPCs) mediate the nucleocytoplasmic transport of macromolecules. Here we provide a structure of the isolated yeast NPC in which the inner ring is resolved by cryo-EM at sub-nanometer resolution to show how flexible connectors tie together different structural and functional layers. These connectors may be targets for phosphorylation and regulated disassembly in cells with an open mitosis. Moreover, some nucleoporin pairs and transport factors have similar interaction motifs, which suggests an evolutionary and mechanistic link between assembly and transport.

We provide evidence for three major NPC variants that may foreshadow functional specializations at the nuclear periphery. Cryo-electron tomography extended these studies, providing a model of the in situ NPC with a radially expanded inner ring. Our comprehensive model reveals features of the nuclear basket and central transporter, suggests a role for the lumenal Pom152 ring in restricting dilation, and highlights structural plasticity that may be required for transport.

[more...]

Mechanism of Bloom syndrome complex assembly required for double Holliday junction dissolution and genome stability

PNAS. 2022. Hodson, Charlotte et al. Univ Melbourne, Dept Med St Vincents, Fitzroy, Vic 3065, Australia; St Vincents Inst Med Res, Genome Stabil Unit, Fitzroy, Vic 3065, Australia

ABSTRACT: The RecQ-like helicase BLM cooperates with topoisomerase IIIa, RMI1, and RMI2 in a heterotetrameric complex (the "Bloom syndrome complex") for dissolution of double Holliday junctions, key intermediates in homologous recombination. Mutations in any component of the Bloom syndrome complex can cause genome instability and a highly cancer-prone disorder called Bloom syndrome. Some heterozygous carriers are also predisposed to breast cancer. To understand how the activities of BLM helicase and topoisomerase IIIa are coupled, we purified the active four-subunit complex.

Chemical cross-linking and mass spectrometry revealed a unique architecture that links the helicase and topoisomerase domains. Using biochemical experiments, we demonstrated dimerization mediated by the N terminus of BLM with a 2:2:2:2 stoichiometry within the Bloom syndrome complex. We identified mutations that independently abrogate dimerization or association of BLM with RMI1, and we show that both are dysfunctional for dissolution using in vitro assays and cause genome instability and synthetic lethal interactions with GEN1/MUS81 in cells. Truncated BLM can also inhibit the activity of full-length BLM in mixed dimers, suggesting a putative mechanism of dominant-negative action in carriers of BLM truncation alleles. Our results identify critical molecular determinants of Bloom syndrome complex assembly required for double Holliday junction dissolution and maintenance of genome stability.

[more...]

Structural basis for assembly of TRAPPII complex and specific activation of GTPase Ypt31/32

Science Advances. 2022. Mi, Chenchen et al. Southern Univ Sci & Technol, Dept Biol, Shenzhen 518055, Guangdong, Peoples R China; Tsinghua Univ, Beijing Adv Innovat Ctr Struct Biol, Beijing Frontier Res Ctr Biol Struct, Sch Life Sci,State Key Lab Membrane Biol, Beijing 100084, Peoples R China

ABSTRACT: Transport protein particle (TRAPP) complexes belong to the multiprotein tethering complex and exist in three forms-core TRAPP/TRAPPI, TRAPPII, and TRAPPIII. TRAPPII activates GTPase Ypt31/Ypt32 as the guanine nucleotide exchange factor in the trans-Golgi network to determine the maturation of Golgi cisternae into post-Golgi carriers in yeast. Here, we present cryo-EM structures of yeast TRAPPII in apo and Ypt32-bound states. All the structures show a dimeric architecture assembled by two triangle-shaped monomers, while the monomer in the apo state exhibits both open and closed conformations, and the monomer in the Ypt32-bound form only captures the closed conformation.

Located in the interior of the monomer, Ypt32 binds with both core TRAPP/TRAPPI and Trs120 via its nucleotide-binding domain and binds with Trs31 via its hypervariable domain. Combined with functional analysis, the structures provide insights into the assembly of TRAPPII and the mechanism of the specific activation of Ypt31/Ypt32 by TRAPPII.

[more...]

SpotLink enables sensitive and precise identification of site nonspecific cross-links at the proteome scale

Briefings in Bioinformatics. 2022. Zhang, Weijie et al. Beihang Univ, Sch Biol Sci & Med Engn, Beijing 10010, Peoples R China; Beihang Univ, Sch Engn Med, Beijing 10010, Peoples R China; Chinese Acad Sci, Key Lab Separat Sci Analyt Chem, Dalian Inst Chem Phys, Dalian 116023, Peoples R China

ABSTRACT: Nonspecific cross-linker can provide distance restraints between surface residues of any type, which could be used to investigate protein structure construction and protein-protein interaction (PPI). However, the vast number of potential combinations of cross-linked residues or sites obtained with such a cross-linker makes the data challenging to analyze, especially for the proteome-wide applications. Here, we developed SpotLink software for identifying site nonspecific cross-links at the proteome scale.

Contributed by the dual pointer dynamic pruning algorithm and the quality control of cross-linking sites, SpotLink identified > 3000 cross-links from human cell samples within a short period of days. We demonstrated that SpotLink outperformed other approaches in terms of sensitivity and precision on the datasets of the simulated succinimidyl 4,4 '-azipentanoate dataset and the condensin complexes with known structures. In addition, some valuable PPI were discovered in the datasets of the condensin complexes and the HeLa dataset, indicating the unique identification advantages of site nonspecific cross-linking. These findings reinforce the importance of SpotLink as a fundamental characteristic of site nonspecific cross-linking technologies.

[more...]

Structures of transcription preinitiation complex engaged with the+ 1 nucleosome

Nature Structural & Molecular Biology. 2022. Wang, HB et al. Max Planck Inst Multidisciplinary Sci, Dept Mol Biol, Gottingen, Germany

ABSTRACT: The preinitiation complex (PIC) assembles on promoters of protein-coding genes to position RNA polymerase II (Pol II) for transcription initiation. Previous structural studies revealed the PIC on different promoters, but did not address how the PIC assembles within chromatin. In the yeast Saccharomyces cerevisiae, PIC assembly occurs adjacent to the +1 nucleosome that is located downstream of the core promoter. Here we present cryo-EM structures of the yeast PIC bound to promoter DNA and the +1 nucleosome located at three different positions.

The general transcription factor TFIIH engages with the incoming downstream nucleosome and its translocase subunit Ssl2 (XPB in human TFIIH) drives the rotation of the +1 nucleosome leading to partial detachment of nucleosomal DNA and intimate interactions between TFIIH and the nucleosome. The structures provide insights into how transcription initiation can be influenced by the +1 nucleosome and may explain why the transcription start site is often located roughly 60 base pairs upstream of the dyad of the +1 nucleosome in yeast.Cryo-EM structures of the transcription preinitiation complex in the presence of the +1 nucleosome show how the general transcription factor TFIIH can interact with the nucleosome at several positions.

[more...]

A toxin-deformation dependent inhibition mechanism in the T7SS toxin-antitoxin system of Gram-positive bacteria

Nature Communications. 2022. Wang, YJ et al. Jinan Univ, Coll Pharm, Int Cooperat Lab Tradit Chinese Med Modernizat &, Chinese Minist Educ MOE, Guangzhou 510632, Peoples R China; Weill Cornell Med, Dept Physiol & Biophys, New York, NY 10065 USA; Guangdong Youmei Inst Intelligent Biomfg, Foshan 528200, Guangdong, Peoples R China

ABSTRACT: Toxin EsaD secreted by some S. aureus strains through the type VII secretion system (T7SS) specifically kills those strains lacking the antitoxin EsaG. Here we report the structures of EsaG, the nuclease domain of EsaD and their complex, which together reveal an inhibition mechanism that relies on significant conformational change of the toxin. To inhibit EsaD, EsaG breaks the nuclease domain of EsaD protein into two independent fragments that, in turn, sandwich EsaG. The originally well-folded beta beta alpha-metal finger connecting the two fragments is stretched to become a disordered loop, leading to disruption of the catalytic site of EsaD and loss of nuclease activity.

This mechanism is distinct from that of the other Type II toxin-antitoxin systems, which utilize an intrinsically disordered region on the antitoxins to cover the active site of the toxins. This study paves the way for developing therapeutic approaches targeting this antagonism.Antimicrobial toxins are secreted by bacteria to kill rival species. Here the authors report the mechanism of inhibition of EsaD, a toxin secreted by some S. aureus strains to kill competitors that lack the antitoxin EsaG, showing marked mechanistic differences to other Type II toxin-antitoxin systems.

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Mapping of the plant SnRK1 kinase signalling network reveals a key regulatory role for the class II T6P synthase-like proteins

Nature Plants. 2022. Van Leene, J et al. Univ Ghent, Dept Plant Biotechnol & Bioinformat, Ghent, Belgium; VIB Ctr Plant Syst Biol, Ghent, Belgium

ABSTRACT: The central metabolic regulator SnRK1 controls plant growth and survival upon activation by energy depletion, but detailed molecular insight into its regulation and downstream targets is limited. Here we used phosphoproteomics to infer the sucrose-dependent processes targeted upon starvation by kinases as SnRK1, corroborating the relation of SnRK1 with metabolic enzymes and transcriptional regulators, while also pointing to SnRK1 control of intracellular trafficking. Next, we integrated affinity purification, proximity labelling and crosslinking mass spectrometry to map the protein interaction landscape, composition and structure of the SnRK1 heterotrimer, providing insight in its plant-specific regulation.

At the intersection of this multi-dimensional interactome, we discovered a strong association of SnRK1 with class II T6P synthase (TPS)-like proteins. Biochemical and cellular assays show that TPS-like proteins function as negative regulators of SnRK1. Next to stable interactions with the TPS-like proteins, similar intricate connections were found with known regulators, suggesting that plants utilize an extended kinase complex to fine-tune SnRK1 activity for optimal responses to metabolic stress.SnRK1 is a key metabolic sensor that controls plant development and stress responses. This study integrates phosphoproteomics, affinity purification coupled to mass spectrometry, proximity labelling and crosslinking mass spectrometry to obtain more insight into its upstream regulation and downstream target processes.

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Subcellular Interactomes Revealed by Merging APEX with Cross-Linking Mass Spectrometry

Analytical Chemistry. 2022. Sun, MZ et al. Chinese Inst Brain Res CIBR, Beijing 102206, Peoples R China; Peking Univ, Synthet & Funct Biomol Ctr, Coll Chem & Mol Engn, Dept Chem Biol,Beijing Natl Lab Mol Sci,Key Lab Bi, Beijing 100871, Peoples R China; Inst Canc Res, Shenzhen Bay Lab, Shenzhen 518107, Peoples R China; Peking Univ, Peking Tsinghua Ctr Life Sci, Beijing 100871, Peoples R China; Peking Univ, PKU IDG McGovern Inst Brain Res, Beijing 100871, Peoples R China

ABSTRACT: Subcellular protein-protein interactions (PPIs) are essential to understanding the mechanism of diverse cellular signaling events and the pathogenesis of diseases. Herein, we report an integrated APEX proximity labeling and chemical cross-linking coupled with mass spectrometry (CXMS) platform named APEX-CXMS for spatially resolved subcellular interactome profiling in a high-throughput manner. APEX proximity labeling rapidly captures subcellular proteomes, and the highly reactive chemical cross-linkers can capture weak and dynamic interactions globally without extra genetic manipulation.

APEX-CXMS was first applied to mitochondria and identified 653 pairs of interprotein cross-links. Six pairs of new interactions were selected and verified by coimmunoprecipitation, the mammalian two-hybrid system, and surface plasmon resonance method. Besides, our approach was further applied to the nucleus, capturing 336 pairs of interprotein cross-links with approximately 94% nuclear specificity. APEX-CXMS thus provides a simple, fast, and general alternative to map diverse subcellular PPIs.

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Genetically encoded chemical crosslinking of carbohydrate

Nature Chemistry. 2022. Li, SS et al. Univ Calif San Francisco, Dept Pharmaceut Chem, San Francisco, CA 94143 USA; Univ Calif San Francisco, Cardiovasc Res Inst, Box 0544, San Francisco, CA 94143 USA

ABSTRACT: Protein-carbohydrate interactions play important roles in various biological processes, such as organism development, cancer metastasis, pathogen infection and immune response, but they remain challenging to study and exploit due to their low binding affinity and non-covalent nature. Here we site-specifically engineered covalent linkages between proteins and carbohydrates under biocompatible conditions. We show that sulfonyl fluoride reacts with glycans via a proximity-enabled reactivity, and to harness this a bioreactive unnatural amino acid (SFY) that contains sulfonyl fluoride was genetically encoded into proteins.

SFY-incorporated Siglec-7 crosslinked with its sialoglycan ligand specifically in vitro and on the surface of cancer cells. Through irreversible cloaking of sialoglycan at the cancer cell surface, SFY-incorporated Siglec-7 enhanced the killing of cancer cells by natural killer cells. Genetically encoding the chemical crosslinking of proteins to carbohydrates (GECX-sugar) offers a solution to address the low affinity and weak strength of protein-sugar interactions.

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Structural basis of SNAPc-dependent snRNA transcription initiation by RNA polymerase II

Nature Structural & Molecular Biology. 2022. Rengachari, S et al. Inst Canc Res, Div Struct Biol, London, England; Human Technopole, Milan, Italy; Max Planck Inst Multidisciplinary Sci, Dept Mol Biol, Gottingen, Germany

ABSTRACT: Rengachari et al. provide a structural investigation of Pol II initiation at snRNA gene promoters and find that the snRNA-activating protein complex enables DNA opening and transcription initiation independent of TFIIE and TFIIH in vitro.RNA polymerase II (Pol II) carries out transcription of both protein-coding and non-coding genes. Whereas Pol II initiation at protein-coding genes has been studied in detail, Pol II initiation at non-coding genes, such as small nuclear RNA (snRNA) genes, is less well understood at the structural level.

Here, we study Pol II initiation at snRNA gene promoters and show that the snRNA-activating protein complex (SNAPc) enables DNA opening and transcription initiation independent of TFIIE and TFIIH in vitro. We then resolve cryo-EM structures of the SNAPc-containing Pol IIpre-initiation complex (PIC) assembled on U1 and U5 snRNA promoters. The core of SNAPc binds two turns of DNA and recognizes the snRNA promoter-specific proximal sequence element (PSE), located upstream of the TATA box-binding protein TBP. Two extensions of SNAPc, called wing-1 and wing-2, bind TFIIA and TFIIB, respectively, explaining how SNAPc directs Pol II to snRNA promoters. Comparison of structures of closed and open promoter complexes elucidates TFIIH-independent DNA opening. These results provide the structural basis of Pol II initiation at non-coding RNA gene promoters.

