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

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Citations Last Updated: Apri. 1, 2024

2024




Immunopeptidomics-based identification of naturally presented non-canonical circRNA-derived peptides
Nature Communications. 2024. Humberto J. Ferreira et al. Ludwig Institute for Cancer Research, University of Lausanne, Lausanne, Switzerland
ABSTRACT: Circular RNAs (circRNAs) are covalently closed non-coding RNAs lacking the 5' cap and the poly-A tail. Nevertheless, it has been demonstrated that certain circRNAs can undergo active translation. Therefore, aberrantly expressed circRNAs in human cancers could be an unexplored source of tumor-specific antigens, potentially mediating anti-tumor T cell responses. This study presents an immunopeptidomics workflow with a specific focus on generating a circRNA-specific protein fasta reference. The main goal of this workflow is to streamline the process of identifying and validating human leukocyte antigen (HLA) bound peptides potentially originating from circRNAs.
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Spatiotemporal and direct capturing global substrates of lysine-modifying enzymes in living cells
Nature Communications. 2024. Hao Hu et al. State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China
ABSTRACT: Protein-modifying enzymes regulate the dynamics of myriad post-translational modification (PTM) substrates. Precise characterization of enzyme-substrate associations is essential for the molecular basis of cellular function and phenotype. Methods for direct capturing global substrates of protein-modifying enzymes in living cells are with many challenges, and yet largely unexplored. Here, we report a strategy to directly capture substrates of lysine-modifying enzymes via PTM-acceptor residue crosslinking in living cells, enabling global profiling of substrates of PTM-enzymes and validation of PTM-sites in a straightforward manner.
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PgxSAVy: A tool for comprehensive evaluation of variant peptide quality in proteogenomics--catching the (un) usual suspects
Computational and Structural Biotechnology Journal. 2024. Anurag Raj  et al. G. N. Ramachandran Knowledge Centre for Genomics Informatics, CSIR – Institute of Genomics and Integrative Biology, New Delhi, India
ABSTRACT: Variant peptides resulting from single nucleotide polymorphisms (SNPs) can lead to aberrant protein functions and have translational potential for disease diagnosis and personalized therapy. Variant peptides detected by proteogenomics are fraught with high number of false positives, but there is no uniform and comprehensive approach to assess variant quality across analysis pipelines. Despite class -specific FDR along with ad -hoc filters, the problem is far from solved. These protocols are typically manual and tedious, and thus not uniform across labs.
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Apoptosis releases hydrogen sulfide to inhibit Th17 cell differentiation
Cell Metabolism. 2024. Qianmin Ou et al. Key Laboratory of Stem Cells and Tissue Engineering (Sun Yat-Sen University), Ministry of Education, Guangzhou 510080, China
ABSTRACT: Over 50 billion cells undergo apoptosis each day in an adult human to maintain immune homeostasis. Hydrogen sulfide (H2S) is also required to safeguard the function of immune response. However, it is unknown whether apoptosis regulates H2S production. Here, we show that apoptosis-deficient MRL/lpr (B6.MRL-Faslpr/J) and Bim-/- (B6.129S1-Bcl2l11tm1.1Ast/J) mice exhibit significantly reduced H2S levels along with aberrant differentiation of Th17 cells, which can be rescued by the additional H2S. Moreover, apoptotic cells and vesicles (apoVs) express key H2S-generating enzymes and generate a significant amount of H2S, indicating that apoptotic metabolism is an important source of H2S.
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The Deconvolution Method for Obtaining Correspondence in Data-Independent Acquisition Mass Spectrometry Data
. 2024. Lyu, Mingming et al. College of Computer Science and Technology, Shandong University of Technology, Zibo, China
ABSTRACT:
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Structure of the plant plastid-encoded RNA polymerase
Cell. 2024. Á Vergara-Cruces et al. Department of Biochemistry and Metabolism, John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK
ABSTRACT: Chloroplast genes encoding photosynthesis-associated proteins are predominantly transcribed by the plastid-encoded RNA polymerase (PEP). PEP is a multi-subunit complex composed of plastid-encoded subunits similar to bacterial RNA polymerases (RNAPs) stably bound to a set of nuclear-encoded PEP-associated proteins (PAPs). PAPs are essential to PEP activity and chloroplast biogenesis, but their roles are poorly defined. Here, we present cryoelectron microscopy (cryo-EM) structures of native 21-subunit PEP and a PEP transcription elongation complex from white mustard (Sinapis alba).
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Cryo-EM structures of the plant plastid-encoded RNA polymerase
Cell. 2024. XX Wu et al. Key Laboratory of Synthetic Biology, Key Laboratory of Plant Design, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai 200032, China
ABSTRACT: Chloroplasts are green plastids in the cytoplasm of eukaryotic algae and plants responsible for photosynthesis. The plastid-encoded RNA polymerase (PEP) plays an essential role during chloroplast biogenesis from proplastids and functions as the predominant RNA polymerase in mature chloroplasts. The PEP-centered transcription apparatus comprises a bacterial-origin PEP core and more than a dozen eukaryotic-origin PEP-associated proteins (PAPs) encoded in the nucleus. Here, we determined the cryo-EM structures of Nicotiana tabacum (tobacco) PEP-PAP apoenzyme and PEP-PAP transcription elongation complexes at near-atomic resolutions.
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TopBP1 utilises a bipartite GINS binding mode to support genome replication
Nature communications. 2024. M Day et al. Molecular Genetics II, Center of Medical Biotechnology, University of Duisburg-Essen, Universitätsstraße 2-5, 45141, Essen, Germany
ABSTRACT: Activation of the replicative Mcm2-7 helicase by loading GINS and Cdc45 is crucial for replication origin firing, and as such for faithful genetic inheritance. Our biochemical and structural studies demonstrate that the helicase activator GINS interacts with TopBP1 through two separate binding surfaces, the first involving a stretch of highly conserved amino acids in the TopBP1-GINI region, the second a surface on TopBP1-BRCT4. The two surfaces bind to opposite ends of the A domain of the GINS subunit Psf1.
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Structural insights into histone exchange by human SRCAP complex
CELL DISCOVERY. 2024. J Yu et al. Fudan University Shanghai Cancer Center, Institutes of Biomedical Sciences, New Cornerstone Science Laboratory, State Key Laboratory of Genetic Engineering and Shanghai Key Laboratory of Medical Epigenetics, Shanghai Medical College of Fudan University, Shanghai, China
ABSTRACT: Histone variant H2A.Z is found at promoters and regulates transcription. The ATP-dependent chromatin remodeler SRCAP complex (SRCAP-C) promotes the replacement of canonical histone H2A-H2B dimer with H2A.Z-H2B dimer. Here, we determined structures of human SRCAP-C bound to H2A-containing nucleosome at near-atomic resolution. The SRCAP subunit integrates a 6-subunit actin-related protein (ARP) module and an ATPase-containing motor module. The ATPase-associated ARP module encircles half of the nucleosome along the DNA and may restrain net DNA translocation, a unique feature of SRCAP-C.
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An extensive disulfide bond network prevents tail contraction in Agrobacteriumtumefaciens phage Milano
. 2024. RR Sonani et al. Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA, 22903, USA
ABSTRACT:
Use: pLink



Multi-scale structures of the mammalian radial spoke and divergence of axonemal complexes in ependymal cilia
Nature Communications. 2024. Meng, Xueming et al. Key Laboratory of RNA Science and Engineering, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, 200031, China
ABSTRACT: Radial spokes (RS) transmit mechanochemical signals between the central pair (CP) and axonemal dynein arms to coordinate ciliary motility. Atomic-resolution structures of metazoan RS and structures of axonemal complexes in ependymal cilia, whose rhythmic beating drives the circulation of cerebrospinal fluid, however, remain obscure. Here, we present near-atomic resolution cryo-EM structures of mouse RS head-neck complex in both monomer and dimer forms and reveal the intrinsic flexibility of the dimer.
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Structural mechanisms of autoinhibition and substrate recognition by the ubiquitin ligase HACE1
Nature Structural & Molecular Biology. 2024. D{\"u}ring, Jonas et al. Research Group ‘Ubiquitin Signaling Specificity’, Max Planck Institute for Multidisciplinary Sciences, Göttingen, Germany
ABSTRACT: Ubiquitin ligases (E3s) are pivotal specificity determinants in the ubiquitin system by selecting substrates and decorating them with distinct ubiquitin signals. However, structure determination of the underlying, specific E3-substrate complexes has proven challenging owing to their transient nature. In particular, it is incompletely understood how members of the catalytic cysteine-driven class of HECT-type ligases (HECTs) position substrate proteins for modification. Here, we report a cryogenic electron microscopy (cryo-EM) structure of the full-length human HECT HACE1, along with solution-based conformational analyses by small-angle X-ray scattering and hydrogen-deuterium exchange mass spectrometry.
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RNA helicase IGHMBP2 regulates THO complex to ensure cellular mRNA homeostasis
Cell Reports. 2024. Prusty et al. Department of Biochemistry 1, Biocenter, University of Würzburg, 97074 Würzburg, Germany
ABSTRACT: RNA helicases constitute a large protein family implicated in cellular RNA homeostasis and disease development. Here, we show that the RNA helicase IGHMBP2, linked to the neuromuscular disorder spinal muscular atrophy with respiratory distress type 1 (SMARD1), associates with polysomes and impacts translation of mRNAs containing short, GC-rich, and structured 5' UTRs. The absence of IGHMBP2 causes ribosome stalling at the start codon of target mRNAs, leading to reduced translation efficiency. The main mRNA targets of IGHMBP2-mediated regulation encode for components of the THO complex (THOC), linking IGHMBP2 to mRNA production and nuclear export.
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Structural basis for RNA polymerase II ubiquitylation and inactivation in transcription-coupled repair
Nature Structural & Molecular Biology. 2024. Kokic, Goran et al. Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands
ABSTRACT: During transcription-coupled DNA repair (TCR), RNA polymerase II (Pol II) transitions from a transcriptionally active state to an arrested state that allows for removal of DNA lesions. This transition requires site-specific ubiquitylation of Pol II by the CRL4CSA ubiquitin ligase, a process that is facilitated by ELOF1 in an unknown way. Using cryogenic electron microscopy, biochemical assays and cell biology approaches, we found that ELOF1 serves as an adaptor to stably position UVSSA and CRL4CSA on arrested Pol II, leading to ligase neddylation and activation of Pol II ubiquitylation.
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ppmFixer: a mass error adjustment for pGlyco3. 0 to correct near-isobaric mismatches
Glycobiology. 2024. Adams, Trevor M et al. Department of Biochemistry and Molecular Biology, Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens 30602, Georgia
ABSTRACT: Modern glycoproteomics experiments require the use of search engines due to the generation of countless spectra. While these tools are valuable, manual validation of search engine results is often required for detailed analysis of glycopeptides as false-discovery rates are often not reliable for glycopeptide data. Near-isobaric mismatches are a common source of misidentifications for the popular glycopeptide-focused search engine pGlyco3.0, and in this technical note we share a strategy and script that improves the accuracy of the search utilizing two manually validated datasets of the glycoproteins CD16a and HIV-1 Env as proof-of-principle.
Use: pGlyco



Catalyst-free late-stage functionalization to assemble $\alpha$-acyloxyenamide electrophiles for selectively profiling conserved lysine residues
Communications Chemistry. 2024. Zhao, Yuanyuan et al. State Key Laboratory of Bioactive Molecules and Druggability Assessment, Jinan University, 601 Huangpu Avenue West, Guangzhou, 510632, China
ABSTRACT: Covalent probes coupled with chemical proteomics represent a powerful method for investigating small molecule and protein interactions. However, the creation of a reactive warhead within various ligands to form covalent probes has been a major obstacle. Herein, we report a convenient and robust process to assemble a unique electrophile, an alpha-acyloxyenamide, through a one-step late-stage coupling reaction. This procedure demonstrates remarkable tolerance towards other functional groups and facilitates ligand-directed labeling in proteins of interest.
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Use: pQuant




2023




Molecular characterization of extracellular vesicles derived from follicular fluid of women with and without PCOS: integrating analysis of differential miRNAs and proteins reveals vital molecules involving in PCOS
Journal of Assisted Reproduction and Genetics. 2023. Yuqin Yang et al. Department of Reproductive Medicine, Women’s Hospital of Nanjing Medical University, Nanjing Maternity and Child Health Care Hospital, Nanjing, China
ABSTRACT:
Use: pFind



Modulating Liquid--Liquid Phase Separation of Nck Adaptor Protein against Enteropathogenic Escherichia coli Infection
ACS Central Science. 2023. Liu Min et al. Department of Chemistry and Center for Cell & Developmental Biology, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China
ABSTRACT: Signaling proteins often form biomolecular condensates through liquid-liquid phase separation (LLPS) during intracellular signal transduction. Modulating the LLPS property of intracellular protein condensates will redirect intracellular signals and provide a potential way to regulate cellular physiology. Phosphorylation of multiple tyrosine residues of the transmembrane receptor nephrin is known to drive the LLPS of the adaptor protein Nck and neuronal Wiskott-Aldrich Syndrome protein (N-WASP) and form the Nck signaling complex.
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Ubiquitination-mediated Golgi-to-endosome sorting determines the toxin-antidote duality of fission yeast wtf meiotic drivers
Nature Communications. 2023. Jin-Xin Zheng et al. Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University, Beijing, 102206, China
ABSTRACT: Killer meiotic drivers (KMDs) skew allele transmission in their favor by killing meiotic progeny not inheriting the driver allele. Despite their widespread presence in eukaryotes, the molecular mechanisms behind their selfish behavior are poorly understood. In several fission yeast species, single-gene KMDs belonging to the wtf gene family exert selfish killing by expressing a toxin and an antidote through alternative transcription initiation. Here we investigate how the toxin and antidote products of a wtf-family KMD gene can act antagonistically.
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Proteome landscapes of human hepatocellular carcinoma and intrahepatic cholangiocarcinoma
Molecular & cellular proteomics : MCP. 2023. Xiao Yi et al. Center for ProtTalks, Westlake Laboratory of Life Sciences and Biomedicine, Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, Zhejiang, China
ABSTRACT: Liver cancer is among the top leading causes of cancer mortality worldwide. Particularly, hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (CCA) have been extensively investigated from the aspect of tumor biology. However, a comprehensive and systematic understanding of the molecular characteristics of HCC and CCA remains absent. Here, we characterized the proteome landscapes of HCC and CCA using the data-independent acquisition (DIA) mass spectrometry (MS) method. By comparing the quantitative proteomes of HCC and CCA, we found several differences between the two cancer types.
[more...]
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Sulfoproteomics Workflow with Precursor Ion Accurate Mass Shift Analysis Reveals Novel Tyrosine Sulfoproteins in the Golgi
Journal of Proteome Research. 2023. Kweon, Hye Kyong et al. Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109-1085, United States
ABSTRACT: Tyrosine sulfation in the Golgi of secreted and membrane proteins is an important post-translational modification (PTM). However, its labile nature has limited analysis by mass spectrometry (MS), a major reason why no sulfoproteome studies have been previously reported. Here, we show that a phosphoproteomics experimental workflow, which includes serial enrichment followed by high resolution, high mass accuracy MS, and tandem MS (MS/MS) analysis, enables sulfopeptide coenrichment and identification via accurate precursor ion mass shift open MSFragger database search.
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Combining Tags of Various Lengths Benefits Peptide Identification in Bottom-up Proteomics
. 2023. Lai, Shengzhi et al. Department of Electronic and Computer Engineering
ABSTRACT:
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O-GlcNAcylation determines the translational regulation and phase separation of YTHDF proteins
Nature Cell Biology. 2023. Yulin Chen et al. Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China
ABSTRACT: N6-methyladenosine (m6A) is the most abundant internal mRNA nucleotide modification in mammals, regulating critical aspects of cell physiology and differentiation. The YTHDF proteins are the primary readers of m6A modifications and exert physiological functions of m6A in the cytosol. Elucidating the regulatory mechanisms of YTHDF proteins is critical to understanding m6A biology. Here we report a mechanism that protein post-translational modifications control the biological functions of the YTHDF proteins.
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Disruption of the productive encounter complex results in dysregulation of DIAPH1 activity
Journal of Biological Chemistry. 2023. Gregory G. Theophall et al. Max Planck Inst Multidisciplinary Sci, D-37077 Gottingen, Germany
ABSTRACT: The diaphanous-related formin, Diaphanous 1 (DIAPH1), is required for the assembly of Filamentous (F)-actin structures. DIAPH1 is an intracellular effector of the receptor for advanced glycation end products (RAGE) and contributes to RAGE signaling and effects such as increased cell migration upon RAGE stimulation. Mutations in DIAPH1, including those in the basic "RRKR" motif of its autoregulatory domain, diaphanous autoinhibitory domain (DAD), are implicated in hearing loss, macrothrombocytopenia, and cardiovascular diseases.
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Current perspectives on mass spectrometry-based immunopeptidomics: the computational angle to tumor antigen discovery
. 2023. Bing Zhang et al. Ludwig Institute for Cancer Research, University of Lausanne, Lausanne, Switzerland
ABSTRACT:
Use: pFind; pDeep



Histones with an unconventional DNA-binding mode in vitro are major chromatin constituents in the bacterium Bdellovibrio bacteriovorus
Nature Microbiology. 2023. Antoine Hocher et al. Antoine Hocher, Karolin Luger, Tobias Warnecke. Medical Research Council London Institute of Medical Sciences, London, UK Institute of Clinical Sciences, Faculty of Medicine, Imperial College London, London, UK
ABSTRACT: Histone proteins bind DNA and organize the genomes of eukaryotes and most archaea, whereas bacteria rely on different nucleoid-associated proteins. Homology searches have detected putative histone-fold domains in a few bacteria, but whether these function like archaeal/eukaryotic histones is unknown. Here we report that histones are major chromatin components in the bacteria Bdellovibrio bacteriovorus and Leptospira interrogans. Patterns of sequence evolution suggest important roles for histones in additional bacterial clades.
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Ubiquitination-mediated Golgi-to-endosome sorting determines the toxin-antidote duality of wtf meiotic drivers
. 2023. Jin-Xin Zheng et al. Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University,
ABSTRACT:
Use: pFind