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Structural basis of Tom20 and Tom22 cytosolic domains as the human TOM complex receptors

PNAS. 2022. Su, JY et al. Southern Univ Sci & Technol, Dept Biol, Shenzhen 518055, Guangdong, Peoples R China; Tsinghua Univ, Beijing Adv Innovat Ctr Struct Biol, Beijing Frontier Res Ctr Biol Struct, State Key Lab Membrane Biol,Sch Life Sci, Beijing 100084, Peoples R China

ABSTRACT: Mitochondrial preproteins synthesized in cytosol are imported into mitochondria by a multisubunit translocase of the outer membrane (TOM) complex. Functioned as the receptor, the TOM complex components, Tom 20, Tom22, and Tom70, recognize the presequence and further guide the protein translocation. Their deficiency has been linked with neurodegenerative diseases and cardiac pathology. Although several structures of the TOM complex have been reported by cryoelectron microscopy (cryo-EM), how Tom22 and Tom20 function as TOM receptors remains elusive.

Here we determined the structure of TOM core complex at 2.53 angstrom and captured the structure of the TOM complex containing Tom22 and Tom20 cytosolic domains at 3.74 angstrom. Structural analysis indicates that Tom20 and Tom22 share a similar three-helix bundle structural feature in the cytosolic domain Further structure-guided biochemical analysis reveals that the Tom22 cytosolic domain is responsible for binding to the presequence, and the helix H1 is critical for this binding. Altogether, our results provide insights into the functional mechanism of the TOM complex recognizing and transferring preproteins across the mitochondrial membrane.

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Structural basis for c-di-AMP--dependent regulation of the bacterial stringent response by receptor protein DarB

Journal of Biological Chemistry. 2022. Heidemann, JL et al. Georg August Univ Gottingen, Dept Mol Struct Biol, Gottingen, Germany

ABSTRACT: The bacterial second messenger c-di-AMP controls essential cellular processes, including potassium and osmolyte homeo-stasis. This makes synthesizing enzymes and components involved in c-di-AMP signal transduction intriguing as poten-tial targets for drug development. The c-di-AMP receptor protein DarB of Bacillus subtilis binds the Rel protein and triggers the Rel-dependent stringent response to stress condi-tions; however, the structural basis for this trigger is unclear. Here, we report crystal structures of DarB in the ligand-free state and of DarB complexed with c-di-AMP, 3'3'-cGAMP, and AMP.

We show that DarB forms a homodimer with a parallel, head-to-head assembly of the monomers. We also confirm the DarB dimer binds two cyclic dinucleotide mole-cules or two AMP molecules; only one adenine of bound c-di -AMP is specifically recognized by DarB, while the second protrudes out of the donut-shaped protein. This enables DarB to bind also 3'3'-cGAMP, as only the adenine fits in the active site. In absence of c-di-AMP, DarB binds to Rel and stimulates (p)ppGpp synthesis, whereas the presence of c-di-AMP abol-ishes this interaction. Furthermore, the DarB crystal structures reveal no conformational changes upon c-di-AMP binding, leading us to conclude the regulatory function of DarB on Rel must be controlled directly by the bound c-di-AMP. We thus derived a structural model of the DarB-Rel complex via in silico docking, which was validated with mass spectrometric analysis of the chemically crosslinked DarB-Rel complex and mutagenesis studies. We suggest, based on the predicted complex structure, a mechanism of stringent response regula-tion by c-di-AMP.

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Juxtaposition of Bub1 and Cdc20 on phosphorylated Mad1 during catalytic mitotic checkpoint complex assembly

Nature communications. 2022. Fischer, ES et al. MRC Lab Mol Biol, Cambridge Biomed Campus,Francis Crick Ave, Cambridge CB2 0QH, England

ABSTRACT: Formation of the mitotic checkpoint complex (MCC) is catalysed by a phosphorylation-dependent scaffold. This work provides structural details of how a tripartite Mad1:Bub1:Cdc20 complex presents Cdc20 to Mad2, triggering open-to-closed conversion of Mad2 to assemble the MCC.In response to improper kinetochore-microtubule attachments in mitosis, the spindle assembly checkpoint (SAC) assembles the mitotic checkpoint complex (MCC) to inhibit the anaphase-promoting complex/cyclosome, thereby delaying entry into anaphase.

The MCC comprises Mad2:Cdc20:BubR1:Bub3. Its assembly is catalysed by unattached kinetochores on a Mad1:Mad2 platform. Mad1-bound closed-Mad2 (C-Mad2) recruits open-Mad2 (O-Mad2) through self-dimerization. This interaction, combined with Mps1 kinase-mediated phosphorylation of Bub1 and Mad1, accelerates MCC assembly, in a process that requires O-Mad2 to C-Mad2 conversion and concomitant binding of Cdc20. How Mad1 phosphorylation catalyses MCC assembly is poorly understood. Here, we characterized Mps1 phosphorylation of Mad1 and obtained structural insights into a phosphorylation-specific Mad1:Cdc20 interaction. This interaction, together with the Mps1-phosphorylation dependent association of Bub1 and Mad1, generates a tripartite assembly of Bub1 and Cdc20 onto the C-terminal domain of Mad1 (Mad1(CTD)). We additionally identify flexibility of Mad1:Mad2 that suggests how the Cdc20:Mad1(CTD) interaction brings the Mad2-interacting motif (MIM) of Cdc20 near O-Mad2. Thus, Mps1-dependent formation of the MCC-assembly scaffold functions to position and orient Cdc20 MIM near O-Mad2, thereby catalysing formation of C-Mad2:Cdc20.

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Activation of the essential kinase PDK1 by phosphoinositide-driven trans-autophosphorylation

Nature communications. 2022. Levina, A et al. Max Perutz Labs, Dept Struct & Computat Biol, Campus Vienna Bioctr 5, A-1030 Vienna, Austria; Med Univ Vienna, Dept Med Biochem, A-1090 Vienna, Austria

ABSTRACT: 3-phosphoinositide-dependent kinase 1 (PDK1) is an essential serine/threonine protein kinase, which plays a crucial role in cell growth and proliferation. It is often referred to as a 'master' kinase due to its ability to activate at least 23 downstream protein kinases implicated in various signaling pathways. In this study, we have elucidated the mechanism of phosphoinositide-driven PDK1 auto-activation. We show that PDK1 trans-autophosphorylation is mediated by a PIP3-mediated face-to-face dimer.

We report regulatory motifs in the kinase-PH interdomain linker that allosterically activate PDK1 autophosphorylation via a linker-swapped dimer mechanism. Finally, we show that PDK1 is autoinhibited by its PH domain and that positive cooperativity of PIP3 binding drives switch-like activation of PDK1. These results imply that the PDK1-mediated activation of effector kinases, including Akt, PKC, Sgk, S6K and RSK, many of whom are not directly regulated by phosphoinositides, is also likely to be dependent on PIP3 or PI(3,4)P-2.The essential protein kinase PDK1 is activated by phospoinositide-mediated dimerization and trans-autophosphorylation. Here, the authors show that in the absence of PIP3 or PI(3,4)P-2 phosphoinositides, PDK1 is maintained in an inactive, autoinhibited conformation in the cytosol.

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Real-time library search increases cross-link identification depth across all levels of sample complexity

Analytical chemistry. 2022. Ruwolt, Max et al. Leibniz Forschungsinst Mol Pharmakol FMP, Dept Struct Biol, D-13125 Berlin, Germany; Charite Univ Med Berlin, D-10117 Berlin, Germany

ABSTRACT: Cross-linking mass spectrometry (XL-MS) is a universal tool for probing structural dynamics and protein-protein interactions in vitro and in vivo. Although cross-linked peptides are naturally less abundant than their unlinked counterparts, recent experimental advances improved cross-link identification by enriching the cross-linker-modified peptides chemically with the use of enrichable cross-linkers. However, mono-links (i.e., peptides modified with a hydrolyzed cross-linker) still hinder efficient cross-link identification since a large proportion of measurement time is spent on their MS2 acquisition.

Currently, cross-links and mono-links cannot be separated by sample preparation techniques or chromatography because they are chemically almost identical. Here, we found that based on the intensity ratios of four diagnostic peaks when using PhoX/tBu-PhoX cross-linkers, cross-links and mono-links can be partially distinguished. Harnessing their characteristic intensity ratios for real-time library search (RTLS)-based triggering of high-resolution MS2 scans increased the number of cross-link identifications from both single protein samples and intact E. coli cells. Specifically, RTLS improves cross-link identification from unenriched samples and short gradients, emphasizing its advantages in high-throughput approaches and when instrument time or sample amount is limited.

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D/E-rich peptides are less suitable than D/E-deficient peptides for identification by negative-ion HCD due to scarce production of sequencing ions from multiply charged precursors

International Journal of Mass Spectrometry. 2022. Zuo, Mei-Qing et al. Natl Inst Biol Sci, Beijing, Peoples R China

ABSTRACT: Highly acidic, D/E-rich peptides or proteins are difficult to identify by positive-ion-mode mass spec-trometry (MS), and negative-ion-mode MS is an attractive but insufficiently explored alternative. Based on high-resolution and accurate-mass MS analysis of 115 synthetic peptides of 5-28 amino acids, we confirmed that higher-energy collisional dissociation (HCD) of deprotonated peptides induced abundant backbone or side-chain neutral losses (NL), and updated the ranking list of NLs by abundance. The most abundant fragment ion types are y-> x-, z-> c-if the NL ions are included, or c-> y-> z-> 6-if not.

The most frequent side-chain NLs involve amino acids C, S, T, D, E, N, Q, and R. Although NL of CO2 is common for all peptides, it is markedly enhanced in D/E-containing peptides. Long peptides and D/E-rich peptides are prone to carrying multiple negative charges. HCD spectra produced from multiply deprotonated peptides have a lower fraction of sequencing ions (i.e., a-, 6-, c-, x-, y-, z -ions) than those produced from 1- ([M -H]-) and 2- ([M -2H]2-) precursors. Based on the above findings, we predict that under negative-ion HCD, D/E-rich peptides should be difficult to identify and that choosing a protease to generate peptides containing fewer D/E residues will improve identification of highly acidic proteins.(c) 2022 Elsevier B.V. All rights reserved.

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Specific binding of Hsp27 and phosphorylated Tau mitigates abnormal Tau aggregation-induced pathology

Elife. 2022. Zhang, SN et al. Shanghai Jiao Tong Univ, Bio X Inst, Key Lab Genet Dev & Neuropsychiat Disorders, Minist Educ, Shanghai, Peoples R China; Shanghai Jiao Tong Univ, Zhangjiang Inst Adv Study, Shanghai, Peoples R China; Univ Miami, Miller Sch Med, Dept Mol & Cellular Pharmacol, Miami, FL 33136 USA

ABSTRACT: Amyloid aggregation of phosphorylated Tau (pTau) into neurofibrillary tangles is closely associated with Alzheimer's disease (AD). Several molecular chaperones have been reported to bind Tau and impede its pathological aggregation. Recent findings of elevated levels of Hsp27 in the brains of patients with AD suggested its important role in pTau pathology. However, the molecular mechanism of Hsp27 in pTau aggregation remains poorly understood. Here, we show that Hsp27 partially co-localizes with pTau tangles in the brains of patients with AD.

Notably, phosphoryla-tion of Tau by microtubule affinity regulating kinase 2 (MARK2), dramatically enhances the binding affinity of Hsp27 to Tau. Moreover, Hsp27 efficiently prevents pTau fibrillation in vitro and mitigates neuropathology of pTau aggregation in a Drosophila tauopathy model. Further mechanistic study reveals that Hsp27 employs its N-terminal domain to directly interact with multiple phosphorylation sites of pTau for specific binding. Our work provides the structural basis for the specific recognition of Hsp27 to pathogenic pTau, and highlights the important role of Hsp27 in preventing abnormal aggregation and pathology of pTau in AD.

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Divergent polo boxes in KKT2 bind KKT1 to initiate the kinetochore assembly cascade in Trypanosoma brucei

Molecular Biology of the Cell. 2022. Ishii, Midori et al. Univ Oxford, Dept Biochem, Oxford OX1 3QU, England

ABSTRACT: Chromosome segregation requires assembly of the macromolecular kinetochore complex onto centromeric DNA. While most eukaryotes have canonical kinetochore proteins that are widely conserved among eukaryotes, evolutionarily divergent kinetoplastids have a unique set of kinetochore proteins. Little is known about the mechanism of kinetochore assembly in kinetoplastids. Here we characterize two homologous kinetoplastid kinetochore proteins, KKT2 and KKT3, that constitutively localize at centromeres. They have three domains that are highly conserved among kinetoplastids: an N-terminal kinase domain of unknown function, the centromere localization domain in the middle, and the C-terminal domain that has weak similarity to polo boxes of Polo-like kinases.

We show that the kinase activity of KKT2 is essential for accurate chromosome segregation, while that of KKT3 is dispensable for cell growth in Trypanosoma brucei. Crystal structures of their divergent polo boxes reveal differences between KKT2 and KKT3. We also show that the divergent polo boxes of KKT3 are sufficient to recruit KKT2 in trypanosomes. Furthermore, we demonstrate that the divergent polo boxes of KKT2 interact directly with KKT1 and that KKT1 interacts with KKT6. These results show that the divergent polo boxes of KKT2 and KKT3 are protein-protein interaction domains that initiate kinetochore assembly in T. brucei.

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Multistate structures of the MLL1-WRAD complex bound to H2B-ubiquitinated nucleosome

PNAS. 2022. Rahman, S et al. Johns Hopkins Univ, Dept Biophys & Biophys Chem, Sch Med, Baltimore, MD 21205 USA

ABSTRACT: The human Mixed Lineage Leukemia-1 (MLL1) complex methylates histone H3K4 to promote transcription and is stimulated by monoubiquitination of histone H2B. Recent structures of the MLL1-WRAD core complex, which comprises the MLL1 methyltransferase, WDR5, RbBp5, Ash2L, and DPY-30, have revealed variability in the docking of MLL1-WRAD on nucleosomes. In addition, portions of the Ash2L structure and the position of DPY30 remain ambiguous. We used an integrated approach combining cryoelectron microscopy (cryo-EM) and mass spectrometry cross-linking to determine a structure of the MLL1-WRAD complex bound to ubiquitinated nucleosomes.

The resulting model contains the Ash2L intrinsically disordered region (IDR), SPRY insertion region, Sdc1-DPY30 interacting region (SDI-motif), and the DPY30 dimer. We also resolved three additional states of MLL1-WRAD lacking one or more subunits, which may reflect different steps in the assembly of MLL1-WRAD. The docking of subunits in all four states differs from structures of MLL1- WRAD bound to unmodified nucleosomes, suggesting that H2B-ubiquitin favors assembly of the active complex. Our results provide a more complete picture of MLL1-WRAD and the role of ubiquitin in promoting formation of the active methyltransferase complex.

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Alkynyl-Enrichable Carboxyl-Selective Crosslinkers to Increase the Crosslinking Coverage for Deciphering Protein Structures

Analytical Chemistry. 2022. Gao, H et al. Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog R&A Ctr, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Liaoning, Peoples R China

ABSTRACT: The coverage of chemical crosslinking coupled with mass spectrometry (CXMS) is of great importance to determine its ability for deciphering protein structures. At present, N- hydroxysuccinimidyl (NHS) ester-based crosslinkers targeting lysines have been predominantly used in CXMS. However, they are not always effective for some proteins with few lysines. Other amino acid residues such as carboxyl could be crosslinked to complement lysines and improve the crosslinking coverage of CXMS, but the low intrinsic chemical reactivity of carboxyl compromises the application of carboxyl-selective crosslinkers for complex samples.

To enhance the crosslinking efficiency targeting acidic residues and realize in-depth crosslinking analysis of complex samples, we developed three new alkynyl-enrichable carboxyl-selective crosslinkers with different reactive groups such as hydrazide, amino, and aminooxy. The crosslinking efficiencies of the three crosslinkers were systematically evaluated, giving the best reactivity of the amino-functionalized crosslinker BAP. Furthermore, BAP was extended to the crosslinking analysis of Escherichia coli lysate in combination with efficient crosslink enrichment. A total of 1291 D/E-D/E crosslinks involved in 392 proteins were identified under a false discovery rate (FDR) of >= 1%. Obvious structural complementarity of BAP was exhibited to the lysine-targeting crosslinker, facilitating the capability of CXMS for protein structure elucidation. To the best of our knowledge, this was the first time for the carboxyl-selective crosslinker to achieve proteome-wide crosslinking analysis of the whole cell lysate. Collectively, we believe that this work not only expands on a promising toolkit of CXMS targeting acidic residues but also provides a valuable guideline to advance the performance of carboxyl-selective crosslinkers.

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DPY30 acts as an ASH2L-specific stabilizer to stimulate the enzyme activity of MLL family methyltransferases on different substrates

Iscience. 2022. Zhao, LJ et al. Chinese Acad Sci, Shanghai Inst Biochem & Cell Biol, Natl Ctr Prot Sci Shanghai, Ctr Excellence Mol Cell Sci, Shanghai 200031, Peoples R China; ShanghaiTech Univ, Sch Life Sci & Technol, 100 Haike Rd, Shanghai 201210, Peoples R China; Univ Chinese Acad Sci, Beijing 100049, Peoples R China

ABSTRACT: Dumpy-30 (DPY30) is a conserved component of the mixed lineage leukemia (MLL) family complex and is essential for robust methyltransferase activity of MLL complexes. However, the biochemical role of DPY30 in stimulating methyl-transferase activity of MLL complexes remains elusive. Here, we demonstrate that DPY30 plays a crucial role in regulating MLL1 activity through two com-plementary mechanisms: A nucleosome-independent mechanism and a nucleo-some-specific mechanism. DPY30 functions as an ASH2L-specific stabilizer to increase the stability of ASH2L and enhance ASH2L-mediated interactions.

As a result, DPY30 promotes the compaction and stabilization of the MLL1 complex, consequently increasing the HKMT activity of the MLL1 complex on diverse sub-strates. DPY30-stabilized ASH2L further acquires additional interfaces with H3 and nucleosomal DNA, thereby boosting the methyltransferase activity of the MLL1 complex on nucleosomes. These results collectively highlight the crucial and conserved roles of DPY30 in the complex assembly and activity regulation of MLL family complexes.

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Characterization of protein complexes in extracellular vesicles by intact extracellular vesicle crosslinking mass spectrometry (iEVXL)

Journal of Extracellular Vesicles. 2022. Bauza-Martinez, J et al. ASTAR, Singapore Immunol Network SIgN, Singapore, Singapore

ABSTRACT: Extracellular vesicles (EVs) are blood-borne messengers that coordinate signalling between different tissues and organs in the body. The specificity of such crosstalk is determined by preferential EV docking to target sites, as mediated through protein-protein interactions. As such, the need to structurally characterize the EV surface precedes further understanding of docking selectivity and recipient-cell uptake mechanisms. Here, we describe an intact extracellular vesicle crosslinking mass spectrometry (iEVXL) method that can be applied for structural characterization of protein complexes in EVs.

By using a partially membrane-permeable disuccinimidyl suberate crosslinker, proteins on the EV outer-surface and inside EVs can be immobilized together with their interacting partners. This not only provides covalent stabilization of protein complexes before extraction from the membrane-enclosed environment, but also generates a set of crosslinking distance restraints that can be used for structural modelling and comparative screening of changes in EV protein assemblies. Here we demonstrate iEVXL as a powerful approach to reveal high-resolution information, about protein determinants that govern EV docking and signalling, and as a crucial aid in modelling docking interactions.

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Structure of Arabidopsis SOQ1 lumenal region unveils C-terminal domain essential for negative regulation of photoprotective qH

Nature Plants. 2022. Yu, GM et al. Umea Univ, Dept Plant Physiol, Umea Plant Sci Ctr UPSC, Umea, Sweden; Chinese Acad Sci, Inst Biophys, CAS Ctr Excellence Biomacromol, Natl Lab Biomacromol, Beijing, Peoples R China

ABSTRACT: Genetic, biochemical and high-resolution structural studies of chloroplast protein SOQ1 reveal the existence of a C-terminal lumenal domain with potential redox function and its essential role for suppressing photoprotection in plants.Non-photochemical quenching (NPQ) plays an important role for phototrophs in decreasing photo-oxidative damage. qH is a sustained form of NPQ and depends on the plastid lipocalin (LCNP). A thylakoid membrane-anchored protein SUPPRESSOR OF QUENCHING1 (SOQ1) prevents qH formation by inhibiting LCNP.

SOQ1 suppresses qH with its lumen-located thioredoxin (Trx)-like and NHL domains. Here we report structural data, genetic modification and biochemical characterization of Arabidopsis SOQ1 lumenal domains. Our results show that the Trx-like and NHL domains are associated together, with the cysteine motif located at their interface. Residue E859, required for SOQ1 function, is pivotal for maintaining the Trx-NHL association. Importantly, the C-terminal region of SOQ1 forms an independent beta-stranded domain that has structural homology to the N-terminal domain of bacterial disulfide bond protein D and is essential for negative regulation of qH. Furthermore, SOQ1 is susceptible to cleavage at the loops connecting the neighbouring lumenal domains both in vitro and in vivo, which could be a regulatory process for its suppression function of qH.

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Cryo-EM structures of human m6A writer complexes

Cell Research. 2022. Su, SC et al. Univ Sci & Technol China, MOE Key Lab Cellular Dynam, Hefei, Anhui, Peoples R China; Fudan Univ, Sch Life Sci, Dept Biochem & Biophys,Multiscale Res Inst Comple, State Key Lab Genet Engn,Collaborat Innovat Ctr G, Shanghai, Peoples R China; Univ Sci & Technol China, Div Life Sci & Med, Hefei, Anhui, Peoples R China

ABSTRACT: N-6-methyladenosine (m(6)A) is the most abundant ribonucleotide modification among eukaryotic messenger RNAs. The m(6)A "writer" consists of the catalytic subunit m(6)A-METTL complex (MAC) and the regulatory subunit m(6)A-METTL-associated complex (MACOM), the latter being essential for enzymatic activity. Here, we report the cryo-electron microscopy (cryo-EM) structures of MACOM at a 3.0-angstrom resolution, uncovering that WTAP and VIRMA form the core structure of MACOM and that ZC3H13 stretches the conformation by binding VIRMA.

Furthermore, the 4.4-angstrom resolution cryo-EM map of the MACOM-MAC complex, combined with crosslinking mass spectrometry and GST pull-down analysis, elucidates a plausible model of the m(6)A writer complex, in which MACOM binds to MAC mainly through WTAP and METTL3 interactions. In combination with in vitro RNA substrate binding and m(6)A methyltransferase activity assays, our results illustrate the molecular basis of how MACOM assembles and interacts with MAC to form an active m(6)A writer complex.

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High-resolution mass spectrometry unveils the molecular changes of ovalbumin induced by heating and their influence on IgE binding capacity

Food Chemistry. 2022. Cherkaoui, M et al. INRAE, UR1268 BIA, F-44316 Nantes, France

ABSTRACT: Ovalbumin (OVA) is a food allergen whose allergenicity is modulated by heating. We aimed to establish a molecular connection between heat-induced structural modifications and the modulation of the IgE binding capacity of OVA. For this, we used model samples of heat-modified OVA with increasing complexity; glycated, aggregated, or glycated and aggregated. Using sera from egg-allergic individuals, we show that both aggregation and glycation strongly impacted IgE binding capacity, despite limited structural changes for glycated OVA.

A molecular exploration at the amino acid level using high-resolution mass spectrometry revealed extensive cross -linking, mostly through disulfide and dehydroprotein bridges, and moderate but significant glycation. Structural modifications affected residues located within or at a few amino acids distance of known human linear IgE epitopes, such as C121, K123, S169, K190, K207, H332 and C368. We thus unveil key amino residues implicated in the changes in IgE binding of OVA induced by heating.

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Structural insights into dsRNA processing by Drosophila Dicer-2--Loqs-PD

Nature. 2022. Su, SC et al. Tsinghua Univ, Beijing Adv Innovat Ctr Struct Biol, Tsinghua Peking Joint Ctr Life Sci, Sch Life Sci,Minist Educ,Key Lab Prot Sci, Beijing, Peoples R China; Fudan Univ, Sch Life Sci, Dept Biochem & Biophys, Collaborat Innovat Ctr Genet & Dev, Shanghai, Peoples R China

ABSTRACT: Small interfering RNAs (siRNAs) are the key components for RNA interference (RNAi), a conserved RNA-silencing mechanism in many eukaryotes(1,2). In Drosophila, an RNase III enzyme Dicer-2 (Dcr-2), aided by its cofactor Loquacious-PD (Loqs-PD), has an important role in generating 21 bp siRNA duplexes from long double-stranded RNAs (dsRNAs)(3,4). ATP hydrolysis by the helicase domain of Dcr-2 is critical to the successful processing of a long dsRNA into consecutive siRNA duplexes(5,6). Here we report the cryo-electron microscopy structures of Dcr-2-Loqs-PD in the apo state and in multiple states in which it is processing a 50 bp dsRNA substrate.

The structures elucidated interactions between Dcr-2 and Loqs-PD, and substantial conformational changes of Dcr-2 during a dsRNA-processing cycle. The N-terminal helicase and domain of unknown function 283 (DUF283) domains undergo conformational changes after initial dsRNA binding, forming an ATP-binding pocket and a 5'-phosphate-binding pocket. The overall conformation of Dcr-2-Loqs-PD is relatively rigid during translocating along the dsRNA in the presence of ATP, whereas the interactions between the DUF283 and RIIIDb domains prevent non-specific cleavage during translocation by blocking the access of dsRNA to the RNase active centre. Additional ATP-dependent conformational changes are required to form an active dicing state and precisely cleave the dsRNA into a 21 bp siRNA duplex as confirmed by the structure in the post-dicing state. Collectively, this study revealed the molecular mechanism for the full cycle ofATP-dependent dsRNA processing by Dcr-2-Loqs-PD.

[more...]

Autologous K63 deubiquitylation within the BRCA1-A complex licenses DNA damage recognition

Journal of Cell Biology. 2022. Jiang, QQ et al. Univ Penn, Penn Ctr Genome Integr, Basser Ctr BRCA, Perelman Sch Med,Dept Canc Biol, Philadelphia, PA 19104 USA; Univ Leeds, Fac Biol Sci, Astbury Ctr Struct Mol Biol, Sch Mol & Cellular Biol, Leeds, W Yorkshire, England

ABSTRACT: The BRCA1-A complex contains matching lysine-63 ubiquitin (K63-Ub) binding and deubiquitylating activities. How these functionalities are coordinated to effectively respond to DNA damage remains unknown. We generated Brcc36 deubiquitylating enzyme (DUB) inactive mice to address this gap in knowledge in a physiologic system. DUB inactivation impaired BRCA1-A complex damage localization and repair activities while causing early lethality when combined with Brca2 mutation. Damage response dysfunction in DUB-inactive cells corresponded to increased K63-Ub on RAP80 and BRCC36.

Chemical cross-linking coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and cryogenic-electron microscopy (cryo-EM) analyses of isolated BRCA1-A complexes demonstrated the RAP80 ubiquitin interaction motifs are occupied by ubiquitin exclusively in the DUB-inactive complex, linking auto-inhibition by internal K63-Ub chains to loss of damage site ubiquitin recognition. These findings identify RAP80 and BRCC36 as autologous DUB substrates in the BRCA1-A complex, thus explaining the evolution of matching ubiquitin-binding and hydrolysis activities within a single macromolecular assembly.

[more...]

Structure of the metastatic factor P-Rex1 reveals a two-layered autoinhibitory mechanism

Nature structural & molecular biology. 2022. Chang, YG et al. Monash Univ, Biomed Discovery Inst, Clayton, Vic, Australia

ABSTRACT: P-Rex (PI(3,4,5)P-3-dependent Rac exchanger) guanine nucleotide exchange factors potently activate Rho GTPases. P-Rex guanine nucleotide exchange factors are autoinhibited, synergistically activated by G beta gamma and PI(3,4,5)P-3 binding and dysregulated in cancer. Here, we use X-ray crystallography, cryogenic electron microscopy and crosslinking mass spectrometry to determine the structural basis of human P-Rex1 autoinhibition. P-Rex1 has a bipartite structure of N- and C-terminal modules connected by a C-terminal four-helix bundle that binds the N-terminal Pleckstrin homology (PH) domain.