Grouping groupers in the Mediterranean: Ecological baselines revealed by ancient proteins
Ecology and Evolution. 2023. Rachel M. Winter et al. Groningen Institute of Archaeology, University of Groningen, Groningen, The Netherlands
ABSTRACT: Marine historical ecology provides a means to establish baselines to inform current fisheries management. Groupers (Epinephelidae) are key species for fisheries in the Mediterranean, which have been heavily overfished. Species abundance and distribution prior to the 20th century in the Mediterranean remains poorly known. To reconstruct the past biogeography of Mediterranean groupers, we investigated whether Zooarchaeology by Mass Spectrometry (ZooMS) can be used for identifying intra-genus grouper bones to species level.
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Autoimmunity to synovial extracellular matrix proteins in patients with postinfectious Lyme arthritis
The Journal of Clinical Investigation. 2023. Korawit Kanjana et al. dmitry.temiakov@jefferson.edu
ABSTRACT: BACKGROUNDAutoimmune diseases often have strong genetic associations with specific HLA-DR alleles. The synovial lesion in chronic inflammatory forms of arthritis shows marked upregulation of HLA-DR molecules, including in postinfectious Lyme arthritis (LA). However, the identity of HLA-DR-presented peptides, and therefore the reasons for these associations, has frequently remained elusive.METHODSUsing immunopeptidomics to detect HLA-DR-presented peptides from synovial tissue, we identified T cell epitopes from 3 extracellular matrix (ECM) proteins in patients with postinfectious LA, identified potential Borreliella burgdorferi-mimic (Bb-mimic) epitopes, and characterized T and B cell responses to these peptides or proteins.RESULTSOf 24 postinfectious LA patients, 58% had CD4+ T cell responses to at least 1 epitope of 3 ECM proteins, fibronectin-1, laminin B2, and/or collagen Valpha1, and 17% of 52 such patients had antibody responses to at least 1 of these proteins.
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Proteogenomic analysis reveals RNA as a source for tumor-agnostic neoantigen identification
Nature Communications. 2023. Celina Tretter et al. TUM
ABSTRACT: RNA variants derived from cancer-associated RNA editing events can be a source of neoantigens. Here, based on a proteogenomic pipeline combining DNA and RNA sequencing with MS-based immunopeptidomics, the authors identity and validate potential neoantigen candidates in patients with different tumor entities, highlighting RNA as important neoantigen source.Systemic pan-tumor analyses may reveal the significance of common features implicated in cancer immunogenicity and patient survival. Here, we provide a comprehensive multi-omics data set for 32 patients across 25 tumor types for proteogenomic-based discovery of neoantigens.
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MetaPep: A core peptide database for faster human gut metaproteomics database searches
Computational and Structural Biotechnology Journal. 2023. Sun, Zhongzhi et al. School of Pharmaceutical Sciences, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada
ABSTRACT: Metaproteomics has increasingly been applied to study functional changes in the human gut microbiome. Peptide identification is an important step in metaproteomics research, with sequence database search (SDS) and spectral library search (SLS) as the two main methods to identify peptides. However, the large search space in metaproteomics studies causes significant challenges for both identification methods. Moreover, with the development of mass spectrometry, it is now feasible to perform metaproteomic projects involving 100-1000 individual microbiomes.
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Conformational cycle of human polyamine transporter ATP13A2
Nature Communications. 2023. Jianqiang Mu et al. So Univ Sci & Technol, Sch Life Sci, Dept Immunol & MicroBiol, Shenzhen 518055, Guangdong, Peoples R China; Int Campus Zhejiang Univ, Prov Int Sci & Technol Cooperat Base Engn Biol, Haining 314400, Peoples R China; Zhejiang Univ, Shanghai Inst Adv Study, Inst Quantitat Biol, Coll Life Sci, Hangzhou 310027, Peoples R China
ABSTRACT: ATP13A2 is a lysosomal polyamine transporter, mutated in several diseases including juvenile-onset Parkinson's disease. Here, the authors report structures of human ATP13A2 in six distinct intermediate states, illustrating most of its conformational cycle.Dysregulation of polyamine homeostasis strongly associates with human diseases. ATP13A2, which is mutated in juvenile-onset Parkinson's disease and autosomal recessive spastic paraplegia 78, is a transporter with a critical role in balancing the polyamine concentration between the lysosome and the cytosol.
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Molecular characterization of extracellular vesicles derived from follicular fluid of women with and without PCOS: integrating analysis of differential miRNAs and proteins reveals vital molecules involving in PCOS
Journal of Assisted Reproduction and Genetics. 2023. Yang, Yuqin et al. Nanjing Med Univ, Womens Hosp, Nanjing Matern & Child Hlth Care Hosp, Dept Reprod Med, Nanjing, Peoples R China
ABSTRACT: PurposeTo elucidate the characterization of extracellular vesicles (EVs) in the follicular fluid-derived extracellular vesicles (FF-EVs) and discover critical molecules and signaling pathways associating with the etiology and pathobiology of PCOS, the differentially expressed miRNAs (DEmiRNAs) and differentially expressed proteins profiles (DEPs) were initially explored and combinedly analyzed.MethodsFirst, the miRNA and protein expression profiles of FF-EVs in PCOS patients and control patients were compared by RNA-sequencing and tandem mass tagging (TMT) proteomic methods.
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Fully integrated on-line strategy for highly sensitive proteome profiling of 10–500 mammalian cells
Analyst. 2023. Yang, Yun et al. Chinese Acad Sci, Inst Neurosci, CAS Ctr Excellence Brain Sci & Intelligence Techno, State Key Lab Neurosci, Shanghai 200031, Peoples R China; Hong Kong Univ Sci &Technol, Dept Chem & Biol Engn, Clear Water Bay, Kowloon, Hong Kong, Peoples R China; Southern Univ Sci & Technol, Sch Sci, Dept Chem, 1088 Xueyuan Ave, Shenzhen 518055, Peoples R China
ABSTRACT: Recent development in proteomic sample preparation using nanofluidic devices has made single-cell proteome profiling possible. However, these nanofluidic devices require special expertise and costly nanopipetting instruments. They are also specially designed for single cells, are not well-suited for profiling rare samples consisting of a few hundred mammalian cells, arguably a more common need that remains a great challenge. Herein, we developed an easy-to-use and scalable device for processing low-input samples, which combined the merits of previously reported rare cell proteomic reactor (RCPR) and mixed-mode simple and integrated spintip-based proteomics technology, as an alternative to nanofluidic devices.
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VCF1 is an unconventional p97/VCP cofactor promoting recognition of ubiquitylated p97-UFD1-NPL4 substrates
. 2023. Mirsanaye, Ann Schirin et al. Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, DK-2200, Copenhagen, Denmark
ABSTRACT:
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DbyDeep: Exploration of MS-Detectable Peptides via Deep Learning
Analytical Chemistry. 2023. Juho Son et al. Hanyang Univ, Dept Comp Sci, Seoul 04763, South Korea; Hanyang Univ, Inst Artificial Intelligence Res, Seoul 04763, South Korea
ABSTRACT: Predicting peptide detectability is useful in a varietyof massspectrometry (MS)-based proteomics applications, particularly targetedproteomics. However, most machine learning-based computational methodshave relied solely on information from the peptide itself, such asits amino acid sequences or physicochemical properties, despite thefact that peptides detected by MS are dependent on many factors, includingprotein sample preparation, digestion, separation, ionization, andprecursor selection during MS experiments.
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Novel Proteoform Discovery by Precise Semi-De Novo Sequencing of Novel Junction Peptides
Analytical Chemistry. 2023. Zhitai Hao et al. Chinese Acad Med Sci & Peking Union Med Coll, Peking Union Med Coll Hosp, State Key Lab Complex Severe & Rare Dis, Dept Med Res Ctr, Beijing 100730, Peoples R China; Peking Univ, Acad Adv Interdisciplinary Studies, Peking Tsinghua Ctr Life Sci, Beijing 100871, Peoples R China; Peking Univ Hlth Sci Ctr, Ctr Precis Med Multi Res, Beijing 100191, Peoples R China
ABSTRACT: Alternative splicing allows a smallnumber of human genesto encodelarge amounts of proteoforms that play essential roles in normal anddisease physiology. Some low-abundance proteoforms may remain undiscovereddue to limited detection and analysis capabilities. Peptides coencodedby novel exons and annotated exons separated by introns are callednovel junction peptides, which are the key to identifying novel proteoforms.Traditional de novo sequencing does not take intoaccount the specificity in the composition of the novel junction peptideand is therefore not as accurate.
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Use: pFind; pNovo



Proteome Landscapes of Human Hepatocellular Carcinoma and Intrahepatic Cholangiocarcinoma
Molecular & cellular proteomics : MCP. 2023. Xiao Yi et al. Center for ProtTalks, Westlake Laboratory of Life Sciences and Biomedicine, Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, Zhejiang, China
ABSTRACT: Liver cancer is among the top leading causes of cancer mortality worldwide. Particularly, hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (CCA) have been extensively investigated from the aspect of tumor biology. However, a comprehensive and systematic understanding of the molecular characteristics of HCC and CCA remains absent. Here, we characterized the proteome landscapes of HCC and CCA using the data-independent acquisition (DIA) mass spectrometry (MS) method. By comparing the quantitative proteomes of HCC and CCA, we found several differences between the two cancer types.
[more...]
Use: pFind