In the N-terminal module, the Dbl homology (DH) domain catalytic surface is occluded by the compact arrangement of the DH-PH-DEP1 domains. Structural analysis reveals a remarkable conformational transition to release autoinhibition, requiring a 126 degrees opening of the DH domain hinge helix. The off-axis position of G beta gamma and PI(3,4,5)P-3 binding sites further suggests a counter-rotation of the P-Rex1 halves by 90 degrees facilitates PH domain uncoupling from the four-helix bundle, releasing the autoinhibited DH domain to drive Rho GTPase signaling.Cryo-EM, X-ray crystallography and crosslinking mass spectrometry are harnessed to solve the structure of the full-length Rho-GEF P-Rex1, uncovering a two-layered mechanism of autoinhibition released upon G beta gamma and PI(3,4,5)P3 binding.

[more...]

Cryo-EM structures reveal the dynamic transformation of human alpha-2-macroglobulin working as a protease inhibitor

Science China Life Sciences. 2022. Huang, XX et al. Univ Chinese Acad Sci, Beijing 100049, Peoples R China; Chinese Acad Sci, CAS Ctr Excellence Biomacromol, Inst Biophys, Natl Lab Biomacromol, Beijing 100101, Peoples R China

ABSTRACT: Human alpha-2-macroglobulin is a well-known inhibitor of a broad spectrum of proteases and plays important roles in immunity, inflammation, and infections. Here, we report the cryo-EM structures of human alpha-2-macroglobulin in its native state, induced state transformed by its authentic substrate, human trypsin, and serial intermediate states between the native and fully induced states. These structures exhibit distinct conformations, which reveal the dynamic transformation of alpha-2-macro-globulin that acts as a protease inhibitor.

The results shed light on the molecular mechanism of alpha-2-macroglobulin in entrapping substrates.

[more...]

In-Depth In Vivo Crosslinking in Minutes by a Compact, Membrane-Permeable, and Alkynyl-Enrichable Crosslinker

Analytical Chemistry. 2022. Gao, H et al. Chinese Acad Sci, CAS Key Lab Separat Sci Analyt Chem, Natl Chromatog R&A Ctr, Dalian Inst Chem Phys, Dalian 116023, Liaoning, Peoples R China

ABSTRACT: Chemical crosslinking coupled with mass spectrometry (CXMS) has emerged as a powerful technique to obtain the dynamic conformations and interaction interfaces of protein complexes. Limited by the poor cell membrane permeability, chemical reactivity, and biocompatibility of crosslinkers, in vivo crosslinking to capture the dynamics of protein complexes with finer temporal resolution and higher coverage is attractive but challenging. In this work, a trifunctional crosslinker bis(succinimidyl) with propargyl tag (BSP), involving compact size, proper amphipathy, and enrichment capacity, was developed to enable better cell membrane permeability and efficient crosslinking in 5 min without obvious cellular interference.

Followed by a two-step enrichment method based on click chemistry at the peptide level, 13,098 crosslinked peptides (5068 inter-crosslinked peptides and 8030 intra-crosslinked peptides) were identified under the data threshold of peptide-spectrum matches (PSMs) >= 2 on the basic of the FDR control of 1%, which was the most comprehensive dataset for homo species cells by a non-cleavable crosslinker. Besides, the interactome network comprising 1519 proteins connected by 2913 interaction edges in various intracellular compartments, as well as 80S ribosome structural dynamics, were characterized, showing the great potential of our in vivo crosslinking approach in minutes. All these results demonstrated that our developed BSP could provide a valuable toolkit for the in-depth in vivo analysis of protein-protein interactions (PPIs) and protein architectures with finer temporal resolution.

[more...]

Structure of human chromatin-remodelling PBAF complex bound to a nucleosome

Nature. 2022. Yuan, JJ et al. Tsinghua Univ, MOE Key Lab Prot Sci, Beijing, Peoples R China; Tsinghua Univ, Sch Life Sci, Beijing, Peoples R China; Beijing Adv Innovat Ctr Struct Biol, Tsinghua Peking Joint Ctr Life Sci, Beijing, Peoples R China

ABSTRACT: DNA wraps around the histone octamer to form nucleosomes(1), the repeating unit of chromatin, which create barriers for accessing genetic information. Snf2-like chromatin remodellers couple the energy of ATP binding and hydrolysis to reposition and recompose the nucleosome, and have vital roles in various chromatin-based transactions(2,3). Here we report the cryo-electron microscopy structure of the 12-subunit human chromatin-remodelling polybromo-associated BRG1-associated factor (PBAF) complex bound to the nucleosome.

The motor subunit SMARCA4 engages the nucleosome in the active conformation, which reveals clustering of multiple disease-associated mutations at the interfaces that are essential for chromatin-remodelling activity. SMARCA4 recognizes the H2A-H2B acidic pocket of the nucleosome through three arginine anchors of the Snf2 ATP coupling (SnAc) domain. PBAF shows notable functional modularity, and most of the auxiliary subunits are interwoven into three lobe-like submodules for nucleosome recognition. The PBAF-specific auxiliary subunit ARID2 acts as the structural core for assembly of the DNA-binding lobe, whereas PBRM1, PHF10 and BRD7 are collectively incorporated into the lobe for histone tail binding. Together, our findings provide mechanistic insights into nucleosome recognition by PBAF and a structural basis for understanding SMARCA4-related human diseases.

[more...]

Architecture of the human NALCN channelosome

CELL DISCOVERY. 2022. Zhou, LN et al. Westlake Univ, Sch Life Sci, Key Lab Struct Biol Zhejiang Prov, Hangzhou, Zhejiang, Peoples R China; Westlake Lab Life Sci & Biomed, Hangzhou, Zhejiang, Peoples R China; Westlake Inst Adv Study, Inst Biol, Hangzhou, Zhejiang, Peoples R China

ABSTRACT: NALCN regulates the resting membrane potential by mediating the Na+ leak current in neurons, and it functions as a channelosome in complex with FAM155A, UNC79, and UNC80. Dysfunction of the NALCN channelosome causes a broad range of neurological and developmental diseases called NALCN channelopathies in humans. How the auxiliary subunits, especially the two large components UNC79 and UNC80, assemble with NALCN and regulate its function remains unclear. Here we report an overall architecture of the human NALCN channelosome.

UNC79 and UNC80 each adopt an S-shape super-helical structure consisting of HEAT and armadillo repeats, forming a super-coiled heterodimeric assembly in the cytoplasmic side, which may provide a scaffold for the binding of other potential modulators of the channelosome. The UNC79-UNC80 assembly specifically associates with the NALCN-FAM155A subcomplex through the intracellular II-III linker of NALCN. Disruptions of the interaction interfaces between UNC79 and UNC80, and between the II-III linker of NALCN and the UNC79-UNC80 assembly, significantly reduce the NALCN-mediated currents in HEK293T system, suggesting the importance of the UNC79-UNC80 assembly in regulating channelosome function. Cross-linking mass spectrometry analysis identified an additional calmodulin (CaM) bound in the carboxyl-terminal domain of NALCN. Our study thus provides a structural basis for understanding the unique assembly mechanism and functional regulation of the NALCN channelosome, and also provides an opportunity for the interpretation of many disease-related mutations in UNC80.

[more...]

The mouse nicotinamide mononucleotide adenylyltransferase chaperones diverse pathological amyloid client proteins

Journal of Biological Chemistry. 2022. Huang, CA et al. Chinese Acad Sci, Shanghai Inst Organ Chem, Interdisciplinary Res Ctr Biol & Chem, Shanghai, Peoples R China; Shanghai Jiao Tong Univ, Biox Renji Hosp Res Ctr, Renji Hosp, Sch Med, Shanghai, Peoples R China; Shanghai Jiao Tong Univ, Biox Inst, Minist Educ, Key Lab Genet Dev & Neuropsychiat Disorders, Shanghai, Peoples R China; Shanghai Jiao Tong Univ, Zhangjiang Inst Adv Study, Shanghai, Peoples R China

ABSTRACT: Molecular chaperones safeguard cellular protein homeostasis and obviate proteotoxicity. In the process of aging, as chaperone networks decline, aberrant protein amyloid aggregation accumulates in a mechanism that underpins neurodegeneration, leading to pathologies such as Alzheimer's disease and Parkinson's disease. Thus, it is important to identify and characterize chaperones for preventing such protein aggregation. In this work, we identified that the NAD(+) synthase-nicotinamide mononucleotide adenylyltransferase (NMNAT) 3 from mouse (mN3) exhibits potent chaperone activity to antagonize aggregation of a wide spectrum of pathological amyloid client proteins including alpha-synuclein, Tau (K19), amyloid beta, and islet amyloid polypeptide.

By combining NMR spectroscopy, cross-linking mass spectrometry, and computational modeling, we further reveal that mN3 uses different region of its amphiphilic surface near the active site to directly bind different amyloid client proteins. Our work demonstrates a client recognition mechanism of NMNAT via which it chaperones different amyloid client proteins against pathological aggregation and implies a potential protective role for NMNAT in different amyloid-associated diseases.

[more...]

Nitrogen Mustard Alkylates and Cross-Links p53 in Human Keratinocytes

Chemical Research in Toxicology. 2022. Jan, YH et al. Rutgers State Univ, Sch Publ Hlth, Dept Environm & Occupat Hlth & Justice, Piscataway, NJ 08854 USA

ABSTRACT: Cytotoxic blistering agents such as sulfur mustard and nitrogen mustard (HN2) were synthesized for chemical warfare. Toxicity is due to reactive chloroethyl side chains that modify and damage cellular macromolecules including DNA and proteins. In response to DNA damage, cells initiate a DNA damage response directed at the recruitment and activation of repair-related proteins. A central mediator of the DNA damage response is p53, a protein that plays a critical role in regulating DNA repair. We found that HN2 causes cytosolic and nuclear accumulation of p53 in HaCaT keratinocytes; HN2 also induced post-translational modifications on p53 including S15 phosphorylation and K382 acetylation, which enhance p53 stability, promote DNA repair, and mediate cellular metabolic responses to stress.

HN2 also cross-linked p53, forming dimers and high-molecular-weight protein complexes in the cells. Cross-linked multimers were also modified by K48-linked ubiquitination indicating that they are targets for proteasome degradation. HN2-induced modifications transiently suppressed the transcriptional activity of p53. Using recombinant human p53, HN2 alkylation was found to be concentration- and redox status-dependent. Dithiothreitol-reduced protein was more efficiently cross-linked indicating that p53 cysteine residues play a key role in protein modification. LC-MS/MS analysis revealed that HN2 directly alkylated p53 at C124, C135, C141, C176, C182, C275, C277, H115, H178, K132, and K139, forming both monoadducts and cross-links. The formation of intermolecular complexes was a consequence of HN2 cross-linked cysteine residues between two molecules of p53. Together, these data demonstrate that p53 is a molecular target for mustard vesicants. Modification of p53 likely mediates cellular responses to HN2 including DNA repair and cell survival contributing to vesicant-induced cytotoxicity.

[more...]

Optimized TMT-Based Quantitative Cross-Linking Mass Spectrometry Strategy for Large-Scale Interactomic Studies

Analytical Chemistry. 2022. Ruwolt, M et al. Leibniz Forschungsinst Mol Pharmakol FMP, Dept Struct Biol, D-13125 Berlin, Germany

ABSTRACT: Cross-linking mass spectrometry (XL-MS) is a powerful method for theinvestigation of protein-protein interactions (PPI) from highly complex samples. XL-MScombined with tandem mass tag (TMT) labeling holds the promise of large-scale PPIquantification. However, a robust and efficient TMT-based XL-MS quantificationmethod has not yet been established due to the lack of a benchmarking dataset andthorough evaluation of various MS parameters. To tackle these limitations, we generate atwo-interactome dataset by spiking in TMT-labeled cross-linkedEscherichia colilysateinto TMT-labeled cross-linked HEK293T lysate using a defined mixing scheme.

Usingthis benchmarking dataset, we assess the efficacy of cross-link identification and accuracyof cross-link quantification using different MS acquisition strategies. For identification,we compare various MS2- and MS3-based XL-MS methods, and optimize stepped higherenergy collisional dissociation (HCD) energies for TMT-labeled cross-links. Weobserved a need for notably higher fragmentation energies compared to unlabeledcross-links. For quantification, we assess the quantification accuracy and dispersion of MS2-, MS3-, and synchronous precursorselection-MS3-based methods. We show that a stepped HCD-MS2 method with stepped collision energies 36-42-48 provides a vastnumber of quantifiable cross-links with high quantification accuracy. This widely applicable method paves the way for multiplexedquantitative PPI characterization from complex biological systems.

[more...]

Hsp multichaperone complex buffers pathologically modified Tau

Nature communications. 2022. Moll, A et al. Max Planck Inst Multidisciplinary Sci, Dept NMR Based Struct Biol, Fassberg 11, D-37077 Gottingen, Germany; German Ctr Neurodegenerat Dis DZNE, Von Siebold Str 3a, D-37075 Gottingen, Germany

ABSTRACT: Alzheimer's disease is a neurodegenerative disorder in which misfolding and aggregation of pathologically modified Tau is critical for neuronal dysfunction and degeneration. The two central chaperones Hsp70 and Hsp90 coordinate protein homeostasis, but the nature of the interaction of Tau with the Hsp70/Hsp90 machinery has remained enigmatic. Here we show that Tau is a high-affinity substrate of the human Hsp70/Hsp90 machinery. Complex formation involves extensive intermolecular contacts, blocks Tau aggregation and depends on Tau's aggregation-prone repeat region.

The Hsp90 co-chaperone p23 directly binds Tau and stabilizes the multichaperone/substrate complex, whereas the E3 ubiquitin-protein ligase CHIP efficiently disassembles the machinery targeting Tau to proteasomal degradation. Because phosphorylated Tau binds the Hsp70/Hsp90 machinery but is not recognized by Hsp90 alone, the data establish the Hsp70/Hsp90 multichaperone complex as a critical regulator of Tau in neurodegenerative diseases.Alzheimer's disease is characterized by the accumulation of aggregated tau protein. Here the authors find that Hsp chaperones, which normally protect cell homeostasis, can assemble with co-chaperones in a "multichaperone machinery" to target tau aggregation.

[more...]

Identification of an autoinhibitory, mitophagy-inducing peptide derived from the transmembrane domain of USP30

Autophagy. 2022. Qin, X et al. Pingshan Translat Med Ctr, Shenzhen Bay Lab, Shenzhen, Peoples R China; Peking Univ, Sch Chem Biol & Biotechnol, State Key Lab Chem Oncogen, Shenzhen Grad Sch, Shenzhen, Peoples R China; Tsinghua Univ, Tsinghua Shenzhen Int Grad Sch, Inst Biopharmaceut & Hlth Engn, Shenzhen, Peoples R China

ABSTRACT: The mitochondrial-anchored deubiquitinating enzyme USP30 (ubiquitin specific peptidase 30) antagonizes PRKN/parkin-mediated mitophagy, making it a potential target for treating Parkinson disease. However, few inhibitors targeting USP30 have been reported. Here, we report a novel peptide (Q14) derived from the transmembrane (TM) domain of USP30 that can target mitochondrial-anchored USP30 directly and increase mitophagy through two intriguing and distinct mechanisms: a novel autoinhibition mechanism in USP30 and accelerated autophagosome formation via the LC3-interacting region (LIR) of the Q14 peptide.

We identified the potential binding sites between the Q14 peptide and USP30 and postulated that an allosteric autoinhibition mechanism regulates USP30 activity. Furthermore, the LIR motif in the Q14 peptide offers additional binding with LC3 and accelerated autophagosome formation. The two mechanisms synergistically enhance mitophagy. Our work provides novel insight and direction to the design of inhibitors for USP30 or other deubiquitinating enzymes (DUBs).

[more...]

Selective Removal of Unhydrolyzed Monolinked Peptides from Enriched Crosslinked Peptides To Improve the Coverage of Protein Complex Analysis

Analytical Chemistry. 2022. An, YX et al. Chinese Acad Sci, Dalian Inst Chem Phys, CAS Key Lab Separat Sci Analyt Chem, Natl Chromatog R&A Ctr, Dalian 116023, Liaoning, Peoples R China

ABSTRACT: Chemical crosslinking combined with mass spectrometry (CXMS) has allowed the global characterization of protein complexes with high throughput and accuracy. Although enrichable crosslinkers have been introduced to exclude the interference of regular peptides, the crosslinked peptide identification is still severely inhibited by a large amount of monolinked peptides. In this work, we proposed a strategy called MoTE (unhydrolyzed Monolinked peptide Targeting Elimination) to remove the unhydrolyzed monolinked peptides, while enriching crosslinked peptides for regular peptide removal.

In this strategy, followed by the crosslinking reaction, an amine biotin reagent was used to block the unreacted reactive groups on the crosslinker, and subsequently, the crosslinked proteins were tagged by a cleavable biotin-azide ligand based on click chemistry for enrichment. The following crosslinked protein digestion, purification by streptavidin beads, and release by chemical cleavage of the biotin-azide ligand were sequentially performed. In this case, the amine biotin-blocked unhydrolyzed monolinked peptides with the unbreakable arm remained on the streptavidin beads, which realized selective removal without any additional steps. By combining in vivo crosslinking with our proposed MoTE strategy for protein complex analysis of the HeLa cell, the number of high reliability (score

[more...]

Identification of Per a 13 as a novel allergen in American cockroach

Molecular immunology. 2022. Xu, ZQ et al. Nanjing Med Univ, Dept Pharm, Jiangsu Canc Hosp, Affiliated Canc Hosp,Jiangsu Inst Canc Res, 42 Baiziting Rd, Nanjing 210029, Peoples R China; Nanjing Med Univ, Childrens Hosp, Dept Resp Med, 72 Guangzhou Rd, Nanjing 210000, Peoples R China; Chinese Acad Med Sci & Peking Union Med Coll, Peking Union Med Coll Hosp, State Key Lab Complex Severe & Rare Dis, Dept Allergy, 1 Shuaifuyuan, Beijing 100730, Peoples R China

ABSTRACT: Background: Cockroaches are an important source of indoor allergens. Environmental exposure to cockroach allergens is closely associated with the development of immunoglobulin E (IgE)-mediated allergic diseases. However, the allergenic components in the American cockroaches are not fully studied yet. In order to develop novel diagnostic and therapeutic strategies for cockroach allergy, it is necessary to comprehensively investigate this undescribed allergen in the American cockroach.Methods: The full-length cDNA of the potential allergen was isolated from the cDNA library of the American cockroach by PCR cloning.

Both the recombinant and natural protein molecules were purified and characterized. The allergenicity was further analyzed by enzyme linked immunosorbent assay, immunoblot, and basophil activation test using sera from cockroach allergic patients.Results: A novel allergen belonging to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was firstly identified in the American cockroach and named as Per a 13. The cDNA of this allergen is 1255 base pairs in length and contains an open reading frame of 999 base pairs, encoding 332 amino acids. The purified Per a 13 was fully characterized and assessed to react with IgEs from 49.3 % of cockroach allergic patients, and patients with allergic rhinitis were more sensitized to it. Moreover, the allergenicity was further confirmed by immunoblot and basophil activation test.Conclusions: We firstly identified GAPDH (Per a 13) in the American cockroach, which is a novel type of inhalant allergen derived from animal species. These findings could be useful in developing novel diagnostic and therapeutic strategies for cockroach allergy.

[more...]

A new cysteine protease allergen from Ambrosia trifida pollen: proforms and mature forms

Molecular Immunology. 2022. Ling, XJ et al. Gannan Med Univ, Dept Blood Transfus, Affiliated Hosp 1, Ganzhou 341000, Jiangxi, Peoples R China; Nanjing Med Univ, Jiangsu Canc Hosp, Dept Pharm, Nanjing 210009, Peoples R China; Peking Union Med Coll Hosp, Chinese Acad Med Sci & Peking Union Med Coll, State Key Lab Complex Severe & Rare Dis, Allergy Dept, Beijing 100730, Peoples R China; Nanjing Med Univ, Affiliated Canc Hosp, Nanjing 210009, Peoples R China; Nanjing Med Univ, Jiangsu Inst Canc Res, Nanjing 210009, Peoples R China

ABSTRACT: Giant ragweed (Ambrosia trifida) pollen is closely associated with respiratory allergy in late summer and autumn, and the prevalence of giant ragweed pollen allergy progressively increases. Compared with short ragweed (Ambrosia artemisiifolia), allergenic components from giant ragweed pollen are poorly investigated. To promote component resolved diagnosis and treatment for giant ragweed pollen allergy, it becomes necessary to identify and characterize unknown allergens from giant ragweed pollen. In the present study, we identified and characterized a new cysteineprotease (CP) allergen from giant ragweed pollen, named as Amb t CP.

The cloned Amb t CP gene encoded 387 amino acids. Recombinant Amb t CP (rAmb t CP) and natural Amb t CP (nAmb t CP) were purified by high-affinity Ni2+ resin and immunoaffinity chromatography respectively. During refolding, purified rAmb t CP could autocatalytically converted to its mature forms displaying a higher enzymatic activity. Moreover, the autocatalytic conversion of proforms to mature forms of nAmb t CP could cause their amount to change in giant ragweed pollen extracts. Then, the allergenicity of Amb t CP was characterized: 23 (33.8%) of 68 Chinese patients with ragweed pollen allergy showed positive IgE binding to nAmb t CP by enzyme-linked immunosorbent assay (ELISA); the result of subsequent ELISA showed that IgE-binding activity of proforms and mature forms of rAmb t CP was different, with positive rate of 39.1% (9/23) and 47.8% (11/23) respectively; Amb t CP showed IgE cross-reactivity with the CP components from short ragweed, Artemisia annua and Artemisia sieversiana pollen. Our findings will help to promote component-resolved diagnosis and treatment for giant ragweed pollen allergy, standardize allergen products and individualize allergen specific immunotherapy.

[more...]

Multiaspect Examinations of Possible Alternative Mappings of Identified Variant Peptides: A Case Study on the HEK293 Cell Line

ACS omega. 2022. Choong, WK et al. Acad Sinica, Inst Informat Sci, Taipei 11529, Taiwan

ABSTRACT: Adopting proteogenomics approach to validatesingle nucleotide variation events by identifying correspondingsingle amino acid variant peptides from mass spectrometry (MS)-based proteomics data facilitates translational and clinical research.Although variant peptides are usually identified from MS data witha stringent false discovery rate (FDR), FDR control could fail toeliminate dubious results caused by several issues; thus,postexamination to eliminate dubious results is required. However,comprehensive postexaminations of identification results are stilllacking.

Therefore, we propose a framework of three bottom-uplevels, peptide-spectrum match, peptide, and variant event levels,that consists of rigorous 11-aspect examinations from the MSperspective to further confirm the reliability of variant events. As aproof of concept and showing feasibility, we demonstrate 11 examinations on the identified variant peptides from an HEK293 cellline data set, where various database search strategies were applied to maximize the number of identified variant PSMs with an FDR<1% for postexaminations. The results showed that only FDR criterion is insufficient to validate identified variant peptides and the11 postexaminations can reveal low-confidence variant events detected by shotgun proteomics experiments. Therefore, we suggestthat postexaminations of identified variant events based on the proposed framework are necessary for proteogenomics studies

[more...]

Highly robust de novo full-length protein sequencing

Analytical Chemistry. 2022. Mai, NB et al. Jinan Univ, Key Lab Funct Prot Res Guangdong Higher Educ Inst, Guangzhou 510632, Peoples R China; Jinan Univ, MOE Key Lab Tumor Mol Biol, Inst Life & Hlth Engn, Guangzhou 510632, Peoples R China

ABSTRACT: Accurate full-length sequencing of a purified unknown protein is still challenging nowadays due to the error-prone mass-spectrometry (MS)-based methods. De novo identified peptide sequence largely contain errors, undermining the accuracy of assembly. Bias on the detectability of the peptides also makes low-coverage regions, resulting in gaps. Although recent advances on multi-enzyme hydrolysis and algorithms showed complete assembly of full-length protein sequences in a few examples, the robustness in practical application is still to be improved.

Here, inspired by genome assembly strategies, we demonstrate a contig-scaffolding strategy to assemble protein sequences with high robustness and accuracy. This strategy integrates multiple unspecific hydrolysis methods to minimize the bias in the hydrolysis process. After de novo identification of the peptides, our assembly algorithm, named Multiple Contigs & Scaffolding (MuCS), assembles the peptide sequences in a multistep, i.e., contig-scaffold manner, with error correction in each step. MS data from different hydrolysis experiments complement each other for robust contig extension and error correction. We demonstrated that our strategy on three proteins and three replications all reached 100% coverage (except one with 98.85%) and 98.69-100% accuracy. It can also efficiently deal with the membrane protein, although the transmembrane region was missing due to the limitation of the MS. The three replicates reached 88.85-92.57% coverage and 97.57-100% accuracy. In sum, we provided a practical, robust, and accurate solution for full-length protein sequencing. The MuCS software is available at http://chi-biotech. com/mucs/.

[more...]

A new cysteine protease allergen from Ambrosia trifida pollen: proforms and mature forms

Molecular Immunology. 2022. Ling, XJ et al. Gannan Med Univ, Dept Blood Transfus, Affiliated Hosp 1, Ganzhou 341000, Jiangxi, Peoples R China; Nanjing Med Univ, Jiangsu Canc Hosp, Dept Pharm, Nanjing 210009, Peoples R China; Peking Union Med Coll Hosp, Chinese Acad Med Sci & Peking Union Med Coll, State Key Lab Complex Severe & Rare Dis, Allergy Dept, Beijing 100730, Peoples R China; Nanjing Med Univ, Affiliated Canc Hosp, Nanjing 210009, Peoples R China; Nanjing Med Univ, Jiangsu Inst Canc Res, Nanjing 210009, Peoples R China

ABSTRACT: Giant ragweed (Ambrosia trifida) pollen is closely associated with respiratory allergy in late summer and autumn, and the prevalence of giant ragweed pollen allergy progressively increases. Compared with short ragweed (Ambrosia artemisiifolia), allergenic components from giant ragweed pollen are poorly investigated. To promote component resolved diagnosis and treatment for giant ragweed pollen allergy, it becomes necessary to identify and characterize unknown allergens from giant ragweed pollen. In the present study, we identified and characterized a new cysteineprotease (CP) allergen from giant ragweed pollen, named as Amb t CP.

The cloned Amb t CP gene encoded 387 amino acids. Recombinant Amb t CP (rAmb t CP) and natural Amb t CP (nAmb t CP) were purified by high-affinity Ni2+ resin and immunoaffinity chromatography respectively. During refolding, purified rAmb t CP could autocatalytically converted to its mature forms displaying a higher enzymatic activity. Moreover, the autocatalytic conversion of proforms to mature forms of nAmb t CP could cause their amount to change in giant ragweed pollen extracts. Then, the allergenicity of Amb t CP was characterized: 23 (33.8%) of 68 Chinese patients with ragweed pollen allergy showed positive IgE binding to nAmb t CP by enzyme-linked immunosorbent assay (ELISA); the result of subsequent ELISA showed that IgE-binding activity of proforms and mature forms of rAmb t CP was different, with positive rate of 39.1% (9/23) and 47.8% (11/23) respectively; Amb t CP showed IgE cross-reactivity with the CP components from short ragweed, Artemisia annua and Artemisia sieversiana pollen. Our findings will help to promote component-resolved diagnosis and treatment for giant ragweed pollen allergy, standardize allergen products and individualize allergen specific immunotherapy.

[more...]

Multiaspect Examinations of Possible Alternative Mappings of Identified Variant Peptides: A Case Study on the HEK293 Cell Line

ACS omega. 2022. Choong, WK et al. Acad Sinica, Inst Informat Sci, Taipei 11529, Taiwan

ABSTRACT: Adopting proteogenomics approach to validatesingle nucleotide variation events by identifying correspondingsingle amino acid variant peptides from mass spectrometry (MS)-based proteomics data facilitates translational and clinical research.Although variant peptides are usually identified from MS data witha stringent false discovery rate (FDR), FDR control could fail toeliminate dubious results caused by several issues; thus,postexamination to eliminate dubious results is required. However,comprehensive postexaminations of identification results are stilllacking.

Therefore, we propose a framework of three bottom-uplevels, peptide-spectrum match, peptide, and variant event levels,that consists of rigorous 11-aspect examinations from the MSperspective to further confirm the reliability of variant events. As aproof of concept and showing feasibility, we demonstrate 11 examinations on the identified variant peptides from an HEK293 cellline data set, where various database search strategies were applied to maximize the number of identified variant PSMs with an FDR<1% for postexaminations. The results showed that only FDR criterion is insufficient to validate identified variant peptides and the11 postexaminations can reveal low-confidence variant events detected by shotgun proteomics experiments. Therefore, we suggestthat postexaminations of identified variant events based on the proposed framework are necessary for proteogenomics studies

[more...]