RNA polymerase drives ribonucleotide excision DNA repair in E. coli
Cell. 2023. Nudler, Evgeny et al. New York Univ, Dept Biochem & Mol Pharmacol, Grossman Sch Med, New York, NY 10016 USA; New York Univ, Howard Hughes Med Inst, Grossman Sch Med, New York, NY 10016 USA
ABSTRACT: Ribonuclease HII (RNaseHII) is the principal enzyme that removes misincorporated ribonucleoside mono -phosphates (rNMPs) from genomic DNA. Here, we present structural, biochemical, and genetic evidence demonstrating that ribonucleotide excision repair (RER) is directly coupled to transcription. Affinity pull -downs and mass-spectrometry-assisted mapping of in cellulo inter-protein cross-linking reveal the majority of RNaseHII molecules interacting with RNA polymerase (RNAP) in E. coli. Cryoelectron microscopy struc-tures of RNaseHII bound to RNAP during elongation, with and without the target rNMP substrate, show spe-cific protein-protein interactions that define the transcription-coupled RER (TC-RER) complex in engaged and unengaged states.
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Characterization of the natural peptidome of four leeches by integrated proteogenomics and pseudotargeted peptidomics
Analytical and Bioanalytical Chemistry. 2023. Jingmei Liao1 et al. Chinese Acad Sci, Shanghai Inst Mat Med, Shanghai Res Ctr Modernizat Tradit Chinese Med, Natl Engn Res Ctr TCM Standardizat Technol, Shanghai 201203, Peoples R China; Nanjing Univ Chinese Med, Sch Chinese Mat Med, Nanjing 210023, Jiangsu, Peoples R China; Univ Chinese Acad Sci, 19A Yuquan Rd, Beijing 100049, Peoples R China
ABSTRACT: Animal-derived drugs are an indispensable part of folk medicine worldwide. However, their chemical constituents are poorly approached, which leads to the low level of the quality standard system of animal-derived drugs and further causes a chaotic market. Natural peptides are ubiquitous throughout the organism, especially in animal-derived drugs. Thus, in this study, we used multi-source leeches, including Hirudo nipponica (HN), Whitmania pigra (WP), Whitmania acranulata (WA), and Poecilobdella manillensis (PM), as a model.
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Large-Scale Profiling of Unexpected Tryptic Cleaved Sites at Ubiquitinated Lysines
Journal of Proteome Research. 2023. Sun, Zhen et al. Anhui Med Univ, Hefei 230032, Peoples R China; Chinese Acad Med Sci, Natl Ctr Prot Sci Beijing, Res Unit Prote Res & Dev New Drug, State Key Lab Prote,Beijing Proteome Res Ctr,Inst, Beijing 102206, Peoples R China; Wuhan Univ, Sch Basic Med Sci, Sch Pharmaceut Sci, Key Lab Combinatorial Biosynth & Drug Discovery,Mi, Wuhan 430071, Peoples R China; Hebei Univ, Coll Life Sci, Hebei Prov Key Lab Res & Applicat Microbial Divers, Baoding 071002, Hebei, Peoples R China
ABSTRACT: Trypsin specifically cleaves the C-terminus of lysine and arginine residues but often fails to cleave modified lysines, such as ubiquitination, therefore resulting in the uncleaved K-e-GG peptides. Therefore, the cleaved ubiquitinated peptide identification was often regarded as false positives and discarded. Interestingly, unexpected cleavage at the K48-linked ubiquitin chain has been reported, suggesting the latent ability of trypsin to cleave ubiquitinated lysine residues. However, it remains unclear whether other trypsin-cleavable ubiquitinated sites are present.
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Linking chromatin acylation mark-defined proteome and genome in living cells
Cell. 2023. Qin, Fangfei et al. Shenzhen Bay Lab, Shenzhen 518055, Peoples R China; Peking Univ, Acad Adv Interdisciplinary Studies, Peking Tsinghua Ctr Life Sci, Beijing 100871, Peoples R China; Peking Univ, Sch Life Sci, Key Lab Cell Proliferat & Differentiat, Minist Educ, Beijing 100871, Peoples R China; Peking Univ, Coll Chem & Mol Engn, Key Lab Bioorgan Chem & Mol Engn, Minist Educ, Beijing 100871, Peoples R China; Peking Univ, Coll Chem & Mol Engn, Synthet & Funct Biomol Ctr, Beijing Natl Lab Mol Sci, Beijing 100871, Peoples R China
ABSTRACT: A generalizable strategy with programmable site specificity for in situ profiling of histone modifications on unperturbed chromatin remains highly desirable but challenging. We herein developed a single-site-resolved multi-omics (SiTomics) strategy for systematic mapping of dynamic modifications and subsequent profiling of chromatinized proteome and genome defined by specific chromatin acylations in living cells. By leveraging the genetic code expansion strategy, our SiTomics toolkit revealed distinct crotonylation (e.g., H3K56cr) and b-hydroxybutyrylation (e.g., H3K56bhb) upon short chain fatty acids stimulation and established linkages for chromatin acylation mark-defined proteome, genome, and functions.
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Mapping the signaling network of BIN2 kinase using TurboID-mediated biotin labeling and phosphoproteomics
The Plant Cell. 2023. Kim, Tae-Wuk et al. Carnegie Inst Sci, Dept Plant Biol, Stanford, CA 94305 USA
ABSTRACT: Combining TurboID-mediated proximity labeling with quantitative phosphoproteomics identifies BIN2 signaling components including kinase substrates in vivo, revealing cellular functions of BIN2.Elucidating enzyme-substrate relationships in posttranslational modification (PTM) networks is crucial for understanding signal transduction pathways but is technically difficult because enzyme-substrate interactions tend to be transient. Here, we demonstrate that TurboID-based proximity labeling (TbPL) effectively and specifically captures the substrates of kinases and phosphatases.
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Identification and characterization of natural PR-1 protein as major allergen from Humulus japonicus pollen
Molecular Immunology. 2023. Wang, Ye et al. Chinese Acad Med Sci, Peking Union Med Coll Hosp, Peking Union Med Coll, Dept Allergy, 1 Shuaifuyuan, Wangfujing 100730, Beijing, Peoples R China; Affiliated Canc Hosp Nanjing Med Univ, Jiangsu Canc Hosp, Jiangsu Inst Canc Res, Dept Pharm, 42,Baiziting Rd, Nanjing 210029, Jiangsu, Peoples R China; Childrens Hosp Nanjing Med Univ, Dept Resp Med, 72 Guangzhou Rd, Nanjing 210008, Jiangsu, Peoples R China
ABSTRACT: Background: The Humulus japonicus pollen is one of the most common allergenic pollens in China. However, little is unveiled regarding the allergenic components in Humulus japonicus pollen. Our study aimed to purify and identify the pathogenesis-related 1 (PR-1) protein from Humulus japonicus pollen, and to characterize the mo-lecular and immunochemical properties of this novel allergen.Methods: The natural PR-1 protein (named as Hum j PR-1) was purified from Humulus japonicus pollen extracts with a combined strategy of chromatography, and identified by mass spectrometry.
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One Size Fits All—Venomics of the Iberian Adder (Vipera seoanei, Lataste 1878) Reveals Low Levels of Venom Variation across Its Distributional Range
Toxins. 2023. Avella et al. CIBIO, BIOPOLIS Program Genom Biodivers & Land Planning, Campus Vairao, P-4485661 Vairao, Portugal; Univ Porto, Ctr Invest Biodiversidade & Recursos Genet, InBIO Lab Associado, CIBIO, Campus Vairao, P-4485661 Vairao, Portugal; Univ Porto, Fac Ciencias, Dept Biol, P-4099002 Porto, Portugal
ABSTRACT: European vipers (genus Vipera) are medically important snakes displaying considerable venom variation, occurring at different levels in this group. The presence of intraspecific venom variation, however, remains understudied in several Vipera species. Vipera seoanei is a venomous snake endemic to the northern Iberian Peninsula and south-western France, presenting notable phenotypic variation and inhabiting several diverse habitats across its range. We analysed the venoms of 49 adult specimens of V.
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A Cosine-Similarity-Based Deconvolution Method for Analyzing Data-Independent Acquisition Mass Spectrometry Data
APPLIED SCIENCES-BASEL. 2023. Xiang Zhang et al. Shandong Univ Technol, Sch Comp Sci & Technol, Zibo 255049, Peoples R China
ABSTRACT: Although data-independent acquisition (DIA) has the ability to identify and quantify all peptides in a sample, highly complex mixed mass spectra present difficulties for accurate peptide and protein identification. Additionally, the correspondence between the precursor and its fragments is broken, making it challenging to perform peptide identification directly using conventional DDA search engines. In this paper, we propose a cosine-similarity-based deconvolution method: CorrDIA. This is achieved by reconstructing the correspondence between precursor and fragment ions based on the consistency of extracted ion chromatograms (XICs).
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IgE recognition and structural analysis of disulfide bond rearrangement and chemical modifications in allergen aggregations in roasted peanuts
Journal of Agricultural and Food Chemistry. 2023. Ying Zhang et al. Nanchang Univ, Sino German Joint Res Inst, Nanchang 330047, Peoples R China; Nanchang Univ, State Key Lab Food Sci & Technol, Nanchang 330047, Peoples R China
ABSTRACT: Giventhat roasting changes the structure and allergenicity ofpeanut allergens, the structural information of peanut allergens mustbe expounded to explain the alteration in their allergenicity. Thiswork focused on allergen aggregations (AAs) in roasted peanuts. IgErecognition capability was assessed via western blot analysis. Thedisulfide bond (DB) rearrangement and chemical modification in AAswere identified by combining mass spectroscopy and software tools,and structural changes induced by cross-links were displayed by moleculardynamics and PyMOL software.
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Structural surfaceomics reveals an AML-specific conformation of integrin $\beta$2 as a CAR T cellular therapy target
Nature Cancer. 2023. Kamal Manda et al. Arun P. Wiita
ABSTRACT: Safely expanding indications for cellular therapies has been challenging given a lack of highly cancer-specific surface markers. Here we explore the hypothesis that tumor cells express cancer-specific surface protein conformations that are invisible to standard target discovery pipelines evaluating gene or protein expression, and these conformations can be identified and immunotherapeutically targeted. We term this strategy integrating cross-linking mass spectrometry with glycoprotein surface capture 'structural surfaceomics'.
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DIAPH1-MFN2 interaction regulates mitochondria-SR/ER contact and modulates ischemic/hypoxic stress
Nature Communications. 2023. Gautham Yepuri et al. Diabetes Research Program, Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, NYU Grossman School of Medicine, New York, New York, 10016, USA
ABSTRACT: Inter-organelle contact and communication between mitochondria and sarco/endoplasmic reticulum (SR/ER) maintain cellular homeostasis and are profoundly disturbed during tissue ischemia. We tested the hypothesis that the formin Diaphanous-1 (DIAPH1), which regulates actin dynamics, signal transduction and metabolic functions, contributes to these processes. We demonstrate that DIAPH1 interacts directly with Mitofusin-2 (MFN2) to shorten mitochondria-SR/ER distance, thereby enhancing mitochondria-ER contact in cells including cardiomyocytes, endothelial cells and macrophages.
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A noncanonical function of SKP1 regulates the switch between autophagy and unconventional secretion
Science Advances. 2023. Li Jie et al. Department of Biochemistry and Molecular Pharmacology, New York University Grossman School of Medicine, New York, NY 10016, USA.
ABSTRACT: Intracellular degradation of proteins and organelles by the autophagy-lysosome system is essential for cellular quality control and energy homeostasis. Besides degradation, endolysosomal organelles can fuse with the plasma membrane and contribute to unconventional secretion. Here, we identify a function for mammalian SKP1 in endolysosomes that is independent of its established role as an essential component of the family of SCF/CRL1 ubiquitin ligases. We found that, under nutrient-poor conditions, SKP1 is phosphorylated on Thr(131), allowing its interaction with V-1 subunits of the vacuolar ATPase (V-ATPase).
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The Intrinsically Disordered N Terminus in Atg12 from Yeast Is Necessary for the Functional Structure of the Protein
International Journal of Molecular Sciences. 2023. Hana Popelka et al. Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA Department of Pathology, Michigan Medicine, University of Michigan, Ann Arbor, MI 48109, USA
ABSTRACT: The Atg12 protein in yeast is an indispensable polypeptide in the highly conserved ubiquitin-like conjugation system operating in the macroautophagy/autophagy pathway. Atg12 is covalently conjugated to Atg5 through the action of Atg7 and Atg10; the Atg12-Atg5 conjugate binds Atg16 to form an E3 ligase that functions in a separate conjugation pathway involving Atg8. Atg12 is comprised of a ubiquitin-like (UBL) domain preceded at the N terminus by an intrinsically disordered protein region (IDPR), a domain that comprises a major portion of the protein but remains elusive in its conformation and function.
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The intracellular helical bundle of human glucose transporter GLUT4 is important for complex formation with ASPL
FEBS Open bio. 2023. Huang Peng et al. Department of Experimental Medical Science, Lund University, Sweden
ABSTRACT: Glucose transporters (GLUTs) are responsible for transporting hexose molecules across cellular membranes. In adipocytes, insulin stimulates glucose uptake by redistributing GLUT4 to the plasma membrane. In unstimulated adipose-like mouse cell lines, GLUT4 is known to be retained intracellularly by binding to TUG protein, while upon insulin stimulation, GLUT4 dissociates from TUG. Here, we report that the TUG homolog in human, ASPL, exerts similar properties, i.e., forms a complex with GLUT4. We describe the structural details of complex formation by combining biochemical assays with cross-linking mass spectrometry and computational modeling.
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Architecture of the baculovirus nucleocapsid revealed by cryo-EM
Nature Communications. 2023. Jia, Xudong et al. State key laboratory of biocontrol, School of Life Sciences, Sun Yat-sen University, 510275, Guangzhou, China
ABSTRACT: Baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) has been widely used as a bioinsecticide and a protein expression vector. Despite their importance, very little is known about the structure of most baculovirus proteins. Here, we show a 3.2A resolution structure of helical cylindrical body of the AcMNPV nucleocapsid, composed of VP39, as well as 4.3A resolution structures of both the head and the base of the nucleocapsid composed of over 100 protein subunits. AcMNPV VP39 demonstrates some features of the HK97-like fold and utilizes disulfide-bonds and a set of interactions at its C-termini to mediate nucleocapsid assembly and stability.
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Recognition and maturation of IL-18 by caspase-4 noncanonical inflammasome
. 2023. Xuyan Shi et al. National Institute of Biological Sciences, Beijing, Beijing, P. R. China National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, P. R. China Research Unit of Pyroptosis and Immunity, Chinese Academy of Medical Sciences and National Institute of Biological Sciences, Beijing, Beijing, P. R. China Changping Laboratory, Beijing, P. R. China Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University, Beijing, P. R. China New Cornerstone Science Laboratory, Shenzhen, P. R. China
ABSTRACT:
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Structure of the transcribing RNA polymerase II--Elongin complex
Nature Structural & Molecular Biology. 2023. Chen, Ying et al. Max Planck Inst Multidisciplinary Sci, Dept Mol Biol, Gottingen,
ABSTRACT: Elongin is a heterotrimeric elongation factor for RNA polymerase (Pol) II transcription that is conserved among metazoa. Here, we report three cryo-EM structures of human Elongin bound to transcribing Pol II. The structures show that Elongin subunit ELOA binds the RPB2 side of Pol II and anchors the ELOB-ELOC subunit heterodimer. ELOA contains a 'latch' that binds between the end of the Pol II bridge helix and funnel helices, thereby inducing a conformational change near the polymerase active center.
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The LIKE SEX FOUR 1--malate dehydrogenase complex functions as a scaffold to recruit $\beta$-amylase to promote starch degradation
The Plant Cell. 2023. Jian Liu et al. Huazhong Agr Univ, Natl Ctr Plant Gene Res, Wuhan 430070, Peoples R China; Huazhong Agr Univ, Natl Key Lab Crop Genet Improvement, Wuhan 430070, Peoples R China
ABSTRACT: In plant leaves, starch is composed of glucan polymers that accumulate in chloroplasts as the products of photosynthesis during the day; starch is mobilized at night to continuously provide sugars to sustain plant growth and development. Efficient starch degradation requires the involvement of several enzymes, including beta-amylase and glucan phosphatase. However, how these enzymes cooperate remains largely unclear. Here, we show that the glucan phosphatase LIKE SEX FOUR 1 (LSF1) interacts with plastid NAD-dependent malate dehydrogenase (MDH) to recruit beta-amylase (BAM1), thus reconstituting the BAM1-LSF1-MDH complex.
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Structure and activation of the RING E3 ubiquitin ligase TRIM72 on the membrane
Nature Structural & Molecular Biology. 2023. Park, Si Hoon et al. Department of Life Sciences, Korea University, Seoul, South Korea
ABSTRACT: Defects in plasma membrane repair can lead to muscle and heart diseases in humans. Tripartite motif-containing protein (TRIM)72 (mitsugumin 53; MG53) has been determined to rapidly nucleate vesicles at the site of membrane damage, but the underlying molecular mechanisms remain poorly understood. Here we present the structure of Mus musculus TRIM72, a complete model of a TRIM E3 ubiquitin ligase. We demonstrated that the interaction between TRIM72 and phosphatidylserine-enriched membranes is necessary for its oligomeric assembly and ubiquitination activity.
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Peroxiredoxin-1 is an H2O2 safe-guard antioxidant and signalling enzyme in M1 macrophages
. 2023. Ezeri{\c{n}}a, Daria et al. Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels B-1050, Belgium
ABSTRACT:
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ECL 3.0: a sensitive peptide identification tool for cross-linking mass spectrometry data analysis
BMC bioinformatics. 2023. Zhou, Chen et al. Myeloid Cell Immunology Lab, VIB Center for Inflammation Research, Pleinlaan 2, Brussels B-1050, Belgium
ABSTRACT: BACKGROUND: Cross-linking mass spectrometry (XL-MS) is a powerful technique for detecting protein-protein interactions (PPIs) and modeling protein structures in a high-throughput manner. In XL-MS experiments, proteins are cross-linked by a chemical reagent (namely cross-linker), fragmented, and then fed into a tandem mass spectrum (MS/MS). Cross-linkers are either cleavable or non-cleavable, and each type requires distinct data analysis tools. However, both types of cross-linkers suffer from imbalanced fragmentation efficiency, resulting in a large number of unidentifiable spectra that hinder the discovery of PPIs and protein conformations.
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4D-diaXLMS: Proteome-wide Four-Dimensional Data-Independent Acquisition Workflow for Cross-Linking Mass Spectrometry
Analytical Chemistry. 2023. Yanhong Hao et al. Suming Chen
ABSTRACT: Cross-linking mass spectrometry (XL-MS) is a powerful tool for examining protein structures and interactions. Nevertheless, analysis of low-abundance cross-linked peptides is often limited in the data-dependent acquisition (DDA) mode due to its semistochastic nature. To address this issue, we introduced a workflow called 4D-diaXLMS, representing the first-ever application of four-dimensional data-independent acquisition for proteome-wide cross-linking analysis. Cross-linking studies of the HeLa cell proteome were evaluated using the classical cross-linker disuccinimidyl suberate as an example.
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CaMKII autophosphorylation can occur between holoenzymes without subunit exchange
Elife. 2023. Lu{\v{c}}i{\'c}, Iva et al. Institute of Biology, Cellular Biophysics, Humboldt Universität zu Berlin
ABSTRACT: The dodecameric protein kinase CaMKII is expressed throughout the body. The alpha isoform is responsible for synaptic plasticity and participates in memory through its phosphorylation of synaptic proteins. Its elaborate subunit organization and propensity for autophosphorylation allow it to preserve neuronal plasticity across space and time. The prevailing hypothesis for the spread of CaMKII activity, involving shuffling of subunits between activated and naive holoenzymes, is broadly termed subunit exchange.
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Structural basis of nucleosome deacetylation and DNA linker tightening by Rpd3S histone deacetylase complex
Cell Research. 2023. Dong, Shuqi et al. CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, GIBH-HKU Guangdong–Hong Kong Stem Cell and Regenerative Medicine Research Centre, GIBH-CUHK Joint Research Laboratory on Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong, China
ABSTRACT: In Saccharomyces cerevisiae, cryptic transcription at the coding region is prevented by the activity of Sin3 histone deacetylase (HDAC) complex Rpd3S, which is carried by the transcribing RNA polymerase II (RNAPII) to deacetylate and stabilize chromatin. Despite its fundamental importance, the mechanisms by which Rpd3S deacetylates nucleosomes and regulates chromatin dynamics remain elusive. Here, we determined several cryo-EM structures of Rpd3S in complex with nucleosome core particles (NCPs), including the H3/H4 deacetylation states, the alternative deacetylation state, the linker tightening state, and a state in which Rpd3S co-exists with the Hho1 linker histone on NCP.
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Proteomic Characterization of Native and Rearranged Disulfide Bonds in Proteins from Thermally Treated and Commercial Milk Samples
Journal of Agricultural and Food Chemistry. 2023. Valentina Ciaravolo et al. CNR, Prote Metabol & Mass Spectrometry Lab, ISPAAM, I-80055 Portici, Italy
ABSTRACT: To investigate thiol-disulfide interchange reactionsin heatedmilk yielding non-native intramolecular rearranged and intermolecularcross-linked proteins, a proteomic study based on nanoLC-ESI-Q-Orbitrap-MS/MSand dedicated bioinformatics was accomplished. Raw milk samples heatedfor different times and various commercial dairy products were analyzed.Qualitative experiments on tryptic digests of resolved protein mixturesassigned the corresponding disulfide-linked peptides. Results confirmedthe limited data available on few milk proteins, generated the widestinventory of components (63 in number) involved in thiol-disulfideexchange processes, and provided novel structural information on S-S-bridgedmolecules.
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Crosslinker Nanocarriers Delivery to Chloroplasts for In Vivo Mapping of Photosynthetic Membrane Protein Complexes in Living Chlamydomonas reinhardtii Cells
Analytical Chemistry. 2023. Xinwei Li et al. Chinese Acad Sci, Dalian Inst Chem Phys, Collaborat Innovat Ctr Chem Energy Mat iChEM, State Key Lab Catalysis, Dalian 116023, Peoples R China; Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog Res & Anal Ctr, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Peoples R China
ABSTRACT: Photosynthesis, as the core of solar energy biotransformation,is driven by photosynthetic membrane protein complexes in plants andalgae. Current methods for intracellular photosynthetic membrane proteincomplex analysis mostly require the separation of specific chloroplastsor the change of the intracellular environment, which causes the missingof real-time and on-site information. Thus, we explored a method for in vivo crosslinking and mapping of photosynthetic membraneprotein complexes in the chloroplasts of living Chlamydomonasreinhardtii (C.
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Heterologous Expression of Plantaricin 423 and Mundticin ST4SA in Saccharomyces cerevisiae
Probiotics and Antimicrobial Proteins. 2023. Michelle Rossouw et al. Stellenbosch Univ, Dept Microbiol, Private Bag X1, ZA-7602 Matieland, South Africa
ABSTRACT: Antimicrobial peptides or bacteriocins are excellent candidates for alternative antimicrobials, but high manufacturing costs limit their applications. Recombinant gene expression offers the potential to produce these peptides more cost-effectively at a larger scale. Saccharomyces cerevisiae is a popular host for recombinant protein production, but with limited success reported on antimicrobial peptides. Individual recombinant S. cerevisiae strains were constructed to secrete two class IIa bacteriocins, plantaricin 423 (PlaX) and mundticin ST4SA (MunX).
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In vivo cross-linking-based affinity purification and mass spectrometry for targeting intracellular protein-protein interactions
Analytica Chimica Acta. 2023. Zhong, Bowen 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: Comprehensive interactome analysis of targeted proteins is important to understand how proteins work together in regulating functions. Commonly, affinity purification followed by mass spectrometry (AP-MS) has been recognized as the most often used technique for studying protein-protein interactions (PPIs). However, some proteins with weak interactions, which are responsible for key roles in regulation, are easily broken during cell lysis and purification through an AP approach. Herein, we have developed an approach termed in vivo cross-linking-based affinity purification and mass spectrometry (ICAP-MS).
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Total Chemical Synthesis of Glycosylated TREM2 Ectodomain
ACS Chemical Neuroscience. 2023. Gayani Wijegunawardena et al. Univ Calif San Francisco, Weill Inst Neurosci, Dept Neurol, San Francisco, CA 94158 USA; Univ Calif San Francisco, Inst Neurodegenerat Dis, San Francisco, CA 94158 USA; Wichita State Univ, Dept Chem & Biochem, Wichita, KS 67260 USA
ABSTRACT: Mutations in a microglia-associated gene TREM2increase the risk of Alzheimer's disease. Currently, structuraland functional studies of TREM2 mainly rely on recombinant TREM2 proteinsexpressed from mammalian cells. However, using this method, it isdifficult to achieve site-specific labeling. Here, we present thetotal chemical synthesis of the 116 amino acid TREM2 ectodomain. Rigorousstructural analysis ensured correct structural fold after refolding.Treating microglial cells with refolded synthetic TREM2 enhanced microglialphagocytosis, proliferation, and survival.
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Near-atomic architecture of Singapore grouper iridovirus and implications for giant virus assembly
Nature Communications. 2023. Zhennan Zhao et al. Chinese Acad Sci, Inst Biophys, Natl Lab Biomacromol, Beijing 100101, Peoples R China; Southern Univ Sci & Technol, Cryo EM Ctr, Dept Biol, Shenzhen 518055, Peoples R China; Chinese Acad Med Sci & Peking Union Med Coll, Peking Union Med Coll Hosp, Clin Res Inst, State Key Lab Complex Severe & Rare Dis, Beijing 100730, Peoples R China; South China Agr Univ, Coll Marine Sci, Guangdong Lab Lingnan Modern Agr, Guangzhou 510642, Peoples R China; Univ Chinese Acad Sci, Beijing 100049, Peoples R China; Chinese Acad Sci, Inst Microbiol, CAS Key Lab Pathogen Microbiol & Immunol, Beijing 100101, Peoples R China
ABSTRACT: High morbidity and mortality in aquatic have been caused by iridovirids worldwide. Here the authors present a near-atomic SGIV capsid structure. Functional assays further reveal the relationships between identified capsid proteins and viral assembly.Singapore grouper iridovirus (SGIV), one of the nucleocytoviricota viruses (NCVs), is a highly pathogenic iridovirid. SGIV infection results in massive economic losses to the aquaculture industry and significantly threatens global biodiversity. In recent years, high morbidity and mortality in aquatic animals have been caused by iridovirid infections worldwide.
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Spt6 directly interacts with Cdc73 and is required for Paf1 complex occupancy at active genes in Saccharomyces cerevisiae
Nucleic Acids Research. 2023. Ellison, Mitchell A. et al. Univ Pittsburgh, Dept Biol Sci, Pittsburgh, PA 15260 USA
ABSTRACT: The Paf1 complex (Paf1C) is a conserved transcription elongation factor that regulates transcription elongation efficiency, facilitates co-transcriptional histone modifications, and impacts molecular processes linked to RNA synthesis, such as polyA site selection. Coupling of the activities of Paf1C to transcription elongation requires its association with RNA polymerase II (Pol II). Mutational studies in yeast identified Paf1C subunits Cdc73 and Rtf1 as important mediators of Paf1C association with Pol II on active genes.
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ERR$\gamma$-DBD undergoes dimerization and conformational rearrangement upon binding to the downstream site of the DR1 element
Biochemical and Biophysical Research Communications. 2023. Xuhui Zhen  et al. Chinese Acad Sci, Guangzhou Inst Hlth, Guangdong Prov Key Lab Biocomp, Guangzhou 510530, Peoples R China; Chinese Acad Sci, Guangzhou Inst Biomed, Guangdong Prov Key Lab Biocomp, Guangzhou 510530, Peoples R China
ABSTRACT: The estrogen-related receptor (ERR) family members are reported to bind DNA elements as either monomer or dimer. However, to date, only one solution NMR structure of ERRI3 in complex with a half -site DNA element has been reported. To better understand the DNA regulation mechanism, we deter-mined the crystal structure of ERRy-DBD bound to a natural DR1 element in Pla2g12b promoter to 2.2 angstrom resolution. Combined with biochemical assays, we show that ERRy acts as a dimer and the C-terminal extension region undergoes conformational rearrangement when binding to the downstream DR1 element.
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Regulation of 3′ splice site selection after step 1 of splicing by spliceosomal C* proteins
Science Advances. 2023. Dybkov, Olexandr et al. Max Planck Inst Multidisciplinary Sci, Cellular Biochem, Fassberg 11, D-37077 Gottingen, Germany; Free Univ Berlin, Inst Chem & Biochem, RNA Biochem, Takustr 6, D-14195 Berlin, Germany
ABSTRACT: Alternative precursor messenger RNA splicing is instrumental in expanding the proteome of higher eukaryotes, and changes in 3 ' splice site (3'ss) usage contribute to human disease. We demonstrate by small interfering RNA-mediated knockdowns, followed by RNA sequencing, that many proteins first recruited to human C* spli-ceosomes, which catalyze step 2 of splicing, regulate alternative splicing, including the selection of alternatively spliced NAGNAG 3 ' ss. Cryo-electron microscopy and protein cross-linking reveal the molecular architecture of these proteins in C* spliceosomes, providing mechanistic and structural insights into how they influence 3'ss usage.
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Structural basis of bacterial effector protein azurin targeting tumor suppressor p53 and inhibiting its ubiquitination
Communications Biology. 2023. Hu, Jianjian et al. Hubei Univ, Coll Life Sci, State Key Lab Biocatalysis & Enzyme Engn, Wuhan 430074, Peoples R China
ABSTRACT: Structural and mutagenic analyses reveal the mechanistic basis of azurin-mediated p53 stabilization and tumor suppression, and several affinity-enhancing azurin mutants are designed.Tumor suppressor p53 prevents tumorigenesis by promoting cell cycle arrest and apoptosis through transcriptional regulation. Dysfunction of p53 occurs frequently in human cancers. Thus, p53 becomes one of the most promising targets for anticancer treatment. A bacterial effector protein azurin triggers tumor suppression by stabilizing p53 and elevating its basal level.
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IgE Recognition and Structural Analysis of Disulfide Bond Rearrangement and Chemical Modifications in Allergen Aggregations in Roasted Peanuts
Journal of Agricultural and Food Chemistry. 2023. Ying Zhang et al. Nanchang Univ, Sino German Joint Res Inst, Nanchang 330047, Peoples R China; Nanchang Univ, State Key Lab Food Sci & Technol, Nanchang 330047, Peoples R China
ABSTRACT: Giventhat roasting changes the structure and allergenicity ofpeanut allergens, the structural information of peanut allergens mustbe expounded to explain the alteration in their allergenicity. Thiswork focused on allergen aggregations (AAs) in roasted peanuts. IgErecognition capability was assessed via western blot analysis. Thedisulfide bond (DB) rearrangement and chemical modification in AAswere identified by combining mass spectroscopy and software tools,and structural changes induced by cross-links were displayed by moleculardynamics and PyMOL software.
[more...]
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Structural basis of amine odorant perception by a mammal olfactory receptor
Nature. 2023. Lulu Guo et al. Shanghai Jiao Tong Univ, Ctr Brain Sci, Shanghai Childrens Med Ctr, Sch Med, Shanghai, Peoples R China; Shandong Univ, Adv Med Res Inst, Cheeloo Coll Med, Meili Lake Translat Res Pk, Jinan, Peoples R China; Shandong Univ, Dept Gen Surg, Qilu Hosp, Jinan, Peoples R China; Shanghai Jiao Tong Univ, Songjiang Inst, Sch Med, Shanghai, Peoples R China; Shandong Univ, Dept Biochem & Mol Biol, Sch Med, Jinan, Peoples R China; Shanghai Jiao Tong Univ, Songjiang Hosp, Sch Med, Shanghai, Peoples R China; Shanghai Res Ctr Brain Sci & Brain Inspired Intel, Shanghai, Peoples R China; Shanghai Jiao Tong Univ, Shanghai Key Lab Childrens Environm Hlth, Dept Anat & Physiol, Minist Educ,Sch Med,Xinhua Hosp, Shanghai, Peoples R China; Peking Univ, Sch Basic Med Sci, Dept Physiol & Pathophysiol, Key Lab Mol Cardiovasc Sci,Minist Educ, Beijing, Peoples R China
ABSTRACT: Odorants are detected as smell in the nasal epithelium of mammals by two G-protein-coupled receptor families, the odorant receptors and the trace amine-associated receptors1,2 (TAARs). TAARs emerged following the divergence of jawed and jawless fish, and comprise a large monophyletic family of receptors that recognize volatile amine odorants to elicit both intraspecific and interspecific innate behaviours such as attraction and aversion3-5. Here we report cryo-electron microscopy structures of mouse TAAR9 (mTAAR9) and mTAAR9-Gs or mTAAR9-Golf trimers in complex with beta-phenylethylamine, N,N-dimethylcyclohexylamine or spermidine.
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Pathway and mechanism of tubulin folding mediated by TRiC/CCT along its ATPase cycle revealed using cryo-EM
Communications Biology. 2023. Caixuan Liu et al. Univ Chinese Acad Sci, Beijing 100049, Peoples R China; Chinese Acad Sci, Ctr Excellence Mol Cell Sci, Shanghai Inst Biochem & Cell Biol, State Key Lab Mol Biol,Natl Ctr Prot Sci Shanghai, Shanghai 200031, Peoples R China
ABSTRACT: The eukaryotic chaperonin TRiC/CCT assists the folding of about 10% of cytosolic proteins through an ATP-driven conformational cycle, and the essential cytoskeleton protein tubulin is the obligate substrate of TRiC. Here, we present an ensemble of cryo-EM structures of endogenous human TRiC throughout its ATPase cycle, with three of them revealing endogenously engaged tubulin in different folding stages. The open-state TRiC-tubulin-S1 and -S2 maps show extra density corresponding to tubulin in the cis-ring chamber of TRiC.
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Large-Scale Qualitative and Quantitative Assessment of Dityrosine Crosslinking Omics in Response to Endogenous and Exogenous Hydrogen Peroxide in Escherichia coli
Antioxidants. 2023. Xiangzhe Zhou et al. Beijing Inst Technol, Sch Life Sci, Beijing 100081, Peoples R China; Beijing Inst Technol, Inst Engn Med, Sch Med Technol, Beijing 100081, Peoples R China
ABSTRACT: Excessive hydrogen peroxide causes oxidative stress in cells. The oxidation of two tyrosine residues in proteins can generate o,o '-dityrosine, a putative biomarker for protein oxidation, which plays critical roles in a variety of organisms. Thus far, few studies have investigated dityrosine crosslinking under endogenous or exogenous oxidative conditions at the proteome level, and its physiological function remains largely unknown. In this study, to investigate qualitative and quantitative dityrosine crosslinking, two mutant Escherichia coli strains and one mutant strain supplemented with H2O2 were used as models for endogenous and exogenous oxidative stress, respectively.
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Diverse modes of H3K36me3-guided nucleosomal deacetylation by Rpd3S
Nature. 2023. Guan, Haipeng et al. State Key Laboratory of Molecular Oncology, MOE Key Laboratory of Protein Sciences, SXMU-Tsinghua Collaborative Innovation Center for Frontier Medicine, School of Medicine, Tsinghua University, Beijing, China
ABSTRACT: Context-dependent dynamic histone modifications constitute a key epigenetic mechanism in gene regulation1-4. The Rpd3 small (Rpd3S) complex recognizes histone H3 trimethylation on lysine 36 (H3K36me3) and deacetylates histones H3 and H4 at multiple sites across transcribed regions5-7. Here we solved the cryo-electron microscopy structures of Saccharomyces cerevisiae Rpd3S in its free and H3K36me3 nucleosome-bound states. We demonstrated a unique architecture of Rpd3S, in which two copies of Eaf3-Rco1 heterodimers are asymmetrically assembled with Rpd3 and Sin3 to form a catalytic core complex.
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Hydrogen sulfide functions as a micro-modulator bound at the copper active site of Cu/Zn-SOD to regulate the catalytic activity of the enzyme
Cell Reports. 2023. Dongdong Wu et al. Univ Shanghai Sci & Technol, Sch Hlth Sci & Engn, Shanghai 200093, Peoples R China; Chinese Acad Sci, Shenzhen Inst Synthet Biol, Shenzhen Inst Adv Technol, CAS Key Lab Quantitat Engn Biol, Shenzhen 518055, Peoples R China; Fudan Univ, Sch Basic Med Sci, Dept Physiol & Pathophysiol, Shanghai Key Lab Bioact Small Mol,Shanghai Med Col, Shanghai 200032, Peoples R China; Chinese Acad Sci, Shanghai Inst Organ Chem, State Key Lab Chem Biol, Shanghai 200032, Peoples R China
ABSTRACT: The present study examines whether there is a mechanism beyond the current concept of post-translational modifications to regulate the function of a protein. A small gas molecule, hydrogen sulfide (H2S), was found to bind at active-site copper of Cu/Zn-SOD using a series of methods including radiolabeled binding assay, X-ray absorption near-edge structure (XANES), and crystallography. Such an H2S binding enhanced the elec-trostatic forces to guide the negatively charged substrate superoxide radicals to the catalytic copper ion, changed the geometry and energy of the frontier molecular orbitals of the active site, and subsequently facil-itated the transfer of an electron from the superoxide radical to the catalytic copper ion and the breakage of the copper-His61 bridge.
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Structural basis of mRNA binding by the human FERRY Rab5 effector complex
Molecular Cell. 2023. Jan Schuhmacher et al. Max Planck Inst Mol Physiol, Dept Struct Biochem, D-44227 Dortmund, Germany; Max Planck Inst Mol Cell Biol & Genet, D-01307 Dresden, Germany
ABSTRACT: The pentameric FERRY Rab5 effector complex is a molecular link between mRNA and early endosomes in mRNA intracellular distribution. Here, we determine the cryo-EM structure of human FERRY. It reveals a unique clamp-like architecture that bears no resemblance to any known structure of Rab effectors. A com-bination of functional and mutational studies reveals that while the Fy-2 C-terminal coiled-coil acts as binding region for Fy-1/3 and Rab5, both coiled-coils and Fy-5 concur to bind mRNA. Mutations causing truncations of Fy-2 in patients with neurological disorders impair Rab5 binding or FERRY complex assembly.
[more...]
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Ynamide Coupling Reagent for the Chemical Cross-Linking of Proteins in Live Cells
ACS Chemical Biology. 2023. Shengrong Li et al. Jinan Univ, Sch Pharm, Int Cooperat Lab Tradit Chinese Med Modernizat & I, MOE Key Lab Tumor Mol Biol, Guangzhou 510632, Peoples R China; Jinan Univ, Sch Pharm, Int Cooperat Lab Tradit Chinese Med Modernizat & I, Guangzhou 510632, Peoples R China
ABSTRACT: Chemicalcross-linking of proteins coupled with massspectrometryanalysis (CXMS) is a powerful method for the study of protein structureand protein-protein interactions (PPIs). However, the chemicalprobes used in the CXMS are limited to bidentate reactive warheads,and the available zero-length cross-linkers are restricted to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS)and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM). To alleviate this issue, an efficient coupling reagent, sulfonylynamide, was developed as a new zero-length cross-linker that canconnect high-abundance carboxyl residues (D/E) with lysine (K) toform amide bonds in the absence of any catalyst.
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Structure of a SIN3--HDAC complex from budding yeast
Nature Structural & Molecular Biology. 2023. Zhouyan Guo et al. Westlake Univ, Sch Life Sci, Key Lab Struct Biol Zhejiang Prov, Hangzhou, Peoples R China; Westlake Lab Life Sci & Biomed, Hangzhou, Peoples R China; Westlake Inst Adv Study, Inst Biol, Hangzhou, Peoples R China
ABSTRACT: SIN3-HDAC (histone deacetylases) complexes have important roles in facilitating local histone deacetylation to regulate chromatin accessibility and gene expression. Here, we present the cryo-EM structure of the budding yeast SIN3-HDAC complex Rpd3L at an average resolution of 2.6 angstrom. The structure reveals that two distinct arms (ARM1 and ARM2) hang on a T-shaped scaffold formed by two coiled-coil domains. In each arm, Sin3 interacts with different subunits to create a different environment for the histone deacetylase Rpd3.
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Mass spectrometry uncovers intermediates and off-pathway complexes for SNARE complex assembly
Communications Biology. 2023. Hesselbarth, Julia et al. Martin Luther Univ Halle Wittenberg, Inst Biochem & Biotechnol, Interdisciplinary Res Ctr HALOmem, Charles Tanford Prot Ctr, Halle, Germany; Johannes Gutenberg Univ Mainz, Dept Chem Biochem, Bioctr 2, Mainz, Germany
ABSTRACT: The SNARE complex assembles from vesicular Synaptobrevin-2 as well as Syntaxin-1 and SNAP25 both anchored to the presynaptic membrane. It mediates fusion of synaptic vesicles with the presynaptic plasma membrane resulting in exocytosis of neurotransmitters. While the general sequence of SNARE complex formation is well-established, our knowledge on possible intermediates and stable off-pathway complexes is incomplete. We, therefore, follow the stepwise assembly of the SNARE complex and target individual SNAREs, binary sub-complexes, the ternary SNARE complex as well as interactions with Complexin-1.
[more...]
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Structural insights into DNA N6-adenine methylation by the MTA1 complex
Cell Discovery. 2023. Yan, Junjun et al. Huazhong Agr Univ, Natl Key Lab Crop Genet Improvement, Wuhan, Hubei, Peoples R China; Huazhong Agr Univ, Natl Ctr Plant Gene Res, Hubei Hongshan Lab, Wuhan, Hubei, Peoples R China; Hubei Univ, Hubei Collaborat Innovat Ctr Green Transformat Bio, Sch Life Sci, State Key Lab Biocatalysis & Enzyme Engn,Hubei Key, Wuhan, Hubei, Peoples R China
ABSTRACT: N-6-methyldeoxyadenine (6mA) has recently been reported as a prevalent DNA modification in eukaryotes. The Tetrahymena thermophila MTA1 complex consisting of four subunits, namely MTA1, MTA9, p1, and p2, is the first identified eukaryotic 6mA methyltransferase (MTase) complex. Unlike the prokaryotic 6mA MTases which have been biochemically and structurally characterized, the operation mode of the MTA1 complex remains largely elusive. Here, we report the cryogenic electron microscopy structures of the quaternary MTA1 complex in S-adenosyl methionine (SAM)-bound (2.6 angstrom) and S-adenosyl homocysteine (SAH)-bound (2.8 angstrom) states.
[more...]
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Fine structure and assembly pattern of a minimal myophage Pam3
Proceedings of the National Academy of Sciences of the United States of America. 2023. Feng Yang et al. School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei 230026, China
ABSTRACT: The myophage possesses a contractile tail that penetrates its host cell envelope. Except for investigations on the bacteriophage T4 with a rather complicated structure, the assembly pattern and tail contraction mechanism of myophage remain largely unknown. Here, we present the fine structure of a freshwater Myoviridae cyanophage Pam3, which has an icosahedral capsid of ~680 A in diameter, connected via a three-section neck to an 840-A-long contractile tail, ending with a three-module baseplate composed of only six protein components.
[more...]
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Improved Analysis of Cross-Linking Mass Spectrometry Data with Kojak 2.0, Advanced by Integration into the Trans-Proteomic Pipeline
Journal of Proteome Research. 2023. Hoopmann, Michael R. et al. Inst Syst Biol, Seattle, WA 98109 USA
ABSTRACT: Fragmentation ion spectral analysis of chemically cross-linked proteins is an established technology in the proteomics research repertoire for determining protein interactions, spatial orientation, and structure. Here we present Kojak version 2.0, a major update to the original Kojak algorithm, which was developed to identify cross-linked peptides from fragment ion spectra using a database search approach. A substantially improved algorithm with updated scoring metrics, support for cleavable cross-linkers, and identification of cross-links between 15N-labeled homomultimers are among the newest features of Kojak 2.0 presented here.
[more...]
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ERR$\gamma$-DBD undergoes dimerization and conformational rearrangement upon binding to a DR1 element in Pla2g12b promoter
. 2023. Zhen, Xuhui et al. Guangdong Provincial Key Laboratory of Biocomputing, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
ABSTRACT:
Use: pLink



Optimized approach for active peptides identification in Cerebrolysin by nanoLC-MS
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES. 2023. Bingkun Yang et al. Hebei Med Univ, Inst Chinese Integrat Med, Sch Chinese Integrat Med, Dept Pharmacol Chinese Mat Med, 361,Zhongshan East Rd, Shijiazhuang 050017, Hebei, Peoples R China; Hebei Med Univ, Sch Pharm, Shijiazhuang 050017, Peoples R China
ABSTRACT: Cerebrolysin (CBL) is a peptide-rich preparation made by hydrolysis and purified extraction of porcine brain. CBL contains various neuroprotective peptides, such as neurotrophic factor, nerve growth factor and ciliary neuro-trophic factor, which can be used to treat neurodegenerative diseases. However, the active peptides in CBL had not been studied in depth. In this study, the following was carried out in order to investigate the active peptides in CBL. First, CBL samples were treated using organic reagents (acetonitrile and acetone) to precipitate the proteins and different solid phase extraction methods (MCX mixed-mode cartridges, C18 SPE cartridge columns and HILIC sorbent).
[more...]
Use: pNovo



Comprehensive evaluation of peptide de novo sequencing tools for monoclonal antibody assembly
Briefings in Bioinformatics. 2023. Beslic, Denis et al. BAM Fed Inst Mat Res & Testing, Richard Willstatter Str 11, D-12489 Berlin, Germany; Univ Potsdam, Digital Engn Fac, Hasso Plattner Inst, Prof Dr Helmert Str 2-3, D-14482 Potsdam, Germany; BAM Fed Inst Mat Res & Testing, Unter Eichen 87, D-12205 Berlin, Germany; Robert Koch Inst, ZKI PH 3,Nordufer 20, D-13353 Berlin, Germany
ABSTRACT: Monoclonal antibodies are biotechnologically produced proteins with various applications in research, therapeutics and diagnostics. Their ability to recognize and bind to specific molecule structures makes them essential research tools and therapeutic agents. Sequence information of antibodies is helpful for understanding antibody-antigen interactions and ensuring their affinity and specificity. De novo protein sequencing based on mass spectrometry is a valuable method to obtain the amino acid sequence of peptides and proteins without a priori knowledge.
[more...]
Use: pNovo; pDeep



GlycoTCFM: Glycoproteomics Based on Two Complementary Fragmentation Methods Reveals Distinctive O-Glycosylation in Human Sperm and Seminal Plasma
Journal of Proteome Research. 2023. Mengqi Luo et al. Department of Nephrology and Institutes for Systems Genetics, Frontiers Science Center for Disease-Related Molecular Network, West China Hospital, Sichuan University, Chengdu 610041,
ABSTRACT: Human semen, consisting of spermatozoa (sperm) and seminal plasma, represents a special clinical sample type in human body fluid. Protein glycosylation in sperm and seminal plasma plays key roles in spermatogenesis, maturation, capacitation, sperm-egg recognition, motility of sperm, and fertilization. In this study, we profiled the most comprehensive O-glycoproteome map of human sperm and seminal plasma using our recently presented Glycoproteomics based on Two Complementary Fragmentation Methods (GlycoTCFM).
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Use: pGlyco



An N-glycopeptide MS/MS data analysis workflow leveraging two complementary glycoproteomic software tools for more confident identification and assignments
Proteomics. 2023. Chu-Wei Kuo et al. Acad Sinica, Inst Biol Chem, 128 Acad Rd Sec 2, Taipei 115, Taiwan
ABSTRACT: Complete coverage of all N-glycosylation sites on the SARS-CoV2 spike protein would require the use of multiple proteases in addition to trypsin. Subsequent identification of the resulting glycopeptides by searching against database often introduces assignment errors due to similar mass differences between different permutations of amino acids and glycosyl residues. By manually interpreting the individual MS2 spectra, we report here the common sources of errors in assignment, especially those introduced by the use of chymotrypsin.
[more...]
Use: pGlyco



Expression of a Siglec-Fc Protein and Its Characterization
BIOLOGY-BASEL. 2023. Kaijun Chi et al. Jiangnan Univ, Sch Biotechnol, Key Lab Carbohydrate Chem & Biotechnol, Minist Educ, Wuxi 214122, Peoples R China; Chinese Acad Sci, Inst Proc Engn, State Key Lab Biochem Engn, Beijing 100190, Peoples R China
ABSTRACT: Simple Summary The Siglec-Fc protein, a fusion protein combining Siglec with the Fc part of a human antibody, is a promising sialic acid-Siglec axis-targeted agent for cancer treatment and is widely used for Siglec ligands discovery. The recombinant Siglec-Fc fusion protein has been expressed in different cell systems. However, its characteristics have not been investigated in detail. In this study, HEK293 and CHO cell lines were used to express the Siglec9-Fc protein, and their adaptability for production was compared.
[more...]
Use: pGlyco



Glycopeptide database search and de novo sequencing with PEAKS GlycanFinder enable highly sensitive glycoproteomics
Nature Communications. 2023. Sun, Weiping et al. Bioinformatics Solutions Inc., Waterloo, Ontario, Canada
ABSTRACT: Here we present GlycanFinder, a database search and de novo sequencing tool for the analysis of intact glycopeptides from mass spectrometry data. GlycanFinder integrates peptide-based and glycan-based search strategies to address the challenge of complex fragmentation of glycopeptides. A deep learning model is designed to capture glycan tree structures and their fragment ions for de novo sequencing of glycans that do not exist in the database. We performed extensive analyses to validate the false discovery rates (FDRs) at both peptide and glycan levels and to evaluate GlycanFinder based on comprehensive benchmarks from previous community-based studies.
[more...]
Use: pGlyco; pDeep



High-coverage four-Dimensional data-independent acquisition proteomics and phosphoproteomics enabled by deep learning-driven multidimensional predictions
Analytical Chemistry. 2023. Moran Chen et al. Wuhan Univ, Inst Adv Studies, Wuhan 430072, Hubei, Peoples R China
ABSTRACT: Four-dimensional (4D) data-independentacquisition (DIA)-basedproteomics is a promising technology. However, its full performanceis restricted by the time-consuming building and limited coverageof a project-specific experimental library. Herein, we developed aversatile multifunctional deep learning model Deep4D based on self-attentionthat could predict the collisional cross section, retention time,fragment ion intensity, and charge state with high accuracies forboth the unmodified and phosphorylated peptides and thus establishedthe complete workflows for high-coverage 4D DIA proteomics and phosphoproteomicsbased on multidimensional predictions.
[more...]
Use: pDeep



AIomics: exploring more of the proteome using mass spectral libraries extended by artificial intelligence
Journal of Proteome Research. 2023. Lewis Y. Geer et al. Natl Inst Stand & Technol, Mass Spectrometry Data Ctr, Biomol Measurement Div, Gaithersburg, MD 20899 USA
ABSTRACT: The unbounded permutations of biological molecules, includingproteinsand their constituent peptides, present a dilemma in identifying thecomponents of complex biosamples. Sequence search algorithms usedto identify peptide spectra can be expanded to cover larger classesof molecules, including more modifications, isoforms, and atypicalcleavage, but at the cost of false positives or false negatives dueto the simplified spectra they compute from sequence records. Spectrallibrary searching can help solve this issue by precisely matchingexperimental spectra to library spectra with excellent sensitivityand specificity.
[more...]
Use: pDeep



Machine learning-based peptide-spectrum match rescoring opens up the immunopeptidome
. 2023. Charlotte Adams et al. Laboratory of Protein Science, Proteomics and Epigenetic Signaling (PPES), Department of Biomedical Sciences, University of Antwerp, Antwerp,
ABSTRACT:
Use: pDeep



High-Coverage Four-Dimensional Data-Independent Acquisition Proteomics and Phosphoproteomics Enabled by Deep Learning-Driven Multidimensional Predictions
Analytical Chemistry. 2023. Moran Chen et al. Wuhan Univ, Inst Adv Studies, Wuhan 430072, Hubei, Peoples R China
ABSTRACT: Four-dimensional (4D) data-independentacquisition (DIA)-basedproteomics is a promising technology. However, its full performanceis restricted by the time-consuming building and limited coverageof a project-specific experimental library. Herein, we developed aversatile multifunctional deep learning model Deep4D based on self-attentionthat could predict the collisional cross section, retention time,fragment ion intensity, and charge state with high accuracies forboth the unmodified and phosphorylated peptides and thus establishedthe complete workflows for high-coverage 4D DIA proteomics and phosphoproteomicsbased on multidimensional predictions.
[more...]
Use: pDeep



AIomics: Exploring More of the Proteome Using Mass Spectral Libraries Extended by Artificial Intelligence
Journal of Proteome Research. 2023. Lewis Y. Geer et al. Natl Inst Stand & Technol, Mass Spectrometry Data Ctr, Biomol Measurement Div, Gaithersburg, MD 20899 USA
ABSTRACT: The unbounded permutations of biological molecules, includingproteinsand their constituent peptides, present a dilemma in identifying thecomponents of complex biosamples. Sequence search algorithms usedto identify peptide spectra can be expanded to cover larger classesof molecules, including more modifications, isoforms, and atypicalcleavage, but at the cost of false positives or false negatives dueto the simplified spectra they compute from sequence records. Spectrallibrary searching can help solve this issue by precisely matchingexperimental spectra to library spectra with excellent sensitivityand specificity.
[more...]
Use: pDeep




2022




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.
[more...]
Use: pFind; pDeep



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.
[more...]
Use: pFind



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.
[more...]
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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.
[more...]
Use: pFind; pDeep



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.
[more...]
Use: pFind



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.
[more...]
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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.
[more...]
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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.
[more...]
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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.
[more...]
Use: pFind



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.
[more...]
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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.
[more...]
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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.
[more...]
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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.
[more...]
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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).
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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).
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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Structure of nucleosome-bound human PBAF complex
Nature Communications. 2022. Wang, Li et al. Fudan University Shanghai Cancer Center, Institutes of Biomedical Sciences, State Key Laboratory of Genetic Engineering and Shanghai Key Laboratory of Medical Epigenetics, Shanghai Medical College of Fudan University, Shanghai, 200032, China The International Co-laboratory of Medical Epigenetics and Metabolism, Ministry of Science and Technology, China, Department of Systems Biology for Medicine, School of Basic Medical Sciences, Shanghai Medical College of Fudan University, Shanghai, 200032, China Human Phenome Institute, Collaborative Innovation Center of Genetics and Development, School of Life Sciences, Fudan University, Shanghai, 200433, China
ABSTRACT: BAF and PBAF are mammalian SWI/SNF family chromatin remodeling complexes that possess multiple histone/DNA-binding subunits and create nucleosome-depleted/free regions for transcription activation. Despite previous structural studies and recent advance of SWI/SNF family complexes, it remains incompletely understood how PBAF-nucleosome complex is organized. Here we determined structure of 13-subunit human PBAF in complex with acetylated nucleosome in ADP-BeF3-bound state. Four PBAF-specific subunits work together with nine BAF/PBAF-shared subunits to generate PBAF-specific modular organization, distinct from that of BAF at various regions.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
[more...]
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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 …
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.
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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.
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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.
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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.
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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.
[more...]
Use: pNovo



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.
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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.
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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.
[more...]
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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.
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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.
[more...]
Use: pNovo; pParse



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.
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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.
[more...]
Use: pGlyco



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.
[more...]
Use: pGlyco



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.
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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.
[more...]
Use: pGlyco



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.
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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.
[more...]
Use: pGlyco



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.
[more...]
Use: pGlyco



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.
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Use: pGlyco



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.
[more...]
Use: pGlyco



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.
[more...]
Use: pGlyco



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.
[more...]
Use: pQuant



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.
[more...]
Use: pQuant



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.
[more...]
Use: pQuant



Comparing top-down proteoform identification: Deconvolution, PrSM overlap, and PTM detection
. 2022. David L. Tabb et al. Université Paris Cité, Institut Pasteur, CNRS UAR 2024, Mass Spectrometry for Biology Unit, Paris 75015, France
ABSTRACT:
Use: pTop



Comparing Top-Down Proteoform Identification: Deconvolution, PrSM Overlap, and PTM Detection
. 2022. David L. Tabb et al. Université Paris Cité, Institut Pasteur, CNRS UAR 2024, Mass Spectrometry for Biology Unit, Paris 75015, France
ABSTRACT:
Use: pTop




2021




ADAP Y571 phosphorylation is required to prime STAT3 for activation in TLR4-stimulated macrophages
JOURNAL OF IMMUNOLOGY. 2021. Yang, Naiqi et al. Soochow Univ, Inst Biol, 199 Renai Rd, Suzhou 215123, Jiangsu, Peoples R China; Soochow Univ, Inst Med Sci, 199 Renai Rd, Suzhou 215123, Jiangsu, Peoples R China
ABSTRACT: Adhesion and degranulation-promoting adapter protein (ADAP), originally identified as an essential adaptor molecule in TCR signaling and T cell adhesion, has emerged as a critical regulator in innate immune cells such as macrophages; however, its role in macrophage polarization and inflammatory responses remains unknown. In this study, we show that ADAP plays an essential role in TLR4-mediated mouse macrophage polarization via modulation of STAT3 activity. Macrophages from ADAP-deficient mice exhibit enhanced M1 polarization, expression of proinflammatory cytokines and capacity in inducing Th1 responses, but decreased levels of anti-inflammatory cytokines in response to TLR4 activation by LPS.
[more...]
Use: pFind



Acyl carrier protein promotes MukBEF action in Escherichia coli chromosome organization-segregation
Nature Communications. 2021. Prince, JP et al. Univ Oxford, Dept Biochem, South Parks Rd, Oxford OX1 3QU, England.
ABSTRACT: Structural Maintenance of Chromosomes (SMC) complexes act ubiquitously to compact DNA linearly, thereby facilitating chromosome organization-segregation. SMC proteins have a conserved architecture, with a dimerization hinge and an ATPase head domain separated by a long antiparallel intramolecular coiled-coil. Dimeric SMC proteins interact with essential accessory proteins, kleisins that bridge the two subunits of an SMC dimer, and HAWK/KITE proteins that interact with kleisins. The ATPase activity of the Escherichia coli SMC protein, MukB, which is essential for its in vivo function, requires its interaction with the dimeric kleisin, MukF that in turn interacts with the KITE protein, MukE.
[more...]
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Proteogenomic analysis provides novel insight into genome annotation and nitrogen metabolism in Nostoc Sp. PCC 7120
Microbiology Spectrum. 2021. Yu, Shengchao et al. Univ Chinese Acad Sci, Beijing, Peoples R China; Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan, Peoples R China
ABSTRACT: Cyanobacteria, capable of oxygenic photosynthesis, play a vital role in nitrogen and carbon cycles. Nostoc sp. PCC 7120 (Nostoc 7120) is a model cyanobacterium commonly used to study cell differentiation and nitrogen metabolism. Although its genome was released in 2002, a high-quality genome annotation remains unavailable for this model cyanobacterium. Therefore, in this study, we performed an in-depth proteogenomic analysis based on high-resolution mass spectrometry (MS) data to refine the genome annotation of Nostoc 7120.
[more...]
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ADAP Y571 Phosphorylation Is Required to Prime STAT3 for Activation in TLR4-Stimulated Macrophages
JOURNAL OF IMMUNOLOGY. 2021. Yang, Naiqi et al. Soochow Univ, Inst Biol, 199 Renai Rd, Suzhou 215123, Jiangsu, Peoples R China; Soochow Univ, Inst Med Sci, 199 Renai Rd, Suzhou 215123, Jiangsu, Peoples R China
ABSTRACT: Adhesion and degranulation-promoting adapter protein (ADAP), originally identified as an essential adaptor molecule in TCR signaling and T cell adhesion, has emerged as a critical regulator in innate immune cells such as macrophages; however, its role in macrophage polarization and inflammatory responses remains unknown. In this study, we show that ADAP plays an essential role in TLR4-mediated mouse macrophage polarization via modulation of STAT3 activity. Macrophages from ADAP-deficient mice exhibit enhanced M1 polarization, expression of proinflammatory cytokines and capacity in inducing Th1 responses, but decreased levels of anti-inflammatory cytokines in response to TLR4 activation by LPS.
[more...]
Use: pFind