Identification of Per a 13 as a novel allergen in American cockroach

Molecular immunology. 2022. Xu, ZQ et al. Nanjing Med Univ, Dept Pharm, Jiangsu Canc Hosp, Affiliated Canc Hosp,Jiangsu Inst Canc Res, 42 Baiziting Rd, Nanjing 210029, Peoples R China; Nanjing Med Univ, Childrens Hosp, Dept Resp Med, 72 Guangzhou Rd, Nanjing 210000, Peoples R China; Chinese Acad Med Sci & Peking Union Med Coll, Peking Union Med Coll Hosp, State Key Lab Complex Severe & Rare Dis, Dept Allergy, 1 Shuaifuyuan, Beijing 100730, Peoples R China

ABSTRACT: Background: Cockroaches are an important source of indoor allergens. Environmental exposure to cockroach allergens is closely associated with the development of immunoglobulin E (IgE)-mediated allergic diseases. However, the allergenic components in the American cockroaches are not fully studied yet. In order to develop novel diagnostic and therapeutic strategies for cockroach allergy, it is necessary to comprehensively investigate this undescribed allergen in the American cockroach.Methods: The full-length cDNA of the potential allergen was isolated from the cDNA library of the American cockroach by PCR cloning.

Both the recombinant and natural protein molecules were purified and characterized. The allergenicity was further analyzed by enzyme linked immunosorbent assay, immunoblot, and basophil activation test using sera from cockroach allergic patients.Results: A novel allergen belonging to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was firstly identified in the American cockroach and named as Per a 13. The cDNA of this allergen is 1255 base pairs in length and contains an open reading frame of 999 base pairs, encoding 332 amino acids. The purified Per a 13 was fully characterized and assessed to react with IgEs from 49.3 % of cockroach allergic patients, and patients with allergic rhinitis were more sensitized to it. Moreover, the allergenicity was further confirmed by immunoblot and basophil activation test.Conclusions: We firstly identified GAPDH (Per a 13) in the American cockroach, which is a novel type of inhalant allergen derived from animal species. These findings could be useful in developing novel diagnostic and therapeutic strategies for cockroach allergy.

[more...]

Highly Robust de Novo Full-Length Protein Sequencing

Analytical Chemistry. 2022. Mai, NB et al. Jinan Univ, Key Lab Funct Prot Res Guangdong Higher Educ Inst, Guangzhou 510632, Peoples R China; Jinan Univ, MOE Key Lab Tumor Mol Biol, Inst Life & Hlth Engn, Guangzhou 510632, Peoples R China

ABSTRACT: Accurate full-length sequencing of a purified unknown protein is still challenging nowadays due to the error-prone mass-spectrometry (MS)-based methods. De novo identified peptide sequence largely contain errors, undermining the accuracy of assembly. Bias on the detectability of the peptides also makes low-coverage regions, resulting in gaps. Although recent advances on multi-enzyme hydrolysis and algorithms showed complete assembly of full-length protein sequences in a few examples, the robustness in practical application is still to be improved.

Here, inspired by genome assembly strategies, we demonstrate a contig-scaffolding strategy to assemble protein sequences with high robustness and accuracy. This strategy integrates multiple unspecific hydrolysis methods to minimize the bias in the hydrolysis process. After de novo identification of the peptides, our assembly algorithm, named Multiple Contigs & Scaffolding (MuCS), assembles the peptide sequences in a multistep, i.e., contig-scaffold manner, with error correction in each step. MS data from different hydrolysis experiments complement each other for robust contig extension and error correction. We demonstrated that our strategy on three proteins and three replications all reached 100% coverage (except one with 98.85%) and 98.69-100% accuracy. It can also efficiently deal with the membrane protein, although the transmembrane region was missing due to the limitation of the MS. The three replicates reached 88.85-92.57% coverage and 97.57-100% accuracy. In sum, we provided a practical, robust, and accurate solution for full-length protein sequencing. The MuCS software is available at http://chi-biotech. com/mucs/.

[more...]

Diversity Matters: Optimal Collision Energies for Tandem Mass Spectrometric Analysis of a Large Set of N-Glycopeptides

Journal of Proteome Research. 2022. Hever, H et al. Res Ctr Nat Sci, Eotvos Lorand Res Network, MS Prote Res Grp, H-1117 Budapest, Hungary

ABSTRACT: Identification and characterization of N-glycopep-tides from complex samples are usually based on tandem mass spectrometric measurements. Experimental settings, especially the collision energy selection method, fundamentally influence the obtained fragmentation pattern and hence the confidence of the database search results ("score"). Using standards of naturally occurring glycoproteins, we mapped the Byonic and pGlyco search engine scores of almost 200 individual N-glycopeptides as a function of collision energy settings on a quadrupole time of flight instrument.

The resulting unprecedented amount of peptide-level information on such a large and diverse set of N-glycopeptides revealed that the peptide sequence heavily influences the energy for the highest score on top of an expected general linear trend with m/z. Search engine dependence may also be noteworthy. Based on the trends, we designed an experimental method and tested it on HeLa, blood plasma, and monoclonal antibody samples. As compared to the literature, these notably lower collision energies in our workflow led to 10-50% more identified N-glycopeptides, with higher scores. We recommend a simple approach based on a small set of reference N-glycopeptides easily accessible from glycoprotein standards to ease the precise determination of optimal methods on other instruments. Data sets can be accessed via the MassIVE repository (MSV000089657 and MSV000090218).

[more...]

Directed evolution of adeno-associated virus 5 capsid enables specific liver tropism

Molecular Therapy-Nucleic Acids. 2022. Wang, YQ et al. East China Univ Sci & Technol, Sch Bioengn, Shanghai 200237, Peoples R China; East China Univ Sci & Technol, Sch Pharm, Shanghai 200237, Peoples R China

ABSTRACT: Impressive achievements in clinical trials to treat hemophilia establish a milestone in the development of gene therapy. highlights the significance of AAV-mediated gene delivery to liver. AAV5 is a unique serotype featured by low neutralizing antibody prevalence. Nevertheless, its liver infectivity is rela-tively weak. Consequently, it is vital to exploit novel AAV5 capsid mutants with robust liver tropism. To this aim, we per formed AAV5-NNK library and barcode screening in mice, from which we identified one capsid variant, called AAVzk2.

AAVzk2 displayed a similar yield but divergent post-transla-tional modification sites compared with wild-type serotypes. Mice intravenously injected with AAVzk2 demonstrated stronger liver transduction than AAV5, roughly comparable with AAV8 and AAV9, with undetectable transduction of other tissues or organs such as heart, lung, spleen, kidney, brain, and skeletal muscle, indicating a liver-specific tropism. Further studies showed a superior human hepatocellular transduction of AAVzk2 to AAV5, AAV8 and AAV9, whereas the seroreac-tivity of AAVzk2 was as low as AAV5. Overall, we provide novel AAV serotype that facilitates a robust and specific liver gene delivery to a large population, especially those unable to be treated by AAV8 and AAV9.

[more...]

TMT-based multiplexed quantitation of N-glycopeptides reveals glycoproteome remodeling induced by oncogenic mutations

ACS omega. 2022. Saraswat, M et al. Mayo Clin, Dept Lab Med & Pathol, Rochester, MN 55905 USA; Inst Bioinformat, Bangalore 560066, Karnataka, India; Manipal Acad Higher Educ MAHE, Manipal 576104, Karnataka, India; Natl Inst Mental Hlth & Neurosci NIMHANS, Ctr Mol Med, Bangalore 560029, Karnataka, India; Mayo Clin, Ctr Individualized Med, Rochester, MN 55905 USA

ABSTRACT: Y Glycoproteomics, or the simultaneous characterization of glycans and their attached peptides, is increasingly being employed to generate catalogs of glycopeptides on a large scale. Nevertheless, quantitative glycoproteomics remains challenging even though isobaric tagging reagents such as tandem mass tags (TMT) are routinely used for quantitative proteomics. Here, we present a workflow that combines the enrichment or fractionation of TMT-labeled glycopeptides with size-exclusion chromatography (SEC) for an in-depth and quantitative analysis of the glycoproteome.

We applied this workflow to study the cellular glycoproteome of an isogenic mammary epithelial cell system that recapitulated oncogenic mutations in the PIK3CA gene, which codes for the phosphatidylinositol-3-kinase catalytic subunit. As compared to the parental cells, cells with mutations in exon 9 (E545K) or exon 20 (H1047R) of the PIK3CA gene exhibited site-specific glycosylation alterations in 464 of the 1999 glycopeptides quantified. Our strategy led to the discovery of site-specific glycosylation changes in PIK3CA mutant cells in several important receptors, including cell adhesion proteins such as integrin beta-6 and CD166. This study demonstrates that the SEC-based enrichment of glycopeptides is a simple and robust method with minimal sample processing that can easily be coupled with TMT-labeling for the global quantitation of glycopeptides.

[more...]

GlycAP, a glycoproteomic analysis platform for site-specific N-glycosylation research

International Journal of Mass Spectrometry. 2022. Wu, Mengxi et al. Fudan Univ, Inst Biomed Sci, Shanghai, Peoples R China; Fudan Univ, Shanghai Peoples Hosp 5, Dept Chem, Shanghai, Peoples R China

ABSTRACT: Protein glycosylation is of great importance for its strong association with various diseases. Mass spectrometry-based site-specific glycoproteome methods with efficient interpretation software tools have become powerful strategies for glycosylation research. However, the lack of bioinformatics tools for automatic analysis of the interpretation data hinders further exploration. Here, we developed a comprehensive N-glycoproteomic analysis platform called GlycAP, which is embedded with different analytical modules, including qualitative analysis, quantitative analysis, functional analysis, and clinical analysis.

The qualitative analysis module was designed for the qualitative statistical analysis and het-erogeneity analysis. The quantitative analysis module supports the discovery of differential glycopeptides between different groups with quantitative results. The functional analysis and clinical analysis modules could help users go a step further and find potential biomarkers from differential glycopeptides. GlycAP, which is freely available online (https://project.omicsolution.com/GlycAP/), is a useful tool for facilitating glycoproteomics in site-specific glycosylation exploration.(c) 2022 Elsevier B.V. All rights reserved.

[more...]

N-glycoproteomics reveals distinct glycosylation alterations in NGLY1-deficient patient-derived dermal fibroblasts

Journal of inherited metabolic disease. 2022. Budhraja, R et al. Mayo Clin, Dept Clin Genom, 200 First St SW, Rochester, MN 55905 USA; Mayo Clin, Dept Lab Med & Pathol, 200 First St SW, Rochester, MN 55905 USA

ABSTRACT: Congenital disorders of glycosylation are genetic disorders that occur due to defects in protein and lipid glycosylation pathways. A deficiency of N-glycanase 1, encoded by the NGLY1 gene, results in a congenital disorder of deglycosylation. The NGLY1 enzyme is mainly involved in cleaving N-glycans from misfolded, retro-translocated glycoproteins in the cytosol from the endoplasmic reticulum before their proteasomal degradation or activation. Despite the essential role of NGLY1 in deglycosylation pathways, the exact consequences of NGLY1 deficiency on global cellular protein glycosylation have not yet been investigated.

We undertook a multiplexed tandem mass tags-labeling-based quantitative glycoproteomics and proteomics analysis of fibroblasts from NGLY1-deficient individuals carrying different biallelic pathogenic variants in NGLY1. This quantitative mass spectrometric analysis detected 8041 proteins and defined a proteomic signature of differential expression across affected individuals and controls. Proteins that showed significant differential expression included phospholipid phosphatase 3, stromal cell-derived factor 1, collagen alpha-1 (IV) chain, hyaluronan and proteoglycan link protein 1, and thrombospondin-1. We further detected a total of 3255 N-glycopeptides derived from 550 glycosylation sites of 407 glycoproteins by multiplexed N-glycoproteomics. Several extracellular matrix glycoproteins and adhesion molecules showed altered abundance of N-glycopeptides. Overall, we observed distinct alterations in specific glycoproteins, but our data revealed no global accumulation of glycopeptides in the patient-derived fibroblasts, despite the genetic defect in NGLY1. Our findings highlight new molecular and system-level insights for understanding NGLY1-CDDG.

[more...]

Multiattribute glycan identification and FDR control for glycoproteomics

Molecular & Cellular Proteomics. 2022. Polasky, Daniel A. et al. Univ Michigan, Dept Computat Med & Bioinformat, Ann Arbor, MI 48109 USA; Univ Michigan, Dept Pathol, Ann Arbor, MI 48109 USA

ABSTRACT: Rapidly improving methods for glycoproteomics have enabled increasingly large-scale analyses of complex glycopeptide samples, but annotating the resulting mass spectrometry data with high confidence remains a major bottleneck. We recently introduced a fast and sensitive glycoproteomics search method in our MSFragger search engine, which reports glycopeptides as a combination of a peptide sequence and the mass of the attached glycan. In samples with complex glycosylation patterns, converting this mass to a specific glycan composition is not straightforward; however, as many glycans have similar or identical masses.

Here, we have developed a new method for determining the glycan composition of N-linked glycopeptides fragmented by collisional or hybrid activation that uses multiple sources of information from the spectrum, including observed glycan B-type (oxonium) and Y-type ions and mass and precursor monoisotopic selection errors to discriminate between possible glycan candidates. Combined with false discovery rate estimation for the glycan assignment, we show that this method is capable of specifically and sensitively identifying glycans in complex glycopeptide analyses and effectively controls the rate of false glycan assignments. The new method has been incorporated into the PTM-Shepherd modification analysis tool to work directly with the MSFragger glyco search in the FragPipe graphical user interface, providing a complete computational pipeline for annotation of N-glycopeptide spectra with false discovery rate control of both peptide and glycan components that is both sensitive and robust against false identifications. As for for

[more...]

Multiattribute Glycan Identification and FDR Control for Glycoproteomics

Molecular & Cellular Proteomics. 2022. Polasky, Daniel A. et al. Univ Michigan, Dept Computat Med & Bioinformat, Ann Arbor, MI 48109 USA; Univ Michigan, Dept Pathol, Ann Arbor, MI 48109 USA

ABSTRACT: Rapidly improving methods for glycoproteomics have enabled increasingly large-scale analyses of complex glycopeptide samples, but annotating the resulting mass spectrometry data with high confidence remains a major bottleneck. We recently introduced a fast and sensitive glycoproteomics search method in our MSFragger search engine, which reports glycopeptides as a combination of a peptide sequence and the mass of the attached glycan. In samples with complex glycosylation patterns, converting this mass to a specific glycan composition is not straightforward; however, as many glycans have similar or identical masses.