Ribosome-amplified metabolism, RAMBO, measured by NMR spectroscopy
Biochemistry. 2021. Yu, JianChao et al. SUNY Albany, Dept Chem, Albany, NY 12222 USA
ABSTRACT: NMR spectroscopy was used to investigate the phenomenon of ribosome-amplified metabolism or RAMBO between pyruvate kinase and ribosomes. Because the concentration of ribosomes increases as the cell grows, ribosome binding interactions may regulate metabolic fluxes by altering the distribution of bound and free enzymes. Pyruvate kinase (PK) catalyzes the last step of glycolysis and represents a major drug target for controlling bacterial infections. The binding of metabolic enzymes to ribosomes creates protein quinary structures with altered catalytic activities.
[more...]
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Insulin signaling regulates longevity through protein phosphorylation in Caenorhabditis elegans
Nature Communications. 2021. Li, WJ et al. Natl Inst Biol Sci, Beijing, Peoples R China.
ABSTRACT: Insulin/IGF-1 Signaling (IIS) is known to constrain longevity by inhibiting the transcription factor FOXO. How phosphorylation mediated by IIS kinases regulates lifespan beyond FOXO remains unclear. Here, we profile IIS-dependent phosphorylation changes in a large-scale quantitative phosphoproteomic analysis of wild-type and three IIS mutant Caenorhabditis elegans strains. We quantify more than 15,000 phosphosites and find that 476 of these are differentially phosphorylated in the long-lived daf-2/insulin receptor mutant.
[more...]
Use: pQuant; pFind



Structure of the activated human minor spliceosome
Science. 2021. Bai, Rui et al. Westlake Lab Life Sci & Biomed, Hangzhou 310024, Zhejiang, Peoples R China; Tsinghua Univ, Sch Life Sci, Tsinghua Peking Joint Ctr Life Sci, Beijing Frontier Res Ctr Biol Struct, Beijing 100084, Peoples R China; Tsinghua Univ, Sch Life Sci, Tsinghua Peking Joint Ctr Life Sci, Beijing Adv Innovat Ctr Struct Biol, Beijing 100084, Peoples R China; Westlake Inst Adv Study, Inst Biol, Hangzhou 310024, Zhejiang, Peoples R China; Westlake Univ, Sch Life Sci, Key Lab Struct Biol Zhejiang Prov, Hangzhou 310024, Zhejiang, Peoples R China
ABSTRACT: The minor spliceosome mediates splicing of the rare but essential U12-type precursor messenger RNA. Here, we report the atomic features of the activated human minor spliceosome determined by cryo-electron microscopy at 2.9-angstrom resolution. The 5' splice site and branch point sequence of the U12-type intron are recognized by the U6atac and U12 small nuclear RNAs (snRNAs), respectively. Five newly identified proteins stabilize the conformation of the catalytic center: The zinc finger protein SCNM1 functionally mimics the SF3a complex of the major spliceosome, the RBM48-ARMC7 complex binds the g-monomethyl phosphate cap at the 5' end of U6atac snRNA, the U-box protein PPIL2 coordinates loop I of U5 snRNA and stabilizes U5 small nuclear ribonucleoprotein (snRNP), and CRIPT stabilizes U12 snRNP.
[more...]
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Proteogenomic characterization of the pathogenic fungus Aspergillus flavus reveals novel genes involved in aflatoxin production
Molecular & Cellular Proteomics. 2021. Yang, Mingkun et al. Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan, Peoples R China; Fujian Agr & Forestry Univ, Key Lab Pathogen Fungi & Mycotoxins Fujian Prov, Fuzhou, Peoples R China; Fujian Agr & Forestry Univ, Sch Life Sci, Fuzhou, Peoples R China
ABSTRACT: Aspergillus flavus (A. flavus), a pathogenic fungus, can produce carcinogenic and toxic aflatoxins that are a serious agricultural and medical threat worldwide. Attempts to decipher the aflatoxin biosynthetic pathway have been hampered by the lack of a high-quality genome annotation for A. flavus. To address this gap, we performed a comprehensive proteogenomic analysis using high-accuracy mass spectrometry data for this pathogen. The resulting high-quality data set confirmed the translation of 8724 previously predicted genes and identified 732 novel proteins, 269 splice variants, 447 single amino acid variants, 188 revised genes.
[more...]
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Antibody-free enrichment method for proteome-wide analysis of endogenous SUMOylation sites
Analytica Chimica Acta. 2021. Li, Y et al. Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog R&A Ctr, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Peoples R China.
ABSTRACT: SUMOylation is a reversible post-translational modification that plays crucial roles in numerous cellular processes. Although anti-SUMO antibodies have been applied to analyze exogenous and endogenous SUMOylation, such immunoprecipitation enrichment strategy is applicable only for the enrichment of one specific SUMO type in mammalian cells, unable to map the global landscape of all endogenous SUMOylation simultaneously. To address this issue, we proposed an antibody-free strategy to enrich and profile endogenous SUMO1/2/3-modified peptides simultaneously.
[more...]
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HSP70 chaperones RNA-free TDP-43 into anisotropic intranuclear liquid spherical shells
Science. 2021. Yu, HY et al. Univ Calif San Diego, Ludwig Inst Canc Res, La Jolla, CA 92093 USA.
ABSTRACT: The RNA binding protein TDP-43 forms intranuclear or cytoplasmic aggregates in age-related neurodegenerative diseases. In this study, we found that RNA binding-deficient TDP-43 (produced by neurodegeneration-causing mutations or posttranslational acetylation in its RNA recognition motifs) drove TDP-43 demixing into intranuclear liquid spherical shells with liquid cores. These droplets, which we named "anisosomes", have shells that exhibit birefringence, thus indicating liquid crystal formation. Guided by mathematical modeling, we identified the primary components of the liquid core to be HSP70 family chaperones, whose adenosine triphosphate (ATP)-dependent activity maintained the liquidity of shells and cores.
[more...]
Use: pFind; pParse; pQuant



DeepLC can predict retention times for peptides that carry as-yet unseen modifications
Nature methods. 2021. Bouwmeester, R et al. VIB, VIB UGent Ctr Med Biotechnol, Ghent, Belgium.
ABSTRACT: DeepLC, a deep learning-based peptide retention time predictor, can predict retention times for unmodified peptides as well as peptides with previously unseen modifications. The inclusion of peptide retention time prediction promises to remove peptide identification ambiguity in complex liquid chromatography-mass spectrometry identification workflows. However, due to the way peptides are encoded in current prediction models, accurate retention times cannot be predicted for modified peptides. This is especially problematic for fledgling open searches, which will benefit from accurate retention time prediction for modified peptides to reduce identification ambiguity.
[more...]
Use: pFind



Global profiling of distinct cysteine redox forms reveals wide-ranging redox regulation in C. elegans
Nature communications. 2021. Meng, J et al. Joslin Diabet Ctr, Div Res, 1 Joslin Pl, Boston, MA 02215 USA.
ABSTRACT: Post-translational changes in the redox state of cysteine residues can rapidly and reversibly alter protein functions, thereby modulating biological processes. The nematode C. elegans is an ideal model organism for studying cysteine-mediated redox signaling at a network level. Here we present a comprehensive, quantitative, and site-specific profile of the intrinsic reactivity of the cysteinome in wild-type C. elegans. We also describe a global characterization of the C. elegans redoxome in which we measured changes in three major cysteine redox forms after H2O2 treatment.
[more...]
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Structure of the Arabidopsis thaliana glutamate receptor-like channel GLR3. 4
Molecular cell. 2021. Green, MN et al. Columbia Univ, Dept Biochem & Mol Biophys, 650 West 168th St, New York, NY 10032 USA.
ABSTRACT: Glutamate receptor-like channels (GLRs) play vital roles in various physiological processes in plants, such as wound response, stomatal aperture control, seed germination, root development, innate immune response, pollen tube growth, and morphogenesis. Despite the importance of GLRs, knowledge about their molecular organization is limited. Here we use X-ray crystallography and single-particle cryo-EM to solve structures of the Arabidopsis thaliana GLR3.4. Our structures reveal the tetrameric assembly of GLR3.4 subunits into a three-layer domain architecture, reminiscent of animal ionotropic glutamate receptors (iGluRs).
[more...]
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Spatiotemporally resolved subcellular phosphoproteomics
PNAS. 2021. Liu, YJ et al. Peking Univ, PKU IDG McGovern Inst Brain Res, AH-100871 Beijing, Peoples R China.
ABSTRACT: Proteome-wide profiling of protein phosphorylation has been widely used to reveal the underlying mechanism of diverse cellular signaling events. Yet, characterizing subcellular phosphoproteome with high spatial-temporal resolution has remained challenging. Herein, we developed a subcellular-specific uncaging-assisted biotinylation and mapping of phosphoproteome (SubMAPP) strategy to monitor the phosphorylation dynamics of subcellular proteome in living cells and animals. Our method capitalizes on the genetically encoded bioorthogonal decaging strategy, which enables the rapid activation of subcellular localized proximity labeling biotin ligase through either light illumination or small-molecule triggers.
[more...]
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Wittig reagents for chemoselective sulfenic acid ligation enables global site stoichiometry analysis and redox-controlled mitochondrial targeting
Nature Chemistry. 2021. Shi, YL et al. Scripps Res Inst, Dept Chem, Jupiter, FL 33458 USA.
ABSTRACT: Triphenylphosphonium ylides, known as Wittig reagents, are one of the most commonly used tools in synthetic chemistry. Despite their considerable versatility, Wittig reagents have not yet been explored for their utility in biological applications. Here we introduce a chemoselective ligation reaction that harnesses the reactivity of Wittig reagents and the unique chemical properties of sulfenic acid, a pivotal post-translational cysteine modification in redox biology. The reaction, which generates a covalent bond between the ylide nucleophilic alpha-carbon and electrophilic gamma-sulfur, is highly selective, rapid and affords robust labelling under a range of biocompatible reaction conditions, which includes in living cells.
[more...]
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Potential use of serum proteomics for monitoring COVID-19 progression to complement RT-PCR detection
Journal of proteome research. 2021. Zhang, Y et al. Wenzhou Med Univ, Taizhou Hosp, Linhai 317000, Zhejiang, Peoples R China; Westlake Univ, Sch Life Sci, Key Lab Struct Biol Zhejiang Prov, Hangzhou 310000, Zhejiang, Peoples R China; Westlake Lab Life Sci & Biomed, Ctr Infect Dis Res, Hangzhou 310000, Zhejiang, Peoples R China; Westlake Inst Adv Study, Inst Basic Med Sci, Hangzhou 310000, Zhejiang, Peoples R China
ABSTRACT: RT-PCR is the primary method to diagnose COVID-19 and is also used to monitor the disease course. This approach, however, suffers from false negatives due to RNA instability and poses a high risk to medical practitioners. Here, we investigated the potential of using serum proteomics to predict viral nucleic acid positivity during COVID19. We analyzed the proteome of 275 inactivated serum samples from 54 out of 144 COVID-19 patients and shortlisted 42 regulated proteins in the severe group and 12 in the non-severe group.
[more...]
Use: pFind; pDeep



Highly Efficient Enrichment of O-GlcNAc Glycopeptides Based on Chemical Oxidation and Reversible Hydrazide Chemistry
Analytical Chemistry. 2021. Chen, Y 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
ABSTRACT: Protein O-GlcNAcylation has been implicated in a broad range of cellular processes, while the functional research is still lagging behind other post-translational modification (PTMs), as a result of the low stoichiometry and limited enrichment efficiency. Herein, a strategy, named CHO-GlcNAc, was developed for O-GlcNAc glycopeptide enrichment. In this strategy, the O-GlcNAc glycopeptides were first enzymatically labeled with a Gal moiety, followed by chemical oxidation to efficiently introduce the aldehyde groups.
[more...]
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Dynamically remodeled hepatic extracellular matrix predicts prognosis of early-stage cirrhosis
Cell Death & Disease. 2021. Wu, YX et al. Chinese Acad Sci, Ctr Excellence Mol Cell Sci, Shanghai Inst Biochem & Cell Biol, State Key Lab Cell Biol, Shanghai 200031, Peoples R China.
ABSTRACT: Liver cirrhosis remains major health problem. Despite the progress in diagnosis of asymptomatic early-stage cirrhosis, prognostic biomarkers are needed to identify cirrhotic patients at high risk developing advanced stage disease. Liver cirrhosis is the result of deregulated wound healing and is featured by aberrant extracellular matrix (ECM) remodeling. However, it is not comprehensively understood how ECM is dynamically remodeled in the progressive development of liver cirrhosis. It is yet unknown whether ECM signature is of predictive value in determining prognosis of early-stage liver cirrhosis.
[more...]
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Determinants of genome-wide distribution and evolution of uORFs in eukaryotes
Nature communications. 2021. Zhang, H et al. Peking Univ, Sch Life Sci, Ctr Bioinformat, State Key Lab Prot & Plant Gene Res, Beijing, Peoples R China.
ABSTRACT: Upstream open reading frames (uORFs) play widespread regulatory functions in modulating mRNA translation in eukaryotes, but the principles underlying the genomic distribution and evolution of uORFs remain poorly understood. Here, we analyze similar to 17 million putative canonical uORFs in 478 eukaryotic species that span most of the extant taxa of eukaryotes. We demonstrate how positive and purifying selection, coupled with differences in effective population size (N-e), has shaped the contents of uORFs in eukaryotes.
[more...]
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Allosteric activation of SARS-CoV-2 RNA-dependent RNA polymerase by remdesivir triphosphate and other phosphorylated nucleotides
MBio. 2021. Wang, B et al. Ohio State Univ, Dept Microbiol, Columbus, OH 43210 USA.
ABSTRACT: The catalytic subunit of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) Nsp12 has a unique nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain that transfers nucleoside monophosphates to the Nsp9 protein and the nascent RNA. The NiRAN and RdRp modules form a dynamic interface distant from their catalytic sites, and both activities are essential for viral replication. We report that codon-optimized (for the pause-free translation in bacterial cells) Nsp12 exists in an inactive state in which NiRAN-RdRp interactions are broken, whereas translation by slow ribosomes and incubation with accessory Nsp7/8 subunits or nucleoside triphosphates (NTPs) partially rescue RdRp activity.
[more...]
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Expression profiles of antimicrobial peptides in Mytilus coruscus
Aquaculture. 2021. Yang, JY et al. Zhejiang Ocean Univ, Marine Sci & Tech Coll, Lab Marine Biol Prot Engn, Zhoushan City 316022, Zhejiang, Peoples R China
ABSTRACT: Antimicrobial peptides (AMPs) play a fundamental role in mussels' innate immunity, preventing the invasion of potential pathogens. Previous research has shown that AMPs are abundant in Mytilus species. A mussel with important economic value and limited distribution in the East China Sea, M. coruscus, also contains abundant AMPs, including the mytichitin and myticusin identified previously in this species. However, the molecular diversity and expression pattern of M. coruscus AMPs remain largely unknown.
[more...]
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ZmMPK5 phosphorylates ZmNAC49 to enhance oxidative stress tolerance in maize
New Phytologist. 2021. Xiang, Y et al. Nanjing Agr Univ, Coll Life Sci, Nanjing 210095, Jiangsu, Peoples R China.
ABSTRACT: Mitogen-activated protein kinase (MPK) is a critical regulator of the antioxidant defence system in response to various stimuli. However, how MPK directly and exactly regulates antioxidant enzyme activities is still unclear. Here, we demonstrated that a NAC transcription factor ZmNAC49 mediated the regulation of antioxidant enzyme activities by ZmMPK5. ZmNAC49 expression is induced by oxidative stress. ZmNAC49 enhances oxidative stress tolerance in maize, and it also reduces superoxide anion generation and increases superoxide dismutase (SOD) activity.
[more...]
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A20/Nrdp1 interaction alters the inflammatory signaling profile by mediating K48-and K63-linked polyubiquitination of effectors MyD88 and TBK1
Journal of Biological chemistry. 2021. Meng, ZY et al. Army Med Univ, Xinqiao Hosp, Dept Neurol, Mil Med Univ 3, Chongqing, Peoples R China.
ABSTRACT: A20 is a potent anti-inflammatory protein that mediates both inflammation and ubiquitination in mammals, but the related mechanisms are not clear. In this study, we performed mass spectrometry (MS) screening, gene ontology (GO) analysis, and coimmunoprecipitation (co-IP) in a lipopolysaccharide (LPS)-induced inflammatory cell model to identify novel A20-interacting proteins. We confirmed that the E3 ubiquitin ligase Nrdp1, also known as ring finger protein 41 (RNF41), interacted with A20 in LPS-stimulated cells.
[more...]
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caAtlas: An immunopeptidome atlas of human cancer
Iscience. 2021. Yi, XP et al. Baylor Coll Med, Lester & Sue Smith Breast Ctr, Houston, TX 77030 USA.
ABSTRACT: Comprehensive characterization of tumor antigens is essential for the design of cancer immunotherapies, and mass spectrometry (MS)-based immunopeptidomics enables high-throughput identification of major histocompatibility complex (MHC)-bound peptide antigens in vivo. Here we construct an immunopeptidome atlas of human cancer through an extensive collection of 43 published immunopeptidomic datasets and standardized analysis of 81.6 million MS/MS spectra using an open search engine. Our analysis greatly expands the current knowledge of MHC-bound antigens, including an unprecedented characterization of post-translationally modified antigens and their cancer-association.
[more...]
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Site-specific N-and O-glycosylation analysis of human plasma fibronectin
frontiers in Chemistry. 2021. Liu, D et al. Georgia State Univ, Dept Chem, Atlanta, GA 30303 USA.
ABSTRACT: Human plasma fibronectin is an adhesive protein that plays a crucial role in wound healing. Many studies had indicated that glycans might mediate the expression and functions of fibronectin, yet a comprehensive understanding of its glycosylation is still missing. Here, we performed a comprehensive N- and O-glycosylation mapping of human plasma fibronectin and quantified the occurrence of each glycoform in a site-specific manner. Intact N-glycopeptides were enriched by zwitterionic hydrophilic interaction chromatography, and N-glycosite sites were localized by the O-18-labeling method.
[more...]
Use: pFind; pGlyco



A semi-tryptic peptide centric metaproteomic mining approach and its potential utility in capturing signatures of gut microbial proteolysis
MICROBIOME. 2021. Yan, ZX et al. Sun Yat Sen Univ, Affiliated Hosp 5, Guangdong Prov Key Lab Biomed Imaging, Zhuhai 519000, Guangdong, Peoples R China.
ABSTRACT: BackgroundProteolysis regulation allows gut microbes to respond rapidly to dynamic intestinal environments by fast degradation of misfolded proteins and activation of regulatory proteins. However, alterations of gut microbial proteolytic signatures under complex disease status such as inflammatory bowel disease (IBD, including Crohn's disease (CD) and ulcerative colitis (UC)), have not been investigated. Metaproteomics holds the potential to investigate gut microbial proteolysis because semi-tryptic peptides mainly derive from endogenous proteolysis.ResultsWe have developed a semi-tryptic peptide centric metaproteomic mining approach to obtain a snapshot of human gut microbial proteolysis signatures.
[more...]
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Exploring the microbiome-wide lysine acetylation, succinylation, and propionylation in human gut microbiota
Analytical Chemistry. 2021. Zhang, X et al. Univ Ottawa, Fac Med, Ottawa Inst Syst Biol, Ottawa, ON K1H 8M5, Canada.
ABSTRACT: Lysine acylations are important post-translational modifications that are present in both eukaryotes and prokaryotes and regulate diverse cellular functions. Our knowledge of the microbiome lysine acylation remains limited due to the lack of efficient analytical and bioinformatics methods for complex microbial communities. Here, we show that the serial enrichment using motif antibodies successfully captures peptides containing lysine acetylation, propionylation, and succinylation from human gut microbiome samples.
[more...]
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Proteogenomics Study of Blastobotrys adeninivorans TMCC 70007—A Dominant Yeast in the Fermentation Process of Pu-erh Tea
Journal of proteome research. 2021. Tian, F et al. Yunnan Univ, Sch Life Sci, Yunnan Inst Microbiol, Minist Educ,Key Lab Microbial Divers Southwest Ch, Kunming 650091, Yunnan, Peoples R China.
ABSTRACT: Blastobotrys adeninivorans plays an essential role in pile-fermenting of Pu-erh tea. Its ability to assimilate various carbon and nitrogen sources makes it available for application in a wide range of industry sectors. The genome of B. adeninivorans TMCC 70007 isolated from pile-fermented Pu-erh tea was sequenced and assembled. Proteomics analysis indicated that 4900 proteins in TMCC 70007 were expressed under various culture conditions. Proteogenomics mapping revealed 48 previously unknown genes and corrected 118 gene models predicted by GeneMark-ES.
[more...]
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Mirror-Cutting-Based Digestion Strategy Enables the In-Depth and Accuracy Characterization of N-Linked Protein Glycosylation
Journal of proteome research. 2021. Chen, Y et al. Chinese Acad Sci, Dalian Inst Chem Phys, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Peoples R China.
ABSTRACT: N-linked glycosylation plays important roles in multiple physiological and pathological processes, while the analysis coverage is still limited due to the insufficient digestion of glycoproteins, as well as incomplete ion fragments for intact glycopeptide determination. Herein, a mirror-cutting-based digestion strategy was proposed by combining two orthogonal proteases of LysargiNase and trypsin to characterize the macro- and micro-heterogeneity of protein glycosylation. Using the above two proteases, the b- or y-ion series of peptide sequences were, respectively, enhanced in MS/MS, generating the complementary spectra for peptide sequence identification.
[more...]
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DIA-based proteomics identifies IDH2 as a targetable regulator of acquired drug resistance in chronic myeloid leukemia
Molecular & Cellular Proteomics. 2021. Liu, W et al. Dalian Med Univ, Coll Pharm, Dept Clin Pharmacol, Dalian, Liaoning, Peoples R China; Westlake Univ, Sch Life Sci, Key Lab Struct Biol Zhejiang Prov, Hangzhou, Zhejiang, Peoples R China; Westlake Lab Life Sci & Biomed, Ctr Infect Dis Res, Hangzhou, Zhejiang, Peoples R China; Westlake Inst Adv Study, Inst Basic Med Sci, Hangzhou, Zhejiang, Peoples R China
ABSTRACT: Drug resistance is a critical obstacle to effective treatment in patients with chronic myeloid leukemia. To understand the underlying resistance mechanisms in response imatinib mesylate (IMA) and adriamycin (ADR), the parental K562 cells were treated with low doses of IMA ADR for 2 months to generate derivative cells with mild, intermediate, and severe resistance to the drugs defined by their increasing resistance index. PulseDIAbased (DIA [data-independent acquisition]) quantitative proteomics was then employed to reveal the proteome changes in these resistant cells.
[more...]
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Proteogenomics Integrating Novel Junction Peptide Identification Strategy Discovers Three Novel Protein Isoforms of Human NHSL1 and EEF1B2
Journal of proteome research. 2021. He, Cuitong et al. Peking-Tsinghua Centre for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, 100871 Beijing, China
ABSTRACT: In eukaryotes, alternative pre-mRNA splicing allows a single gene to encode different protein isoforms that function in many biological processes, and they are used as biomarkers or therapeutic targets for diseases. Although protein isoforms in the human genome are well annotated, we speculate that some low-abundance protein isoforms may still be under-annotated because most genes have a primary coding product and alternative protein isoforms tend to be under-expressed. A peptide coencoded by a novel exon and an annotated exon separated by an intron is known as a novel junction peptide.
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An Integrated Strategy Reveals Complex Glycosylation of Erythropoietin Using Mass Spectrometry
JOURNAL OF PROTEOME RESEARCH. 2021. Guan, YD et al. Univ Med Ctr Hamburg Eppendorf, Inst Clin Chem & Lab Med, Sect Mass Spectrometr Prote, D-20246 Hamburg, Germany.
ABSTRACT: The characterization of therapeutic glycoproteins is challenging due to the structural heterogeneity of the therapeutic protein glycosylation. This study presents an in-depth analytical strategy for glycosylation of first-generation erythropoietin (epoetin beta), including a developed mass spectrometric workflow for N-glycan analysis, bottom-up mass spectrometric methods for site-specific N-glycosylation, and a LC-MS approach for O-glycan identification. Permethylated N-glycans, peptides, and enriched glycopeptides of erythropoietin were analyzed by nanoLC-MS/MS, and de-N-glycosylated erythropoietin was measured by LC-MS, enabling the qualitative and quantitative analysis of glycosylation and different glycan modifications (e.g., phosphorylation and O-acetylation).
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Potential Use of Serum Proteomics for Monitoring COVID-19 Progression to Complement RT-PCR Detection
Journal of proteome research. 2021. Zhang, Y et al. Wenzhou Med Univ, Taizhou Hosp, Linhai 317000, Zhejiang, Peoples R China; Westlake Univ, Sch Life Sci, Key Lab Struct Biol Zhejiang Prov, Hangzhou 310000, Zhejiang, Peoples R China; Westlake Lab Life Sci & Biomed, Ctr Infect Dis Res, Hangzhou 310000, Zhejiang, Peoples R China; Westlake Inst Adv Study, Inst Basic Med Sci, Hangzhou 310000, Zhejiang, Peoples R China
ABSTRACT: RT-PCR is the primary method to diagnose COVID-19 and is also used to monitor the disease course. This approach, however, suffers from false negatives due to RNA instability and poses a high risk to medical practitioners. Here, we investigated the potential of using serum proteomics to predict viral nucleic acid positivity during COVID19. We analyzed the proteome of 275 inactivated serum samples from 54 out of 144 COVID-19 patients and shortlisted 42 regulated proteins in the severe group and 12 in the non-severe group.
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Exploring the Microbiome-Wide Lysine Acetylation, Succinylation, and Propionylation in Human Gut Microbiota
Analytical Chemistry. 2021. Zhang, X et al. Univ Ottawa, Fac Med, Ottawa Inst Syst Biol, Ottawa, ON K1H 8M5, Canada.
ABSTRACT: Lysine acylations are important post-translational modifications that are present in both eukaryotes and prokaryotes and regulate diverse cellular functions. Our knowledge of the microbiome lysine acylation remains limited due to the lack of efficient analytical and bioinformatics methods for complex microbial communities. Here, we show that the serial enrichment using motif antibodies successfully captures peptides containing lysine acetylation, propionylation, and succinylation from human gut microbiome samples.
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Discovery and visualization of uncharacterized drug-protein adducts using mass spectrometry
Analytical chemistry. 2021. Riffle, M et al. Univ Washington, Dept Biochem, Seattle, WA 98195 USA
ABSTRACT: Drugs are often metabolized to reactive intermedi-ates that form protein adducts. Adducts can inhibit protein activity,elicit immune responses, and cause life-threatening adverse drugreactions. The masses of reactive metabolites are frequentlyunknown, rendering traditional mass spectrometry-based proteo-mics approaches incapable of adduct identification. Here, wepresent Magnum, an open-mass search algorithm optimized foradduct identification, and Limelight, a web-based data processingpackage for analysis and visualization of data from all existingalgorithms.
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DIA-based Proteomics Identifies IDH2 as a Targetable Regulator of Acquired Drug Resistance in Chronic Myeloid Leukemia
Molecular & Cellular Proteomics. 2021. Liu, W et al. Dalian Med Univ, Coll Pharm, Dept Clin Pharmacol, Dalian, Liaoning, Peoples R China; Westlake Univ, Sch Life Sci, Key Lab Struct Biol Zhejiang Prov, Hangzhou, Zhejiang, Peoples R China; Westlake Lab Life Sci & Biomed, Ctr Infect Dis Res, Hangzhou, Zhejiang, Peoples R China; Westlake Inst Adv Study, Inst Basic Med Sci, Hangzhou, Zhejiang, Peoples R China
ABSTRACT: Drug resistance is a critical obstacle to effective treatment in patients with chronic myeloid leukemia. To understand the underlying resistance mechanisms in response imatinib mesylate (IMA) and adriamycin (ADR), the parental K562 cells were treated with low doses of IMA ADR for 2 months to generate derivative cells with mild, intermediate, and severe resistance to the drugs defined by their increasing resistance index. PulseDIAbased (DIA [data-independent acquisition]) quantitative proteomics was then employed to reveal the proteome changes in these resistant cells.
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Site-Specific N-and O-Glycosylation Analysis of Human Plasma Fibronectin
frontiers in Chemistry. 2021. Liu, D et al. Georgia State Univ, Dept Chem, Atlanta, GA 30303 USA.
ABSTRACT: Human plasma fibronectin is an adhesive protein that plays a crucial role in wound healing. Many studies had indicated that glycans might mediate the expression and functions of fibronectin, yet a comprehensive understanding of its glycosylation is still missing. Here, we performed a comprehensive N- and O-glycosylation mapping of human plasma fibronectin and quantified the occurrence of each glycoform in a site-specific manner. Intact N-glycopeptides were enriched by zwitterionic hydrophilic interaction chromatography, and N-glycosite sites were localized by the O-18-labeling method.
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Allosteric Activation of SARS-CoV-2 RNA-Dependent RNA Polymerase by Remdesivir Triphosphate and Other Phosphorylated Nucleotides
MBio. 2021. Wang, B et al. Ohio State Univ, Dept Microbiol, Columbus, OH 43210 USA.
ABSTRACT: The catalytic subunit of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) Nsp12 has a unique nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain that transfers nucleoside monophosphates to the Nsp9 protein and the nascent RNA. The NiRAN and RdRp modules form a dynamic interface distant from their catalytic sites, and both activities are essential for viral replication. We report that codon-optimized (for the pause-free translation in bacterial cells) Nsp12 exists in an inactive state in which NiRAN-RdRp interactions are broken, whereas translation by slow ribosomes and incubation with accessory Nsp7/8 subunits or nucleoside triphosphates (NTPs) partially rescue RdRp activity.
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FUT8-mediated aberrant N-glycosylation of B7H3 suppresses the immune response in triple-negative breast cancer
Nature communications. 2021. Huang, Y et al. Sun Yat Sen Univ Canc Ctr, Collaborat Innovat Ctr Canc Med, State Key Lab Oncol South China, Guangdong Key Lab Nasopharyngeal Carcinoma Diag &, Guangzhou, Peoples R China.
ABSTRACT: Most patients with triple negative breast cancer (TNBC) do not respond to anti-PD1/PDL1 immunotherapy, indicating the necessity to explore immune checkpoint targets. B7H3 is a highly glycosylated protein. However, the mechanisms of B7H3 glycosylation regulation and whether the sugar moiety contributes to immunosuppression are unclear. Here, we identify aberrant B7H3 glycosylation and show that N-glycosylation of B7H3 at NXT motif sites is responsible for its protein stability and immunosuppression in TNBC tumors.
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Leep1 interacts with PIP3 and the Scar/WAVE complex to regulate cell migration and macropinocytosis
JOURNAL OF CELL BIOLOGY. 2021. Yang, YH et al. Chinese Acad Sci, Ctr Excellence Biomacromol, Inst Biophys, Natl Lab Biomacromol, Beijing, Peoples R China.
ABSTRACT: Polarity is essential for diverse functions in many cell types. Establishing polarity requires targeting a network of specific signaling and cytoskeleton molecules to different subregions of the cell, yet the full complement of polarity regulators and how their activities are integrated over space and time to form morphologically and functionally distinct domains remain to be uncovered. Here, by using the model system Dictyostelium and exploiting the characteristic chemoattractant-stimulated translocation of polarly distributed molecules, we developed a proteomic screening approach, through which we identified a leucine-rich repeat domain-containing protein we named Leep1 as a novel polarity regulator.
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Carboxypeptidase Y assisted disulfide-bond identification with linearized database search
Analytical Chemistry. 2021. Qiang, Jiali et al. Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 100 Haike Road, Pudong, Shanghai 201210, China
ABSTRACT: A disulfide bond is an important protein post-translational modification and plays a key role in regulating protein oxidation status, protein structure, and stability. Analysis of a disulfide bond using mass spectrometry is challenging because there lacks an efficient method to separate the disulfide-linked peptides from a complex protein digest, and the MS data requires sophisticated interpretation. Here, we developed a novel disulfide bond identification strategy, termed as "carboxypeptidase Y assisted disulfide-bond identification (CADI)".
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Structures of the human Mediator and Mediator-bound preinitiation complex
Science. 2021. Chen, XZ et al. Fudan Univ, Shanghai Canc Ctr, Inst Biomed Sci, State Key Lab Genet Engn,Shanghai Key Lab Radiat, Shanghai, Peoples R China.
ABSTRACT: The 1.3-megadalton transcription factor IID (TFIID) is required for preinitiation complex (PIC) assembly and RNA polymerase II (Pol II)-mediated transcription initiation on almost all genes. The 26-subunit Mediator stimulates transcription and cyclin-dependent kinase 7 (CDK7)-mediated phosphorylation of the Pol II C-terminal domain (CTD). We determined the structures of human Mediator in the Tail module-extended (at near-atomic resolution) and Tail-bent conformations and structures of TFIID-based PICMediator (76 polypeptides, similar to 4.1 megadaltons) in four distinct conformations.
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Covalently engineered nanobody chimeras for targeted membrane protein degradation
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY. 2021. Zhang, H et al. Peking Univ, Synthet & Funct Biomol Ctr, Beijing Natl Lab Mol Sci, Coll Chem & Mol Engn, Beijing 100871, Peoples R China.
ABSTRACT: The targeted degradation of membrane proteins would afford an attractive and general strategy for treating various diseases that remain difficult with the current proteolysis-targeting chimera (PROTAC) methodology. We herein report a covalent nanobody-based PROTAC strategy, termed GlueTAC, for targeted membrane protein degradation with high specificity and efficiency. We first established a mass-spectrometry-based screening platform for the rapid development of a covalent nanobody (GlueBody) that allowed proximity-enabled cross-linking with surface antigens on cancer cells.
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Highly synergistic combinations of nanobodies that target SARS-CoV-2 and are resistant to escape
eLife. 2021. Mast, FD et al. Seattle Childrens Res Inst, Ctr Global Infect Dis Res, Seattle, WA 98101 USA; Rockefeller Univ, Lab Cellular & Struct Biol, 1230 York Ave, New York, NY 10021 USA; Rockefeller Univ, Lab Mass Spectrometry & Gaseous Ion Chem, 1230 York Ave, New York, NY 10021 USA; Univ Washington, Dept Pediat, Seattle, WA 98195 USA; Univ Washington, Dept Biochem, Seattle, WA 98195 USA
ABSTRACT: The emergence of SARS-CoV-2 variants threatens current vaccines and therapeutic antibodies and urgently demands powerful new therapeutics that can resist viral escape. We therefore generated a large nanobody repertoire to saturate the distinct and highly conserved available epitope space of SARS-CoV-2 spike, including the S1 receptor binding domain, N-terminal domain, and the S2 subunit, to identify new nanobody binding sites that may reflect novel mechanisms of viral neutralization. Structural mapping and functional assays show that indeed these highly stable monovalent nanobodies potently inhibit SARS-CoV-2 infection, display numerous neutralization mechanisms, are effective against emerging variants of concern, and are resistant to mutational escape.
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The structure of a virus-encoded nucleosome
NATURE STRUCTURAL & MOLECULAR BIOLOGY. 2021. Valencia-Sanchez, MI et al. New York Univ Grossman Sch Med, Skirball Inst Biomol Med, Dept Biochem & Mol Pharmacol, New York, NY 10016 USA.
ABSTRACT: The cryo-EM structure of DNA-assembled histone pairs H beta-H alpha and H delta-H gamma from Marseillevirus, a nucleocytoplasmic large DNA virus, reveals that these proteins form viral nucleosomes with highly conserved features when compared to canonical eukaryotic nucleosomes. Certain large DNA viruses, including those in the Marseilleviridae family, encode histones. Here we show that fused histone pairs H beta-H alpha and H delta-H gamma from Marseillevirus are structurally analogous to the eukaryotic histone pairs H2B-H2A and H4-H3.
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Chemical synthesis of activity‐based E2‐ubiquitin probes for the structural analysis of E3 ligase‐catalyzed transthiolation
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION. 2021. Liang, LJ et al. Tsinghua Univ, Tsinghua Peking Ctr Life Sci, Minist Educ,Dept Chem, Key Lab Bioorgan Phosphorus Chem & Chem Biol,Ctr, Beijing 100084, Peoples R China.
ABSTRACT: Activity-based E2 conjugating enzyme (E2)-ubiquitin (Ub) probes have recently emerged as effective tools for studying the molecular mechanism of E3 ligase (E3)-catalyzed ubiquitination. However, the preparation of existing activity-based E2-Ub probes depends on recombination technology and bioconjugation chemistry, limiting their structural diversity. Herein we describe an expedient total chemical synthesis of an E2 enzyme variant through a hydrazide-based native chemical ligation, which enabled the construction of a structurally new activity-based E2-Ub probe to covalently capture the catalytic site of Cys-dependent E3s.
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Diethynyl Phosphinates for Cysteine‐Selective Protein Labeling and Disulfide Rebridging
Angewandte Chemie International Edition. 2021. Stieger, CE et al. Forsch Verbund Berlin EV FMP, Leibniz Forschungsinst Mol Pharmakol, Chem Biol Dept, Campus Berlin Buch,Robert Roessle Str 10, D-13125 Berlin, Germany.
ABSTRACT: Diethynyl phosphinates were developed as bisfunctional electrophiles for the site-selective modification of peptides, proteins and antibodies. One of their electron-deficient triple bonds reacts selectively with a thiol and positions an electrophilic moiety for a subsequent intra- or intermolecular reaction with another thiol. The obtained conjugates were found to be stable in human plasma and in the presence of small thiols. We further demonstrate that this method is suitable for the generation of functional protein conjugates for intracellular delivery.
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Molecular architecture of the endocytic TPLATE complex
Science Advances. 2021. Yperman, K et al. Univ Ghent, Dept Plant Biotechnol & Bioinformat, Technol Pk 71, B-9052 Ghent, Belgium.
ABSTRACT: Eukaryotic cells rely on endocytosis to regulate their plasma membrane proteome and lipidome. Most eukaryotic groups, except fungi and animals, have retained the evolutionary ancient TSET complex as an endocytic regulator. Unlike other coatomer complexes, structural insight into TSET is lacking. Here, we reveal the molecular architecture of plant TSET [TPLATE complex (TPC)] using an integrative structural approach. We identify crucial roles for specific TSET subunits in complex assembly and membrane interaction.
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Molecular characterization of a complex of apoptosis-inducing factor 1 with cytochrome c oxidase of the mitochondrial respiratory chain
PNAS. 2021. Hevler, Johannes F. et al. Univ Utrecht, Utrecht Inst Pharmaceut Sci, NL-3584 CH Utrecht, Netherlands; Univ Utrecht, Bijvoet Ctr Biomol Res, Netherlands Prote Ctr, NL-3584 CH Utrecht, Netherlands; Univ Cologne, Cologne Excellence Cluster Cellular Stress Respon, D-50931 Cologne, Germany; Radboud Univ Nijmegen, Med Ctr, Radboud Inst Mol Life Sci, NL-6525 GA Nijmegen, Netherlands; Univ Utrecht, Utrecht Inst Pharmaceut Sci, Bijvoet Ctr Biomol Res, Biomol Mass Spectrometry & Prote, NL-3584 CH Utrecht, Netherlands
ABSTRACT: Combining mass spectrometry-based chemical cross-linking and complexome profiling, we analyzed the interactome of heart mitochondria. We focused on complexes of oxidative phosphorylation and found that dimeric apoptosis-inducing factor 1 (AIFM1) forms a defined complex with similar to 10% of monomeric cytochrome c oxidase (COX) but hardly interacts with respiratory chain supercomplexes. Multiple AIFM1 intercross-links engaging six different COX subunits provided structural restraints to build a detailed atomic model of the COX-AIFM1(2) complex (PDBDEV_00000092).
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Structural basis of soluble membrane attack complex packaging for clearance
Nature Communications. 2021. Menny, A et al. Imperial Coll London, Dept Life Sci, Sir Ernst Chain Bldg, London SW7 2AZ, England.
ABSTRACT: To prevent unregulated complement activation, extracellular chaperones capture soluble precursors to the membrane attack complex (sMAC). Here, structural analysis of sMAC reveals how clusterin recognizes heterogeneous sMAC complexes and inhibits polymerization of complement protein C9. Unregulated complement activation causes inflammatory and immunological pathologies with consequences for human disease. To prevent bystander damage during an immune response, extracellular chaperones (clusterin and vitronectin) capture and clear soluble precursors to the membrane attack complex (sMAC).
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Retention time prediction using neural networks increases identifications in crosslinking mass spectrometry
Nature communications. 2021. Giese, SH et al. Tech Univ Berlin, Inst Biotechnol, Bioanalyt, Berlin, Germany.
ABSTRACT: Crosslinking mass spectrometry has developed into a robust technique that is increasingly used to investigate the interactomes of organelles and cells. However, the incomplete and noisy information in the mass spectra of crosslinked peptides limits the numbers of protein-protein interactions that can be confidently identified. Here, we leverage chromatographic retention time information to aid the identification of crosslinked peptides from mass spectra. Our Siamese machine learning model xiRT achieves highly accurate retention time predictions of crosslinked peptides in a multi-dimensional separation of crosslinked E.
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MS Annika: A new cross-linking search engine
Journal of Proteome Research. 2021. Pirklbauer, GJ et al. Univ Appl Sci Upper Austria, Bioinformat Res Grp, A-4232 Hagenberg, Austria.
ABSTRACT: Cross-linking mass spectrometry (XL-MS) has become a powerful technique that enables insights into protein structures and protein interactions. The development of cleavable cross-linkers has further promoted XL-MS through search space reduction, thereby allowing for proteome-wide studies. These new analysis possibilities foster the development of new cross-linkers, which not every search engine can deal with out of the box. In addition, some search engines for XL-MS data also struggle with the validation of identified cross-linked peptides, that is, false discovery rate (FDR) estimation, as FDR calculation is hampered by the fact that not only one but two peptides in a single spectrum have to be correct.
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Repurposing of synaptonemal complex proteins for kinetochores in Kinetoplastida
Open Biology. 2021. Tromer, EC et al. Univ Cambridge, Dept Biochem, Cambridge, England.
ABSTRACT: Chromosome segregation in eukaryotes is driven by the kinetochore, a macromolecular complex that connects centromeric DNA to microtubules of the spindle apparatus. Kinetochores in well-studied model eukaryotes consist of a core set of proteins that are broadly conserved among distant eukaryotic phyla. By contrast, unicellular flagellates of the class Kinetoplastida have a unique set of 36 kinetochore components. The evolutionary origin and history of these kinetochores remain unknown. Here, we report evidence of homology between axial element components of the synaptonemal complex and three kinetoplastid kinetochore proteins KKT16-18.
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Allosteric transcription stimulation by RNA polymerase II super elongation complex
Molecular Cell. 2021. Chen, Y et al. Max Planck Inst Biophys Chem, Dept Mol Biol, Fassberg 11, D-37077 Gottingen, Germany.
ABSTRACT: The super elongation complex (SEC) contains the positive transcription elongation factor b (P-TEFb) and the subcomplex ELL2-EAF1, which stimulates RNA polymerase II (RNA Pol II) elongation. Here, we report the cryoelectron microscopy (cryo-EM) structure of ELL2-EAF1 bound to a RNA Pol II elongation complex at 2.8 A resolution. The ELL2-EAF1 dimerization module directly binds the RNA Pol II lobe domain, explaining how SEC delivers P-TEFb to RNA Pol II. The same site on the lobe also binds the initiation factor TFIIF, consistent with SEC binding only after the transition from transcription initiation to elongation.
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Integrative proteomics identifies thousands of distinct, multi-epitope, and high-affinity nanobodies
Cell Systems. 2021. Xiang, YF et al. Univ Pittsburgh, Dept Cell Biol, Pittsburgh, PA 15260 USA.
ABSTRACT: The antibody immune response is essential for the survival of mammals. However, we still lack a systematic understanding of the antibody repertoire. Here, we developed a proteomic strategy to survey, at an unprecedented scale, the landscape of antigen-engaged, circulating camelid heavy-chain antibodies, whose minimal binding fragments are called VHH antibodies or nanobodies. The sensitivity and robustness of this approach were validated with three antigens spanning orders of magnitude in immune responses; thousands of distinct, high-affinity nanobody families were reliably identified and quantified.
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Large-scale ratcheting in a bacterial DEAH/RHA-type RNA helicase that modulates antibiotics susceptibility
PNAS. 2021. Grass, LM et al. Free Univ Berlin, Inst Chem & Biochem, Lab Struct Biochem, D-14195 Berlin, Germany.
ABSTRACT: Many bacteria harbor RNA-dependent nucleoside-triphosphatases of the DEAH/RHA family, whose molecular mechanisms and cellular functions are poorly understood. Here, we show that the Escherichia coli DEAH/RHA protein, HrpA, is an ATP-dependent 3 to 5 ' RNA helicase and that the RNA helicase activity of HrpA influences bacterial survival under antibiotics treatment. Limited proteolysis, crystal structure analysis, and functional assays showed that HrpA contains an N-terminal DEAH/RHA helicase cassette preceded by a unique N-terminal domain and followed by a large C-terminal region that modulates the helicase activity.
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A resource of high-quality and versatile nanobodies for drug delivery
Iscience. 2021. Shen, ZL et al. Univ Pittsburgh, Dept Cell Biol, Pittsburgh, PA 15260 USA.
ABSTRACT: Therapeutic and diagnostic efficacies of small biomolecules and chemical compounds are hampered by suboptimal pharmacokinetics. Here, we developed a repertoire of robust and high-affinity antihuman serum albumin nanobodies (Nb-HSA) that can be readily fused to small biologics for half-life extension. We characterized the thermostability, binding kinetics, and cross-species reactivity of Nb(HSA)s, mapped their epitopes, and structurally resolved a tetrameric HSA-Nb complex. We parallelly determined the half-lives of a cohort of selected Nb(HSA)s in an HSA mouse model by quantitative proteomics.
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Reverse chemical ecology suggests putative primate pheromones
Molecular Biology and Evolution. 2021. Zaremska, V et al. Austrian Inst Technol GmbH, Biosensor Technol, Tulln, Austria
ABSTRACT: Pheromonal communication is widespread among living organisms, but in apes and particularly in humans there is currently no strong evidence for such phenomenon. Among primates, lemurs use pheromones to communicate within members of the same species, whereas in some monkeys such capabilities seem to be lost. Chemical communication in humans appears to be impaired by the lack or malfunctioning of biochemical tools and anatomical structures mediating detection of pheromones. Here, we report on a pheromone-carrier protein (SAL) adopting a "reverse chemical ecology" approach to get insights on the structures of potential pheromones in a representative species of lemurs (Microcebus murinus) known to use pheromones, Old-World monkeys (Cercocebus atys) for which chemical communication has been observed, and humans (Homo sapiens), where pheromones and chemical communication are still questioned.
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Cryo-EM structure of the yeast TREX complex and coordination with the SR-like protein Gbp2
eLife. 2021. Xie, YH et al. Vanderbilt Univ, Dept Biochem, Sch Med, Nashville, TN 37232 USA.
ABSTRACT: The evolutionarily conserved TRanscript-EXport (TREX) complex plays central roles during mRNP (messenger ribonucleoprotein) maturation and export from the nucleus to the cytoplasm. In yeast, TREX is composed of the THO sub-complex (Tho2, Hpr1, Text, Mft1, and Thp2), the DEAD box ATPase Sub2, and Yra1. Here we present a 3.7 angstrom cryo-EM structure of the yeast THO center dot Sub2 complex. The structure reveals the intimate assembly of THO revolving around its largest subunit Tho2. THO stabilizes a semi-open conformation of the Sub2 ATPase via interactions with Tho2.
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A novel recognition site for polyubiquitin and ubiquitin-like signals in an unexpected region of proteasomal subunit Rpn1
Journal of Biological Chemistry. 2021. Boughton, AJ et al. Univ Maryland, Ctr Biomol Struct & Org, Dept Chem & Biochem, College Pk, MD 20742 USA.
ABSTRACT: The ubiquitin (Ub)-proteasome system is the primary mechanism for maintaining protein homeostasis in eukaryotes, yet the underlying signaling events and specificities of its components are poorly understood. Proteins destined for degradation are tagged with covalently linked polymeric Ub chains and subsequently delivered to the proteasome, often with the assistance of shuttle proteins that contain Ub-like domains. This degradation pathway is riddled with apparent redundancy-in the form of numerous polyubiquitin chains of various lengths and distinct architectures, multiple shuttle proteins, and at least three proteasomal receptors.
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Identification of Native Cross-Links in Bacillus subtilis Spore Coat Proteins
Journal of Proteome Research. 2021. Ursem, R et al. Univ Amsterdam, Dept Mol Biol & Microbial Food Safety, NL-1098 XH Amsterdam, Netherlands.
ABSTRACT: The resistance properties of the bacterial spores are partially due to spore surface proteins, similar to 30% of which are said to form an insoluble protein fraction. Previous research has also identified a group of spore coat proteins affected by spore maturation, which exhibit an increased level of interprotein cross-linking. However, the proteins and the types of cross-links involved, previously proposed based on indirect evidence, have yet to be confirmed experimentally. To obtain more insight into the structural basis of the proteinaceous component of the spore coat, we attempted to identify coat cross-links and the proteins involved using new peptide fractionation and bioinformatic methods.
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The Odorant-Binding Proteins of the Spider Mite Tetranychus urticae
International Journal of Molecular Sciences. 2021. Zhu, J et al. Austrian Inst Technol GmbH, Biosensor Technol, Konrad Lorenz Str 24, A-3430 Tulln, Austria.
ABSTRACT: Spider mites are one of the major agricultural pests, feeding on a large variety of plants. As a contribution to understanding chemical communication in these arthropods, we have characterized a recently discovered class of odorant-binding proteins (OBPs) in Tetranychus urticae. As in other species of Chelicerata, the four OBPs of T. urticae contain six conserved cysteines paired in a pattern (C1-C6, C2-C3, C4-C5) differing from that of insect counterparts (C1-C3, C2-C5, C4-C6). Proteomic analysis uncovered a second family of OBPs, including twelve members that are likely to be unique to T.
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Redox-sensitive CDC-42 clustering promotes wound closure in C. elegans