Here, we have developed a new method for determining the glycan composition of N-linked glycopeptides fragmented by collisional or hybrid activation that uses multiple sources of information from the spectrum, including observed glycan B-type (oxonium) and Y-type ions and mass and precursor monoisotopic selection errors to discriminate between possible glycan candidates. Combined with false discovery rate estimation for the glycan assignment, we show that this method is capable of specifically and sensitively identifying glycans in complex glycopeptide analyses and effectively controls the rate of false glycan assignments. The new method has been incorporated into the PTM-Shepherd modification analysis tool to work directly with the MSFragger glyco search in the FragPipe graphical user interface, providing a complete computational pipeline for annotation of N-glycopeptide spectra with false discovery rate control of both peptide and glycan components that is both sensitive and robust against false identifications. As for for

[more...]

Exploration of quantitative site-specific serum O-glycoproteomics with isobaric labeling for the discovery of putative O-glycoprotein biomarkers

PROTEOMICS--Clinical Applications. 2022. Zhang, Zihan et al. Shanghai Jiao Tong Univ, Sch Med, Shanghai Peoples Hosp 9, Dept Plast & Reconstruct Surg, Shanghai 200011, Peoples R China; Tongji Univ, Shanghai Key Lab Chem Assessment & Sustainabil, Sch Chem Sci & Engn, Shanghai 200092, Peoples R China

ABSTRACT: Purpose Exploration study of site-specific isobaric-TMT-labeling quantitative serum O-glycoproteomics for the discovery of putative O-glycoprotein cancer biomarkers. Experimental design Sera of 10 breast cancer patients was used as the exploration cohort. More abundant N-glycosylation was first removed with PNGase F. After tryptic digestion of de-N-glycosylated serum proteome, the TMT-labeled O-glycopeptides mixture was prepared and analyzed with RPLC-MS/MS. Site-specific qualitative and quantitative database search of O-glycopeptides was carried out with pGlyco 3.0.

The same raw datasets were also searched with intact N-glycopeptide search engine GPSeeker to exclude possible interference of N-glycosylation. The final IDs were checked manually with GlcNAc-containing glycosite-determining fragment ions for confirmation. Results With the control of spectrum-level FDR <= 1% and manual validation, 299 O-glycopeptides corresponding to 83 O-glycosites and 66 O-glycoproteins were identified, and 13 O-glycopeptides were found differentially expressed. Most interestingly, differential O-glycosylation was observed for IgG1 and IgG3, which is an interesting putative biomarker panel. Conclusion and clinical relevance Isobaric-labeling site-specific quantitative O-glycoproteomics is currently a state-of-the-art instrumental platform for discovery of putative seral cancer biomarkers. Differential seral O-glycosylation was observed in the IgG1 and IgG3.

[more...]

Diversity Matters: Optimal Collision Energies for Tandem Mass Spectrometric Analysis of a Large Set of N-Glycopeptides

Journal of Proteome Research. 2022. Hever, H et al. Res Ctr Nat Sci, Eotvos Lorand Res Network, MS Prote Res Grp, H-1117 Budapest, Hungary

ABSTRACT: Identification and characterization of N-glycopep-tides from complex samples are usually based on tandem mass spectrometric measurements. Experimental settings, especially the collision energy selection method, fundamentally influence the obtained fragmentation pattern and hence the confidence of the database search results ("score"). Using standards of naturally occurring glycoproteins, we mapped the Byonic and pGlyco search engine scores of almost 200 individual N-glycopeptides as a function of collision energy settings on a quadrupole time of flight instrument.

The resulting unprecedented amount of peptide-level information on such a large and diverse set of N-glycopeptides revealed that the peptide sequence heavily influences the energy for the highest score on top of an expected general linear trend with m/z. Search engine dependence may also be noteworthy. Based on the trends, we designed an experimental method and tested it on HeLa, blood plasma, and monoclonal antibody samples. As compared to the literature, these notably lower collision energies in our workflow led to 10-50% more identified N-glycopeptides, with higher scores. We recommend a simple approach based on a small set of reference N-glycopeptides easily accessible from glycoprotein standards to ease the precise determination of optimal methods on other instruments. Data sets can be accessed via the MassIVE repository (MSV000089657 and MSV000090218).

[more...]

N-glycoproteomics reveals distinct glycosylation alterations in NGLY1-deficient patient-derived dermal fibroblasts

Journal of inherited metabolic disease. 2022. Budhraja, R et al. Mayo Clin, Dept Clin Genom, 200 First St SW, Rochester, MN 55905 USA; Mayo Clin, Dept Lab Med & Pathol, 200 First St SW, Rochester, MN 55905 USA

ABSTRACT: Congenital disorders of glycosylation are genetic disorders that occur due to defects in protein and lipid glycosylation pathways. A deficiency of N-glycanase 1, encoded by the NGLY1 gene, results in a congenital disorder of deglycosylation. The NGLY1 enzyme is mainly involved in cleaving N-glycans from misfolded, retro-translocated glycoproteins in the cytosol from the endoplasmic reticulum before their proteasomal degradation or activation. Despite the essential role of NGLY1 in deglycosylation pathways, the exact consequences of NGLY1 deficiency on global cellular protein glycosylation have not yet been investigated.

We undertook a multiplexed tandem mass tags-labeling-based quantitative glycoproteomics and proteomics analysis of fibroblasts from NGLY1-deficient individuals carrying different biallelic pathogenic variants in NGLY1. This quantitative mass spectrometric analysis detected 8041 proteins and defined a proteomic signature of differential expression across affected individuals and controls. Proteins that showed significant differential expression included phospholipid phosphatase 3, stromal cell-derived factor 1, collagen alpha-1 (IV) chain, hyaluronan and proteoglycan link protein 1, and thrombospondin-1. We further detected a total of 3255 N-glycopeptides derived from 550 glycosylation sites of 407 glycoproteins by multiplexed N-glycoproteomics. Several extracellular matrix glycoproteins and adhesion molecules showed altered abundance of N-glycopeptides. Overall, we observed distinct alterations in specific glycoproteins, but our data revealed no global accumulation of glycopeptides in the patient-derived fibroblasts, despite the genetic defect in NGLY1. Our findings highlight new molecular and system-level insights for understanding NGLY1-CDDG.

[more...]

GlycAP, a glycoproteomic analysis platform for site-specific N-glycosylation research

International Journal of Mass Spectrometry. 2022. Wu, Mengxi et al. Fudan Univ, Inst Biomed Sci, Shanghai, Peoples R China; Fudan Univ, Shanghai Peoples Hosp 5, Dept Chem, Shanghai, Peoples R China

ABSTRACT: Protein glycosylation is of great importance for its strong association with various diseases. Mass spectrometry-based site-specific glycoproteome methods with efficient interpretation software tools have become powerful strategies for glycosylation research. However, the lack of bioinformatics tools for automatic analysis of the interpretation data hinders further exploration. Here, we developed a comprehensive N-glycoproteomic analysis platform called GlycAP, which is embedded with different analytical modules, including qualitative analysis, quantitative analysis, functional analysis, and clinical analysis.

The qualitative analysis module was designed for the qualitative statistical analysis and het-erogeneity analysis. The quantitative analysis module supports the discovery of differential glycopeptides between different groups with quantitative results. The functional analysis and clinical analysis modules could help users go a step further and find potential biomarkers from differential glycopeptides. GlycAP, which is freely available online (https://project.omicsolution.com/GlycAP/), is a useful tool for facilitating glycoproteomics in site-specific glycosylation exploration.(c) 2022 Elsevier B.V. All rights reserved.

[more...]

N-glycoproteomics reveals distinct glycosylation alterations in NGLY1 deficient patient-derived dermal fibroblasts

Journal of Inherited Metabolic Disease. 2022. Budhraja, Rohit et al. Mayo Clin, Dept Clin Genom, 200 First St SW, Rochester, MN 55905 USA; Mayo Clin, Dept Lab Med & Pathol, 200 First St SW, Rochester, MN 55905 USA

ABSTRACT: Congenital disorders of glycosylation are genetic disorders that occur due to defects in protein and lipid glycosylation pathways. A deficiency of N-glycanase 1, encoded by the NGLY1 gene, results in a congenital disorder of deglycosylation. The NGLY1 enzyme is mainly involved in cleaving N-glycans from misfolded, retro-translocated glycoproteins in the cytosol from the endoplasmic reticulum before their proteasomal degradation or activation. Despite the essential role of NGLY1 in deglycosylation pathways, the exact consequences of NGLY1 deficiency on global cellular protein glycosylation have not yet been investigated.

We undertook a multiplexed tandem mass tags-labeling-based quantitative glycoproteomics and proteomics analysis of fibroblasts from NGLY1-deficient individuals carrying different biallelic pathogenic variants in NGLY1. This quantitative mass spectrometric analysis detected 8041 proteins and defined a proteomic signature of differential expression across affected individuals and controls. Proteins that showed significant differential expression included phospholipid phosphatase 3, stromal cell-derived factor 1, collagen alpha-1 (IV) chain, hyaluronan and proteoglycan link protein 1, and thrombospondin-1. We further detected a total of 3255 N-glycopeptides derived from 550 glycosylation sites of 407 glycoproteins by multiplexed N-glycoproteomics. Several extracellular matrix glycoproteins and adhesion molecules showed altered abundance of N-glycopeptides. Overall, we observed distinct alterations in specific glycoproteins, but our data revealed no global accumulation of glycopeptides in the patient-derived fibroblasts, despite the genetic defect in NGLY1. Our findings highlight new molecular and system-level insights for understanding NGLY1-CDDG.

[more...]

Exploration of quantitative site-specific serum O-glycoproteomics with isobaric labelling for the discovery of putative O-glycoprotein biomarkers

PROTEOMICS--Clinical Applications. 2022. Zhang, ZH et al. Tongji Univ, Shanghai Key Lab Chem Assessment & Sustainabil, Sch Chem Sci & Engn, Shanghai 200092, Peoples R China; Shanghai Jiao Tong Univ, Sch Med, Shanghai Peoples Hosp 9, Dept Plast & Reconstruct Surg, Shanghai 200011, Peoples R China

ABSTRACT: Purpose Exploration study of site-specific isobaric-TMT-labeling quantitative serum O-glycoproteomics for the discovery of putative O-glycoprotein cancer biomarkers. Experimental design Sera of 10 breast cancer patients was used as the exploration cohort. More abundant N-glycosylation was first removed with PNGase F. After tryptic digestion of de-N-glycosylated serum proteome, the TMT-labeled O-glycopeptides mixture was prepared and analyzed with RPLC-MS/MS. Site-specific qualitative and quantitative database search of O-glycopeptides was carried out with pGlyco 3.0.

The same raw datasets were also searched with intact N-glycopeptide search engine GPSeeker to exclude possible interference of N-glycosylation. The final IDs were checked manually with GlcNAc-containing glycosite-determining fragment ions for confirmation. Results With the control of spectrum-level FDR <= 1% and manual validation, 299 O-glycopeptides corresponding to 83 O-glycosites and 66 O-glycoproteins were identified, and 13 O-glycopeptides were found differentially expressed. Most interestingly, differential O-glycosylation was observed for IgG1 and IgG3, which is an interesting putative biomarker panel. Conclusion and clinical relevance Isobaric-labeling site-specific quantitative O-glycoproteomics is currently a state-of-the-art instrumental platform for discovery of putative seral cancer biomarkers. Differential seral O-glycosylation was observed in the IgG1 and IgG3.

[more...]

TMT-Based Multiplexed Quantitation of N-Glycopeptides Reveals Glycoproteome Remodeling Induced by Oncogenic Mutations

ACS omega. 2022. Saraswat, M et al. Mayo Clin, Dept Lab Med & Pathol, Rochester, MN 55905 USA; Inst Bioinformat, Bangalore 560066, Karnataka, India; Manipal Acad Higher Educ MAHE, Manipal 576104, Karnataka, India; Natl Inst Mental Hlth & Neurosci NIMHANS, Ctr Mol Med, Bangalore 560029, Karnataka, India; Mayo Clin, Ctr Individualized Med, Rochester, MN 55905 USA

ABSTRACT: Y Glycoproteomics, or the simultaneous characterization of glycans and their attached peptides, is increasingly being employed to generate catalogs of glycopeptides on a large scale. Nevertheless, quantitative glycoproteomics remains challenging even though isobaric tagging reagents such as tandem mass tags (TMT) are routinely used for quantitative proteomics. Here, we present a workflow that combines the enrichment or fractionation of TMT-labeled glycopeptides with size-exclusion chromatography (SEC) for an in-depth and quantitative analysis of the glycoproteome.

We applied this workflow to study the cellular glycoproteome of an isogenic mammary epithelial cell system that recapitulated oncogenic mutations in the PIK3CA gene, which codes for the phosphatidylinositol-3-kinase catalytic subunit. As compared to the parental cells, cells with mutations in exon 9 (E545K) or exon 20 (H1047R) of the PIK3CA gene exhibited site-specific glycosylation alterations in 464 of the 1999 glycopeptides quantified. Our strategy led to the discovery of site-specific glycosylation changes in PIK3CA mutant cells in several important receptors, including cell adhesion proteins such as integrin beta-6 and CD166. This study demonstrates that the SEC-based enrichment of glycopeptides is a simple and robust method with minimal sample processing that can easily be coupled with TMT-labeling for the global quantitation of glycopeptides.

[more...]

Directed evolution of adeno-associated virus 5 capsid enables specific liver tropism

Molecular Therapy-Nucleic Acids. 2022. Wang, YQ et al. East China Univ Sci & Technol, Sch Bioengn, Shanghai 200237, Peoples R China; East China Univ Sci & Technol, Sch Pharm, Shanghai 200237, Peoples R China

ABSTRACT: Impressive achievements in clinical trials to treat hemophilia establish a milestone in the development of gene therapy. highlights the significance of AAV-mediated gene delivery to liver. AAV5 is a unique serotype featured by low neutralizing antibody prevalence. Nevertheless, its liver infectivity is rela-tively weak. Consequently, it is vital to exploit novel AAV5 capsid mutants with robust liver tropism. To this aim, we per formed AAV5-NNK library and barcode screening in mice, from which we identified one capsid variant, called AAVzk2.

AAVzk2 displayed a similar yield but divergent post-transla-tional modification sites compared with wild-type serotypes. Mice intravenously injected with AAVzk2 demonstrated stronger liver transduction than AAV5, roughly comparable with AAV8 and AAV9, with undetectable transduction of other tissues or organs such as heart, lung, spleen, kidney, brain, and skeletal muscle, indicating a liver-specific tropism. Further studies showed a superior human hepatocellular transduction of AAVzk2 to AAV5, AAV8 and AAV9, whereas the seroreac-tivity of AAVzk2 was as low as AAV5. Overall, we provide novel AAV serotype that facilitates a robust and specific liver gene delivery to a large population, especially those unable to be treated by AAV8 and AAV9.

[more...]

Structure-Specific N-Glycoproteomics Characterization of NIST Monoclonal Antibody Reference Material 8671

Journal of Proteome Research. 2022. Bi, M et al. Tongji Univ, Sch Chem Sci & Engn, Shanghai 200092, Peoples R China; Nanjing Univ, Ctr Precis Med, Dept Lab Med, Affiliated Hosp,Med Sch,Nanjing Drum Tower Hosp, Nanjing 210008, Jiangsu, Peoples R China

ABSTRACT: The characteristics of monoclonal antibodies (mAbs) cohering various function effectors show great expectation in therapy. Glycosylation, one of the common post-translational modifications, deeply influences cohesion. It is necessary to grasp monosaccharide composition/sequence and glycan structures in mAbs. There has been comprehensive mass spectrometry characterization of N-glycosylation of mAbs, and monosaccharide compositions are deduced according to known biosynthetic rules. Our recently developed intact N-glycopeptide search engine GPSeeker has made structure-specific characterization of N-glycosylation possible with structure-diagnostic fragment ions from selective fragmentation of N-glycan moieties.

Here, we report our structure-specific N-glycoproteomics characterization of NIST monoclonal antibody reference material 8671 using GPSeeker, and 59 N-glycan structures (including 16 pairs of isomers) are characterized.

[more...]

A novel role for GalNAc-T2 dependent glycosylation in energy homeostasis

Molecular metabolism. 2022. Verzijl, CRC et al. Univ Med Ctr Groningen, Dept Pediat, Sect Mol Genet, Antonius Deusinglaan 1, NL-9713 AV Groningen, Netherlands

ABSTRACT: Objective: GALNT2, encoding polypeptide N-acetylgalactosaminyltransferase 2 (GalNAc-T2), was initially discovered as a regulator of highdensity lipoprotein metabolism. GalNAc-T2 is known to exert these effects through post-translational modification, i.e., O-linked glycosylation of secreted proteins with established roles in plasma lipid metabolism. It has recently become clear that loss of GALNT2 in rodents, cattle, nonhuman primates, and humans should be regarded as a novel congenital disorder of glycosylation that affects development and body weight.

The role of GALNT2 in metabolic abnormalities other than plasma lipids, including insulin sensitivity and energy homeostasis, is poorly understood.Methods: GWAS data from the UK Biobank was used to study variation in the GALNT2 locus beyond changes in high-density lipoprotein metabolism. Experimental data were obtained through studies in Galnt2-/- mice and wild-type littermates on both control and high-fat diet.Results: First, we uncovered associations between GALNT2 gene variation, adiposity, and body mass index in humans. In mice, we identify the insulin receptor as a novel substrate of GalNAc-T2 and demonstrate that Galnt2-/- mice exhibit decreased adiposity, alterations in insulin signaling and a shift in energy substrate utilization in the inactive phase.Conclusions: This study identifies a novel role for GALNT2 in energy homeostasis, and our findings suggest that the local effects of GalNAc-T2 are mediated through posttranslational modification of the insulin receptor.(c) 2022 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

[more...]

Reaction-based fluorogenic probes for detecting protein cysteine oxidation in living cells

Nature Communications. 2022. Ferreira, RB et al. UF Scripps Biomed Res, Dept Chem, Jupiter, FL 33458 USA

ABSTRACT: Fluorogenic detection of H2O2 in cells is established, but equivalent tools to monitor its cellular targets remain in their infancy. Here authors develop fluorogenic probes for detecting cysteine sulfenic acid, a redox modification inextricably linked to H2O2 signalling and oxidative stress.'Turn-on' fluorescence probes for detecting H2O2 in cells are established, but equivalent tools to monitor the products of its reaction with protein cysteines have not been reported. Here we describe fluorogenic probes for detecting sulfenic acid, a redox modification inextricably linked to H2O2 signaling and oxidative stress.

The reagents exhibit excellent cell permeability, rapid reactivity, and high selectivity with minimal cytotoxicity. We develop a high-throughput assay for measuring S-sulfenation in cells and use it to screen a curated kinase inhibitor library. We reveal a positive association between S-sulfenation and inhibition of TK, AGC, and CMGC kinase group members including GSK3, a promising target for neurological disorders. Proteomic mapping of GSK3 inhibitor-treated cells shows that S-sulfenation sites localize to the regulatory cysteines of antioxidant enzymes. Our studies highlight the ability of kinase inhibitors to modulate the cysteine sulfenome and should find broad application in the rapidly growing field of redox medicine.

[more...]

Identification of NLRP3 as a covalent target of 1, 6-O, O-diacetylbritannilactone against neuroinflammation by quantitative thiol reactivity profiling (QTRP)

Bioorganic Chemistry. 2022. Wang, MR et al. Northwest A&F Univ, Coll Chem & Pharm, Shaanxi Key Lab Nat Prod & Chem Biol, 3 Taicheng Rd, Yangling 712100, Shaanxi, Peoples R China; Hainan Univ, Sch Pharmaceut Sci, Key Lab Trop Biol Resources, Minist Educ, Haikou 570228, Hainan, Peoples R China

ABSTRACT: Neuroinflammation plays a key etiological role in the progressive neuronal damage of neurodegenerative dis-eases. Our phenotypic-based screening discovered 1,6-O,O-diacetylbritannilactone (OABL, 1) from Inula bri-tannica exhibited the potential anti-neuroinflammatory activity as well as a favorable blood-brain barrier penetration. 1 and its active derivative Br-OABL (2) with insert of Br at the C-14 position both modulated TLR4/ NF-kB/MAPK pathways. However, proteome-wide identification of 1 binding proteins remains unclear.

Here, we employed an adapted isoTOP-ABPP, quantitative thiol reactivity profiling (QTRP) approach, to identify and quantify thiol reactivity binding proteins in murine microglia BV-2 cells. We screened out 15 proteins co-targeted by 1 and 2, which are involved in cellular response to oxidative stress and negative regulation NF-icB tran-scription factor in biological processes. In site-specific profiling, NLRP3 was identified as a covalent target of 1 and 2 for the first time, and the Cys483 of NLRP3 NACHT domain was identified as one active-site of NLRP3 cysteine residues that can be covalently modified by the alpha-methylene-gamma-lactone moiety. Furthermore, NLRP3 was validated to be directly binded by 1 and 2 by cellular thermo shift assay (CETSA) and activity-based protein profiling (ABPP), and NLRP3 functions were also verified by small interfering RNA approach. Notably, OABL treatment (i.p., 20 mg/kg/day) for 21 days reduced inflammation in 5XFAD mice brain. Together, we applied the QTRP to uncover the binding proteins of OABL in BV-2 cells, among which NLRP3 was revealed as a new co-valent target of 1 and 2 against neuroinflammation.

[more...]

Nascent glycoproteome reveals that N-linked Glycosylation Inhibitor-1 Suppresses Expression of Glycosylated Lysosome-Associated Membrane Protein-2

Frontiers in Molecular Biosciences. 2022. Cao, Xinyi et al. Fudan Univ, Dept Chem, Shanghai, Peoples R China; Guangxi Med Univ Canc Hosp, Dept Clin Lab, Nanning, Peoples R China; Fudan Univ, Inst Biomed Sci, Shanghai Canc Ctr, Shanghai, Peoples R China; Fudan Univ, NHC Key Lab Glycoconjugates Res, Shanghai, Peoples R China

ABSTRACT: Glycosylation inhibition has great potential in cancer treatment. However, the corresponding cellular response, protein expression and glycosylation changes remain unclear. As a cell-permeable small-molecule inhibitor with reduced cellular toxicity, N-linked glycosylation inhibitor-1 (NGI-1) has become a great approach to regulate glycosylation in mammalian cells. Here for the first time, we applied a nascent proteomic method to investigate the effect of NGI-1 in hepatocellular carcinoma (HCC) cell line.

Besides, hydrophilic interaction liquid chromatography (HILIC) was adopted for the enrichment of glycosylated peptides. Glycoproteomic analysis revealed the abundance of glycopeptides from LAMP2, NICA, and CEIP2 was significantly changed during NGI-1 treatment. Moreover, the alterations of LAMP2 site-specific intact N-glycopeptides were comprehensively assessed. NGI-1 treatment also led to the inhibition of Cathepsin D maturation and the induction of autophagy. In summary, we provided evidence that NGI-1 repressed the expression of glycosylated LAMP2 accompanied with the occurrence of lysosomal defects and autophagy.

[more...]

Nascent Glycoproteome Reveals That N-Linked Glycosylation Inhibitor-1 Suppresses Expression of Glycosylated Lysosome-Associated Membrane Protein-2

Frontiers in Molecular Biosciences. 2022. Cao, Xinyi et al. Fudan Univ, Dept Chem, Shanghai, Peoples R China; Guangxi Med Univ Canc Hosp, Dept Clin Lab, Nanning, Peoples R China; Fudan Univ, Inst Biomed Sci, Shanghai Canc Ctr, Shanghai, Peoples R China; Fudan Univ, NHC Key Lab Glycoconjugates Res, Shanghai, Peoples R China

ABSTRACT: Glycosylation inhibition has great potential in cancer treatment. However, the corresponding cellular response, protein expression and glycosylation changes remain unclear. As a cell-permeable small-molecule inhibitor with reduced cellular toxicity, N-linked glycosylation inhibitor-1 (NGI-1) has become a great approach to regulate glycosylation in mammalian cells. Here for the first time, we applied a nascent proteomic method to investigate the effect of NGI-1 in hepatocellular carcinoma (HCC) cell line.

Besides, hydrophilic interaction liquid chromatography (HILIC) was adopted for the enrichment of glycosylated peptides. Glycoproteomic analysis revealed the abundance of glycopeptides from LAMP2, NICA, and CEIP2 was significantly changed during NGI-1 treatment. Moreover, the alterations of LAMP2 site-specific intact N-glycopeptides were comprehensively assessed. NGI-1 treatment also led to the inhibition of Cathepsin D maturation and the induction of autophagy. In summary, we provided evidence that NGI-1 repressed the expression of glycosylated LAMP2 accompanied with the occurrence of lysosomal defects and autophagy.

[more...]

Reaction-based fluorogenic probes for detecting protein cysteine oxidation in living cells

Nature Communications. 2022. Ferreira, RB et al. UF Scripps Biomed Res, Dept Chem, Jupiter, FL 33458 USA

ABSTRACT: Fluorogenic detection of H2O2 in cells is established, but equivalent tools to monitor its cellular targets remain in their infancy. Here authors develop fluorogenic probes for detecting cysteine sulfenic acid, a redox modification inextricably linked to H2O2 signalling and oxidative stress.'Turn-on' fluorescence probes for detecting H2O2 in cells are established, but equivalent tools to monitor the products of its reaction with protein cysteines have not been reported. Here we describe fluorogenic probes for detecting sulfenic acid, a redox modification inextricably linked to H2O2 signaling and oxidative stress.

The reagents exhibit excellent cell permeability, rapid reactivity, and high selectivity with minimal cytotoxicity. We develop a high-throughput assay for measuring S-sulfenation in cells and use it to screen a curated kinase inhibitor library. We reveal a positive association between S-sulfenation and inhibition of TK, AGC, and CMGC kinase group members including GSK3, a promising target for neurological disorders. Proteomic mapping of GSK3 inhibitor-treated cells shows that S-sulfenation sites localize to the regulatory cysteines of antioxidant enzymes. Our studies highlight the ability of kinase inhibitors to modulate the cysteine sulfenome and should find broad application in the rapidly growing field of redox medicine.

[more...]

Identification of NLRP3 as a covalent target of 1, 6-O, O-diacetylbritannilactone against neuroinflammation by quantitative thiol reactivity profiling (QTRP)

Bioorganic Chemistry. 2022. Wang, MR et al. Northwest A&F Univ, Coll Chem & Pharm, Shaanxi Key Lab Nat Prod & Chem Biol, 3 Taicheng Rd, Yangling 712100, Shaanxi, Peoples R China; Hainan Univ, Sch Pharmaceut Sci, Key Lab Trop Biol Resources, Minist Educ, Haikou 570228, Hainan, Peoples R China

ABSTRACT: Neuroinflammation plays a key etiological role in the progressive neuronal damage of neurodegenerative dis-eases. Our phenotypic-based screening discovered 1,6-O,O-diacetylbritannilactone (OABL, 1) from Inula bri-tannica exhibited the potential anti-neuroinflammatory activity as well as a favorable blood-brain barrier penetration. 1 and its active derivative Br-OABL (2) with insert of Br at the C-14 position both modulated TLR4/ NF-kB/MAPK pathways. However, proteome-wide identification of 1 binding proteins remains unclear.

Here, we employed an adapted isoTOP-ABPP, quantitative thiol reactivity profiling (QTRP) approach, to identify and quantify thiol reactivity binding proteins in murine microglia BV-2 cells. We screened out 15 proteins co-targeted by 1 and 2, which are involved in cellular response to oxidative stress and negative regulation NF-icB tran-scription factor in biological processes. In site-specific profiling, NLRP3 was identified as a covalent target of 1 and 2 for the first time, and the Cys483 of NLRP3 NACHT domain was identified as one active-site of NLRP3 cysteine residues that can be covalently modified by the alpha-methylene-gamma-lactone moiety. Furthermore, NLRP3 was validated to be directly binded by 1 and 2 by cellular thermo shift assay (CETSA) and activity-based protein profiling (ABPP), and NLRP3 functions were also verified by small interfering RNA approach. Notably, OABL treatment (i.p., 20 mg/kg/day) for 21 days reduced inflammation in 5XFAD mice brain. Together, we applied the QTRP to uncover the binding proteins of OABL in BV-2 cells, among which NLRP3 was revealed as a new co-valent target of 1 and 2 against neuroinflammation.

[more...]

2021