Cell reports. 2021. Xu, JX et al. Zhejiang Univ, Ctr Stem Cell & Regenerat Med, Sch Med, Affiliated Hosp 2, Hangzhou 310058, Peoples R China.
ABSTRACT: Tissue damage induces immediate-early signals, activating Rho small GTPases to trigger actin polymerization essential for later wound repair. However, how tissue damage is sensed to activate Rho small GTPases locally remains elusive. Here, we found that wounding the C. elegans epidermis induces rapid relocalization of CDC-42 into plasma membrane-associated clusters, which subsequently recruits WASP/WSP-1 to trigger actin polymerization to close the wound. In addition, wounding induces a local transient increase and subsequent reduction of H2O2, which negatively regulates the clustering of CDC-42 and wound closure.
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HSP-90/kinase complexes are stabilized by the large PPIase FKB-6
Scientific Reports. 2021. Sima, S et al. Tech Univ Munich, Ctr Integrated Prot Res, Dept Chem, Lichtenbergstr 4, D-85748 Garching, Germany.
ABSTRACT: Protein kinases are important regulators in cellular signal transduction. As one major type of Hsp90 client, protein kinases rely on the ATP-dependent molecular chaperone Hsp90, which maintains their structure and supports their activation. Depending on client type, Hsp90 interacts with different cofactors. Here we report that besides the kinase-specific cofactor Cdc37 large PPIases of the Fkbp-type strongly bind to kinase center dot Hsp90 center dot Cdc37 complexes. We evaluate the nucleotide regulation of these assemblies and identify prominent interaction sites in this quaternary complex.
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Unraveling the surface glycoprotein interaction network by integrating chemical crosslinking with MS-based proteomics
Chemical Science. 2021. Sun, FX et al. Georgia Inst Technol, Sch Chem & Biochem, Atlanta, GA 30332 USA.
ABSTRACT: The cell plasma membrane provides a highly interactive platform for the information transfer between the inside and outside of cells. The surface glycoprotein interaction network is extremely important in many extracellular events, and aberrant protein interactions are closely correlated with various diseases including cancer. Comprehensive analysis of cell surface protein interactions will deepen our understanding of the collaborations among surface proteins to regulate cellular activity. In this work, we developed a method integrating chemical crosslinking, an enzymatic reaction, and MS-based proteomics to systematically characterize proteins interacting with surface glycoproteins, and then constructed the surfaceome interaction network.
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Binding of cytochrome P450 27C1, a retinoid desaturase, to its accessory protein adrenodoxin
Archives of Biochemistry and Biophysics. 2021. Glass, Sarah M et al. Department of Biochemistry, Vanderbilt University School of Medicine, 638 Robinson Research Building, 2200 Pierce Avenue, Nashville, Tennessee, 37232-0146, United States.
ABSTRACT: Of the 57 human cytochrome P450 (P450) enzymes, seven are mitochondrial: 11A1, 11B1, 11B2, 24A1, 27A1, 27B1, and 27C1. Mitochondrial P450s utilize an electron transport system with adrenodoxin (Adx) and NADPH-adrenodoxin reductase (AdR). AdR reduces Adx, which then transfers electrons to the P450. The interactions between proteins in the mitochondrial P450 system are largely driven by electrostatic interactions, though the specifics vary depending on the P450. Unlike other mitochondrial P450s, the interaction between P450 27C1, a retinoid 3,4-desaturase expressed in the skin, and Adx remains largely uncharacterized.
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A new non-classical fold of varroa odorant-binding proteins reveals a wide open internal cavity
Scientific Reports. 2021. Amigues, B et al. Aix Marseille Univ AMU, CNRS, Architecture & Fonct Macromol Biol AFMB, UMR 6098, Campus Luminy,Case 932, F-13288 Marseille 09, France.
ABSTRACT: Odorant-binding proteins (OBPs), as they occur in insects, form a distinct class of proteins that apparently has no closely related representatives in other animals. However, ticks, mites, spiders and millipedes contain genes encoding proteins with sequence similarity to insect OBPs. In this work, we have explored the structure and function of such non-insect OBPs in the mite Varroa destructor, a major pest of honey bee. Varroa OBPs present six cysteines paired into three disulphide bridges, but with positions in the sequence and connections different from those of their insect counterparts.
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Structural characterization of KKT4, an unconventional microtubule-binding kinetochore protein
Structure. 2021. Ludzia, P et al. Univ Oxford, Dept Biochem, Oxford OX1 3QU, England.
ABSTRACT: The kinetochore is the macromolecular machinery that drives chromosome segregation by interacting with spindle microtubules. Kinetoplastids (such as Trypanosoma brucei), a group of evolutionarily divergent eukaryotes, have a unique set of kinetochore proteins that lack any significant homology to canonical kinetochore components. To date, KKT4 is the only kinetoplastid kinetochore protein that is known to bind microtubules. Here we use X-ray crystallography, NMR spectroscopy, and crosslinking mass spectrometry to characterize the structure and dynamics of KKT4.
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A composite filter for low FDR of protein-protein interactions detected by in vivo cross-linking
Journal of Proteomics. 2021. de Jong, L et al. Univ Amsterdam, Swammerdam Inst Life Sci, Mass Spectrometry Biomol, Sci Pk 904, NL-1098 XH Amsterdam, Netherlands.
ABSTRACT: In vivo chemical cross-linking combined with LCMSMS of digested extracts (in vivo CX-MS) can reveal stable and dynamic protein-protein interactions at proteome-wide scale and at peptide level. In vivo CX-MS requires a membrane permeable and cleavable cross-linker and a fast and sensitive search engine to identify the linked peptides. Here we explore the use of the search engine pLink 2 to identify cross-links induced in exponentially growing Bacillus subtilis cells treated in culture with bis(succinimidyl)-3-azidomethyl-glutarate (BAMG).
[more...]
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Synthesis, LC-MS/MS analysis, and biological evaluation of two vaccine candidates against ticks based on the antigenic P0 peptide from R. sanguineus linked to the p64K carrier protein from Neisseria meningitidis
Analytical and Bioanalytical Chemistry. 2021. Luis Javier González et al. Ctr Genet Engn & Biotechnol CIGB, Dept Prote, Ave 31,E 158 & 190, Havana 10600, Cuba; Ctr Genet Engn & Biotechnol CIGB, Anim Biotechnol Dept, Ave 31,E 158 & 190, Havana 10600, Cuba
ABSTRACT: A peptide from the P0 acidic ribosomal protein (pP0) of ticks conjugated to keyhole limpet hemocyanin from Megathura crenulata has shown to be effective against different tick species when used in host vaccination. Turning this peptide into a commercial anti-tick vaccine will depend on finding the appropriate, technically and economically feasible way to present it to the host immune system. Two conjugates (p64K-Cys(1)pP0 and p64K-beta Ala(1)pP0) were synthesized using the p64K carrier protein from Neisseria meningitidis produced in Escherichia coli, the same cross-linking reagent, and two analogues of pP0.
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Nematode CDC-37 and DNJ-13 form complexes and can interact with HSP-90
Scientific reports. 2021. Schmauder, Lukas et al. Department of Chemistry, Center for Integrated Protein Research, Technische Universität München, Lichtenbergstr. 4, 85748, Garching, Germany
ABSTRACT: The molecular chaperones Hsc70 and Hsp90 are required for proteostasis control and specific folding of client proteins in eukaryotic and prokaryotic organisms. Especially in eukaryotes these ATP-driven molecular chaperones are interacting with cofactors that specify the client spectrum and coordinate the ATPase cycles. Here we find that a Hsc70-cofactor of the Hsp40 family from nematodes, DNJ-13, directly interacts with the kinase-specific Hsp90-cofactor CDC-37. The interaction is specific for DNJ-13, while DNJ-12 another DnaJ-like protein of C.
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Accurate Retention Time Prediction Based on Monolinked Peptide Information to Confidently Identify Cross-Linked Peptides
Journal of the American Society for Mass Spectrometry. 2021. Huang, R et al. ShanghaiTech Univ, Shanghai Inst Adv Immunochem Studies, Shanghai 201210, Peoples R China
ABSTRACT: Cross-linking mass spectrometry methods have not been successfully applied to protein-protein interaction discovery at a proteome- wide level mainly due to the computation complexity (O (n(2))) issue. In a previous report, we proposed a decision tree searching strategy (DTSS), which can reduce complexity by orders of magnitude. In this study, we further found that the monolinked peptides carry out the information on the retention time of the corresponding cross-linked pairs; therefore, the retention time of cross-linked peptide pairs can be predicted accurately.
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Spontaneous protein--protein crosslinking at glutamine and glutamic acid residues in long-lived proteins
Biochemical Journal. 2021. Friedrich, MG et al. Univ Wollongong, Illawarra Hlth & Med Res Inst, Wollongong, NSW 2522, Australia.
ABSTRACT: Long-lived proteins (LLPs) are susceptible to the accumulation of both enzymatic and spontaneous post-translational modifications (PTMs). A prominent PTM observed in LLPs is covalent protein-protein crosslinking. In this study, we examined aged human lenses and found several proteins to be crosslinked at Glu and Gln residues. This new covalent bond involves the amino group of Lys or an alpha-amino group. A number of these crosslinks were found in intermediate filament proteins. Such crosslinks could be reproduced experimentally by incubation of Glu- or Gln-containing peptides and their formation was consistent with an amino group attacking a glutarimide intermediate.
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Molecular characterization of a complex of Apoptosis Inducing Factor 1 (AIFM1) with cytochrome c oxidase of the mitochondrial respiratory chain
PNAS. 2021. Hevler, JF et al. Univ Utrecht, Utrecht Inst Pharmaceut Sci, Bijvoet Ctr Biomol Res, Biomol Mass Spectrometry & Prote, NL-3584 CH Utrecht, Netherlands.
ABSTRACT: Combining mass spectrometry-based chemical cross-linking and complexome profiling, we analyzed the interactome of heart mitochondria. We focused on complexes of oxidative phosphorylation and found that dimeric apoptosis-inducing factor 1 (AIFM1) forms a defined complex with similar to 10% of monomeric cytochrome c oxidase (COX) but hardly interacts with respiratory chain supercomplexes. Multiple AIFM1 intercross-links engaging six different COX subunits provided structural restraints to build a detailed atomic model of the COX-AIFM1(2) complex (PDBDEV_00000092).
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The SARS-CoV-2 nucleocapsid phosphoprotein forms mutually exclusive condensates with RNA and the membrane-associated M protein
Nature communications. 2021. Lu, S et al. Univ Calif San Diego, Dept Cellular & Mol Med, San Diego, CA 92093 USA.
ABSTRACT: The multifunctional nucleocapsid (N) protein in SARS-CoV-2 binds the similar to 30kb viral RNA genome to aid its packaging into the 80-90nm membrane-enveloped virion. The N protein is composed of N-terminal RNA-binding and C-terminal dimerization domains that are flanked by three intrinsically disordered regions. Here we demonstrate that the N protein's central disordered domain drives phase separation with RNA, and that phosphorylation of an adjacent serine/arginine rich region modulates the physical properties of the resulting condensates.
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Structural insights into preinitiation complex assembly on core promoters
Science. 2021. Chen, XZ et al. Fudan Univ, Shanghai Canc Ctr, Inst Biomed Sci, State Key Lab Genet Engn, Shanghai 200032, Peoples R China.
ABSTRACT: Transcription factor IID (TFIID) recognizes core promoters and supports preinitiation complex (PIC) assembly for RNA polymerase II (Pol II)-mediated eukaryotic transcription. We determined the structures of human TFIID-based PIC in three stepwise assembly states and revealed two-track PIC assembly: stepwise promoter deposition to Pol II and extensive modular reorganization on track I (on TATA-TFIID-binding element promoters) versus direct promoter deposition on track II (on TATA-only and TATA-less promoters).
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Structural basis of Integrator-mediated transcription regulation
Science. 2021. Fianu, I et al. Max Planck Inst Biophys Chem, Dept Mol Biol, D-37077 Gottingen, Germany.
ABSTRACT: Integrator and protein phosphatase 2A (PP2A) form a complex that dephosphorylates paused RNA polymerase II (Pol II), cleaves the nascent RNA, and terminates transcription. We report the structure of the pretermination complex containing a human Integrator-PP2A complex bound to paused Pol II. Integrator binds Pol II and the pausing factors DSIF and NELF to exclude binding of the elongation factors SPT6 and PAF1 complex. Integrator also binds the C-terminal domain of Pol II and positions PP2A to counteract Pol II phosphorylation and elongation.
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Structural basis of human transcription–DNA repair coupling
Nature. 2021. Kokic, G et al. Max Planck Inst Biophys Chem, Dept Mol Biol, Gottingen, Germany.
ABSTRACT: Transcription-coupled DNA repair removes bulky DNA lesions from the genome(1,2) and protects cells against ultraviolet (UV) irradiation(3). Transcription-coupled DNA repair begins when RNA polymerase II (Pol II) stalls at a DNA lesion and recruits the Cockayne syndrome protein CSB, the E3 ubiquitin ligase, CRL4(CSA) and UV-stimulated scaffold protein A (UVSSA)(3). Here we provide five high-resolution structures of Pol II transcription complexes containing human transcription-coupled DNA repair factors and the elongation factors PAF1 complex (PAF) and SPT6.
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DNA-driven condensation assembles the meiotic DNA break machinery
NATURE. 2021. Bouuaert, CC et al. Mem Sloan Kettering Canc Ctr, Program Mol Biol, New York, NY 10021 USA.
ABSTRACT: The accurate segregation of chromosomes during meiosis-which is critical for genome stability across sexual cycles-relies on homologous recombination initiated by DNA double-strand breaks (DSBs) made by the Spo11 protein(1,2). The formation of DSBs is regulated and tied to the elaboration of large-scale chromosome structures(3-5), but the protein assemblies that execute and control DNA breakage are poorly understood. Here we address this through the molecular characterization of Saccharomyces cerevisiae RMM (Rec114, Mei4 and Mer2) proteins-essential, conserved components of the DSB machinery(2).
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The cryo-EM structure of the human neurofibromin dimer reveals the molecular basis for neurofibromatosis type 1
Nature Structural & Molecular Biology. 2021. Lupton, CJ et al. Monash Univ, Biomed Discovery Inst, Clayton, Vic, Australia.
ABSTRACT: Neurofibromin (NF1) mutations cause neurofibromatosis type 1 and drive numerous cancers, including breast and brain tumors. NF1 inhibits cellular proliferation through its guanosine triphosphatase-activating protein (GAP) activity against rat sarcoma (RAS). In the present study, cryo-electron microscope studies reveal that the human similar to 640-kDa NF1 homodimer features a gigantic 30 x 10 nm array of alpha-helices that form a core lemniscate-shaped scaffold. Three-dimensional variability analysis captured the catalytic GAP-related domain and lipid-binding SEC-PH domains positioned against the core scaffold in a closed, autoinhibited conformation.
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Mapping protein interactions in the active TOM-TIM23 supercomplex
Nature Communications. 2021. Gomkale, R et al. Univ Med Ctr Gottingen, Dept Cellular Biochem, Gottingen, Germany.
ABSTRACT: Nuclear-encoded mitochondrial proteins destined for the matrix have to be transported across two membranes. The TOM and TIM23 complexes facilitate the transport of precursor proteins with N-terminal targeting signals into the matrix. During transport, precursors are recognized by the TIM23 complex in the inner membrane for handover from the TOM complex. However, we have little knowledge on the organization of the TOM-TIM23 transition zone and on how precursor transfer between the translocases occurs.
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Structural and functional characterization of the Spo11 core complex
nature structural & molecular biology. 2021. Bouuaert, CC et al. Mem Sloan Kettering Canc Ctr, Mol Biol Program, 1275 York Ave, New York, NY 10021 USA.
ABSTRACT: Spo11, which makes DNA double-strand breaks (DSBs) that are essential for meiotic recombination, has long been recalcitrant to biochemical study. We provide molecular analysis of Saccharomycescerevisiae Spo11 purified with partners Rec102, Rec104 and Ski8. Rec102 and Rec104 jointly resemble the B subunit of archaeal topoisomerase VI, with Rec104 occupying a position similar to the Top6B GHKL-type ATPase domain. Unexpectedly, the Spo11 complex is monomeric (1:1:1:1 stoichiometry), consistent with dimerization controlling DSB formation.
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Structural insights into how Prp5 proofreads the pre-mRNA branch site
Nature. 2021. Zhang, ZW et al. MPI Biophys Chem, Dept Struct Dynam, Gottingen, Germany.
ABSTRACT: During the splicing of introns from precursor messenger RNAs (pre-mRNAs), the U2 small nuclear ribonucleoprotein (snRNP) must undergo stable integration into the spliceosomal A complex-a poorly understood, multistep process that is facilitated by the DEAD-box helicase Prp5 (refs. (1-4)). During this process, the U2 small nuclear RNA (snRNA) forms an RNA duplex with the pre-mRNA branch site (the U2-BS helix), which is proofread by Prp5 at this stage through an unclear mechanism(5). Here, by deleting the branch-site adenosine (BS-A) or mutating the branch-site sequence of an actin pre-mRNA, we stall the assembly of spliceosomes in extracts from the yeast Saccharomyces cerevisiae directly before the A complex is formed.
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Accurate and automated high-coverage identification of chemically cross-linked peptides with MaxLynx
Analytical chemistry. 2021. Yilmaz, S et al. Max Planck Inst Biochem, Computat Syst Biochem Res Grp, D-82152 Martinsried, Germany; Univ Bergen, Dept Biol & Med Psychol, N-5007 Bergen, Norway
ABSTRACT: Cross-linking combined with mass spectrometry (XL-MS) provides a wealth of information about the three-dimensional (3D) structure of proteins and their interactions. We introduce MaxLynx, a novel computational proteomics workflow for XL-MS integrated into the MaxQuant environment. It is applicable to noncleavable and MS-cleavable cross-linkers. For both, we have generalized the Andromeda peptide database search engine to efficiently identify cross-linked peptides. For noncleavable peptides, we implemented a novel dipeptide Andromeda score, which is the basis for a computationally efficient N-squared search engine.
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The Cdk8 kinase module regulates interaction of the mediator complex with RNA polymerase II
Journal of Biological Chemistry. 2021. Osman, S et al. Max Planck Inst Biophys Chem, Dept Mol Biol, Gottingen, Germany.
ABSTRACT: The Cdk8 kinase module (CKM) is a dissociable part of the coactivator complex mediator, which regulates gene transcription by RNA polymerase II. The CKM has both negative and positive functions in gene transcription that remain poorly understood at the mechanistic level. In order to reconstitute the role of the CKM in transcription initiation, we prepared recombinant CKM from the yeast Saccharomyces cerevisiae. We showed that CKM bound to the core mediator (cMed) complex, sterically inhibiting cMed from binding to the polymerase II preinitiation complex (PIC) in vitro.
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Cryo-EM of mammalian PA28αβ-iCP immunoproteasome reveals a distinct mechanism of proteasome activation by PA28αβ
Nature communications. 2021. Chen, JH et al. Chinese Acad Sci, Ctr Excellence Mol Cell Sci, Shanghai Inst Biochem & Cell Biol, State Key Lab Mol Biol Natl Ctr Prot Sci,Shanghai, Shanghai 200031, Peoples R China
ABSTRACT: The proteasome activator PA28 alpha beta affects MHC class I antigen presentation by associating with immunoproteasome core particles (iCPs). However, due to the lack of a mammalian PA28 alpha beta -iCP structure, how PA28 alpha beta regulates proteasome remains elusive. Here we present the complete architectures of the mammalian PA28 alpha beta -iCP immunoproteasome and free iCP at near atomic-resolution by cryo-EM, and determine the spatial arrangement between PA28 alpha beta and iCP through XL-MS.
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Molecular basis of F-actin regulation and sarcomere assembly via myotilin
PLoS biology. 2021. Kostan, J et al. Univ Vienna, Dept Struct & Computat Biol, Max Perutz Labs, Vienna, Austria.
ABSTRACT: Sarcomeres, the basic contractile units of striated muscle cells, contain arrays of thin (actin) and thick (myosin) filaments that slide past each other during contraction. The Ig-like domain-containing protein myotilin provides structural integrity to Z-discs-the boundaries between adjacent sarcomeres. Myotilin binds to Z-disc components, including F-actin and alpha-actinin-2, but the molecular mechanism of binding and implications of these interactions on Z-disc integrity are still elusive. To illuminate them, we used a combination of small-angle X-ray scattering, cross-linking mass spectrometry, and biochemical and molecular biophysics approaches.
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Cross‐linking mass spectrometry reveals the structural topology of peripheral NuRD subunits relative to the core complex
The Federation of European Biochemical Societies Journal. 2021. Spruijt, CG et al. Radboud Univ Nijmegen, Oncode Inst, Radboud Inst Mol Life Sci, Fac Sci,Dept Mol Biol, NL-6525 GA Nijmegen, Netherlands.
ABSTRACT: The multi-subunit nucleosome remodeling and deacetylase (NuRD) complex consists of seven subunits, each of which comprises two or three paralogs in vertebrates. These paralogs define mutually exclusive and functionally distinct complexes. In addition, several proteins in the complex are multimeric, which complicates structural studies. Attempts to purify sufficient amounts of endogenous complex or recombinantly reconstitute the complex for structural studies have proven quite challenging. Until now, only substructures of individual domains or proteins and low-resolution densities of (partial) complexes have been reported.
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Identification of protein direct interactome with genetic code expansion and search engine OpenUaa
Advanced Biology. 2021. Liu, C et al. Beihang Univ, Sch Med & Engn, Beijing Adv Innovat Ctr Big Data Based Precis Med, Beijing 100191, Peoples R China; Beihang Univ, Key Lab Big Data Based Precis Med, Minist Ind & Informat Technol, Beijing 100191, Peoples R China; Zhejiang Univ, MOE Lab Biosyst Homeostasis & Protect, Hangzhou 310058, Peoples R China; Zhejiang Univ, Life Sci Inst, Hangzhou 310058, Peoples R China; Tsinghua Univ, Tsinghua Inst Multidisciplinary Biomed Res, Beijing 102206, Peoples R China; Natl Inst Biol Sci NIBS, Beijing 102206, Peoples R China; Univ Calif San Francisco, Dept Pharmaceut Chem, San Francisco, CA 94158 USA; Univ Calif San Francisco, Cardiovasc Res Inst, San Francisco, CA 94158 USA; Chinese Acad Agr Sci, Inst Anim Sci, Beijing 100193, Peoples R China
ABSTRACT: Protein crosslinks occur endogenously such as modifications by ubiquitin-like proteins for signaling, or exogenously through genetically encoded chemical crosslinkers (GECX) for studying elusive protein-protein interactions. However, it remains challenging to identify these protein crosslinks efficiently at the proteomic scale. Herein, software OpenUaa is developed for identifying protein crosslinks generated by genetically encoded unnatural amino acids and endogenous protein conjugation. OpenUaa features inclusive and open search capability, dramatically improving identification sensitivity and coverage.
[more...]
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Membrane interactions of α-Synuclein revealed by multiscale molecular dynamics simulations, Markov state models, and NMR
JOURNAL OF PHYSICAL CHEMISTRY B. 2021. Amos, SBTA et al. Univ Oxford, Dept Biochem, Oxford OX1 3QU, England.
ABSTRACT: alpha-Synuclein (alpha S) is a presynaptic protein that binds to cell membranes and is linked to Parkinson's disease (PD). Binding of alpha S to membranes is a likely first step in the molecular pathophysiology of PD. The alpha S molecule can adopt multiple conformations, being largely disordered in water, adopting a beta-sheet conformation when present in amyloid fibrils, and forming a dynamic multiplicity of alpha-helical conformations when bound to lipid bilayers and related membrane-mimetic surfaces.
[more...]
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Elucidating the molecular mechanism of dynamic photodamage of photosystem II membrane protein complex by integrated proteomics strategy
CCS Chemistry. 2021. Zhou, Y et al. CAS Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023
ABSTRACT: Photosystem II (PSII), as a multiple-subunit chloroplast membrane-associated pigment-protein complex on the thylakoid membrane, is a primary target of light-induced photodamage. However, the overall molecular details of the conformation and composition dynamics of PSII photodamage are still controversial. In this study, we investigated systematically the dynamic conformation, degradation, and oxidation processes of PSII photodamage by integrating chemical cross-linking and top-down proteomics strategies.
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Colicin-mediated transport of DNA through the iron transporter FepA
MBio. 2021. Cohen-Khait, R et al. Univ Oxford, Dept Biochem, Oxford, England
ABSTRACT: Colicins are protein antibiotics deployed by Escherichia coli to eliminate competing strains. Colicins frequently exploit outer membrane (OM) nutrient transporters to penetrate the selectively permeable bacterial cell envelope. Here, by applying live-cell fluorescence imaging, we were able to monitor the entry of the pore-forming toxin colicin B (ColB) into E. coli and localize it within the periplasm. We further demonstrate that single-stranded DNA coupled to ColB can also be transported to the periplasm, emphasizing that the import routes of colicins can be exploited to carry large cargo molecules into bacteria.
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TAF8 regions important for TFIID lobe B assembly or for TAF2 interactions are required for embryonic stem cell survival
The Journal of biological chemistry. 2021. Scheer, Elisabeth et al. Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Centre National de la Recherche Scientifique, UMR7104, Institut National de la Santé et de la Recherche Médicale, U964, Université de Strasbourg, Illkirch, France
ABSTRACT: The human general transcription factor TFIID is composed of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs). In eukaryotic cells, TFIID is thought to nucleate RNA polymerase II (Pol II) preinitiation complex formation on all protein coding gene promoters and thus, be crucial for Pol II transcription. TFIID is composed of three lobes, named A, B, and C. A 5TAF core complex can be assembled invitro constituting a building block for the further assembly of either lobe A or B in TFIID.
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Distinct architecture and composition of mouse axonemal radial spoke head revealed by cryo-EM
PNAS. 2021. Zheng, W et al. Chinese Acad Sci, Ctr Excellence Mol Cell Sci, Shanghai Inst Biochem & Cell Biol, State Key Lab Mol Biol,Natl Ctr Prot Sci Shanghai, Shanghai 200031, Peoples R China.
ABSTRACT: The radial spoke (RS) heads of motile cilia and flagella contact projections of the central pair (CP) apparatus to coordinate motility, but the morphology is distinct for protozoa and metazoa. Here we show the murine RS head is compositionally distinct from that of Chlamydomonas. Our reconstituted murine RS head core complex consists of Rsph1, Rsph3b, Rsph4a, and Rsph9, lacking Rsph6a and Rsph10b, whose orthologs exist in the protozoan RS head. We resolve its cryo-electron microscopy (cryo-EM) structure at 3.2-angstrom resolution.
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Structural analysis of the PTEN: P-Rex2 signaling complex reveals how cancer-associated mutations coordinate to hyperactivate Rac1
Science Signaling. 2021. D'Andrea, L et al. Monash Univ, Biomed Discovery Inst, Clayton, Vic 3800, Australia; Monash Univ, Monash Inst Pharmaceut Sci, Drug Discovery Biol Theme, Parkville, Vic 3052, Australia
ABSTRACT: The dual-specificity phosphatase PTEN functions as a tumor suppressor by hydrolyzing PI(3,4,5)P-3 to PI(4,5)P-2 to inhibit PI3K-AKT signaling and cellular proliferation. P-Rex2 is a guanine nucleotide exchange factor for Rho GTPases and can be activated by G beta gamma subunits downstream of G protein-coupled receptor signaling and by PI(3,4,5)P-3 downstream of receptor tyrosine kinases. The PTEN:P-Rex2 complex is a commonly mutated signaling node in metastatic cancer. Assembly of the PTEN:P-Rex2 complex inhibits the activity of both proteins, and its dysregulation can drive PI3K-AKT signaling and cellular proliferation.
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Solution structure and conformational flexibility of a polyketide synthase module
JACS. 2021. Klaus, M et al. Institute of Organic Chemistry and Chemical Biology, Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, Max-von-Laue Strasse 15, Frankfurt am Main 60438, Germany
ABSTRACT: Polyketide synthases (PKSs) are versatile C–C bond-forming enzymes that are broadly distributed in bacteria and fungi. The polyketide compound family includes many clinically useful drugs such as the antibiotic erythromycin, the antineoplastic epothilone, and the cholesterol-lowering lovastatin. Harnessing PKSs for custom compound synthesis remains an open challenge, largely because of the lack of knowledge about key structural properties. Particularly, the domains—well characterized on their own—are poorly understood in their arrangement, conformational dynamics, and interplay in the intricate quaternary structure of modular PKSs.
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NeissLock provides an inducible protein anhydride for covalent targeting of endogenous proteins
Nature communications. 2021. Scheu, AHA et al. Univ Oxford, Dept Biochem, South Parks Rd, Oxford OX1 3QU, England.
ABSTRACT: The Neisseria meningitidis protein FrpC contains a self-processing module (SPM) undergoing autoproteolysis via an aspartic anhydride. Herein, we establish NeissLock, using a binding protein genetically fused to SPM. Upon calcium triggering of SPM, the anhydride at the C-terminus of the binding protein allows nucleophilic attack by its target protein, ligating the complex. We establish a computational tool to search the Protein Data Bank, assessing proximity of amines to C-termini. We optimize NeissLock using the Ornithine Decarboxylase/Antizyme complex.
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Carboxypeptidase Y Assisted Disulfide-Bond Identification with Linearized Database Search
Analytical Chemistry. 2021. Qiang, Jiali et al. Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 100 Haike Road, Pudong, Shanghai 201210, China
ABSTRACT: A disulfide bond is an important protein post-translational modification and plays a key role in regulating protein oxidation status, protein structure, and stability. Analysis of a disulfide bond using mass spectrometry is challenging because there lacks an efficient method to separate the disulfide-linked peptides from a complex protein digest, and the MS data requires sophisticated interpretation. Here, we developed a novel disulfide bond identification strategy, termed as "carboxypeptidase Y assisted disulfide-bond identification (CADI)".
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SAGA and SAGA-like SLIK transcriptional coactivators are structurally and biochemically equivalent
Journal of Biological Chemistry. 2021. Adamus, K et al. Monash Univ, Dept Biochem & Mol Biol, Biomed Discovery Inst, Clayton, Vic, Australia.
ABSTRACT: The SAGA-like complex SLIK is a modified version of the Spt-Ada-Gcn5-Acetyltransferase (SAGA) complex. SLIK is formed through C-terminal truncation of the Spt7 SAGAsubunit, causing loss of Spt8, one of the subunits that interacts with the TATA-binding protein (TBP). SLIK and SAGA are both coactivators of RNA polymerase II transcription in yeast, and both SAGA and SLIK perform chromatin modifications. The two complexes have been speculated to uniquely contribute to transcriptional regulation, but their respective contributions are not clear.
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Characterization of the Fc–III–4C-based recombinant protein expression system by using carbonic anhydrase as the model protein
Protein Expression and Purification. 2021. Gong, YY et al. Tsinghua Univ, Ctr Synthet & Systemat Biol, Sch Life Sci, MOE Key Lab Bioinformat, Beijing 100084, Peoples R China.
ABSTRACT: Development of new affinity tags is important for recombinant protein expression and purification. Based on our earlier work, we devised an affinity tag by addition of two cysteine residues onto the N- and C-termini of the Fc-III peptide and designated as the Fc-III-4C tag, in which four cysteine residues form two disulfide linkages. The binding affinity of Fc-III-4C tag to human IgG is measured as 2.28 nM (K-d) and is 100 times higher than that of the Fc-III tag to IgG. Fc-III-4C tagged carbonic anhydrase (CA) can be effectively purified with IgG-immobilized beads, and Fc-III-4C tag does not possess adverse effects on the structure and stability of CA.
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Elucidating the Molecular Mechanism of Dynamic Photodamage of Photosystem II Membrane Protein Complex by Integrated Proteomics Strategy
CCS Chemistry. 2021. Zhou, Y et al. CAS Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023
ABSTRACT: Photosystem II (PSII), as a multiple-subunit chloroplast membrane-associated pigment-protein complex on the thylakoid membrane, is a primary target of light-induced photodamage. However, the overall molecular details of the conformation and composition dynamics of PSII photodamage are still controversial. In this study, we investigated systematically the dynamic conformation, degradation, and oxidation processes of PSII photodamage by integrating chemical cross-linking and top-down proteomics strategies.
[more...]
Use: pLink



Cryo-EM of mammalian PA28$\alpha$$\beta$-iCP immunoproteasome reveals a distinct mechanism of proteasome activation by PA28$\alpha$$\beta$
Nature communications. 2021. Chen, JH et al. Chinese Acad Sci, Ctr Excellence Mol Cell Sci, Shanghai Inst Biochem & Cell Biol, State Key Lab Mol Biol Natl Ctr Prot Sci,Shanghai, Shanghai 200031, Peoples R China
ABSTRACT: The proteasome activator PA28 alpha beta affects MHC class I antigen presentation by associating with immunoproteasome core particles (iCPs). However, due to the lack of a mammalian PA28 alpha beta -iCP structure, how PA28 alpha beta regulates proteasome remains elusive. Here we present the complete architectures of the mammalian PA28 alpha beta -iCP immunoproteasome and free iCP at near atomic-resolution by cryo-EM, and determine the spatial arrangement between PA28 alpha beta and iCP through XL-MS.
[more...]
Use: pLink



Membrane Interactions of α-Synuclein Revealed by Multiscale Molecular Dynamics Simulations, Markov State Models, and NMR
JOURNAL OF PHYSICAL CHEMISTRY B. 2021. Amos, SBTA et al. Univ Oxford, Dept Biochem, Oxford OX1 3QU, England.
ABSTRACT: alpha-Synuclein (alpha S) is a presynaptic protein that binds to cell membranes and is linked to Parkinson's disease (PD). Binding of alpha S to membranes is a likely first step in the molecular pathophysiology of PD. The alpha S molecule can adopt multiple conformations, being largely disordered in water, adopting a beta-sheet conformation when present in amyloid fibrils, and forming a dynamic multiplicity of alpha-helical conformations when bound to lipid bilayers and related membrane-mimetic surfaces.
[more...]
Use: pLink; pParse



Identification of Protein Direct Interactome with Genetic Code Expansion and Search Engine OpenUaa
Advanced Biology. 2021. Liu, C et al. Beihang Univ, Sch Med & Engn, Beijing Adv Innovat Ctr Big Data Based Precis Med, Beijing 100191, Peoples R China; Beihang Univ, Key Lab Big Data Based Precis Med, Minist Ind & Informat Technol, Beijing 100191, Peoples R China; Zhejiang Univ, MOE Lab Biosyst Homeostasis & Protect, Hangzhou 310058, Peoples R China; Zhejiang Univ, Life Sci Inst, Hangzhou 310058, Peoples R China; Tsinghua Univ, Tsinghua Inst Multidisciplinary Biomed Res, Beijing 102206, Peoples R China; Natl Inst Biol Sci NIBS, Beijing 102206, Peoples R China; Univ Calif San Francisco, Dept Pharmaceut Chem, San Francisco, CA 94158 USA; Univ Calif San Francisco, Cardiovasc Res Inst, San Francisco, CA 94158 USA; Chinese Acad Agr Sci, Inst Anim Sci, Beijing 100193, Peoples R China
ABSTRACT: Protein crosslinks occur endogenously such as modifications by ubiquitin-like proteins for signaling, or exogenously through genetically encoded chemical crosslinkers (GECX) for studying elusive protein-protein interactions. However, it remains challenging to identify these protein crosslinks efficiently at the proteomic scale. Herein, software OpenUaa is developed for identifying protein crosslinks generated by genetically encoded unnatural amino acids and endogenous protein conjugation. OpenUaa features inclusive and open search capability, dramatically improving identification sensitivity and coverage.
[more...]
Use: pLink



Solution Structure and Conformational Flexibility of a Polyketide Synthase Module
JACS. 2021. Klaus, M et al. Institute of Organic Chemistry and Chemical Biology, Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, Max-von-Laue Strasse 15, Frankfurt am Main 60438, Germany
ABSTRACT: Polyketide synthases (PKSs) are versatile C–C bond-forming enzymes that are broadly distributed in bacteria and fungi. The polyketide compound family includes many clinically useful drugs such as the antibiotic erythromycin, the antineoplastic epothilone, and the cholesterol-lowering lovastatin. Harnessing PKSs for custom compound synthesis remains an open challenge, largely because of the lack of knowledge about key structural properties. Particularly, the domains—well characterized on their own—are poorly understood in their arrangement, conformational dynamics, and interplay in the intricate quaternary structure of modular PKSs.
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Use: pLink



Covalently Engineered Nanobody Chimeras for Targeted Membrane Protein Degradation
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY. 2021. Zhang, H et al. Peking Univ, Synthet & Funct Biomol Ctr, Beijing Natl Lab Mol Sci, Coll Chem & Mol Engn, Beijing 100871, Peoples R China.
ABSTRACT: The targeted degradation of membrane proteins would afford an attractive and general strategy for treating various diseases that remain difficult with the current proteolysis-targeting chimera (PROTAC) methodology. We herein report a covalent nanobody-based PROTAC strategy, termed GlueTAC, for targeted membrane protein degradation with high specificity and efficiency. We first established a mass-spectrometry-based screening platform for the rapid development of a covalent nanobody (GlueBody) that allowed proximity-enabled cross-linking with surface antigens on cancer cells.
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Cysteine inducing formation and reshuffling of disulfide bonds in cold-extruded whey protein molecules: From structural and functional characteristics to cytotoxicity
Food Chemistry. 2021. Yang, N et al. Northeast Agr Univ, Coll Food Sci & Technol, Key Lab Dairy Sci, Minist Educ, Harbin 150030, Heilongjiang, Peoples R China.
ABSTRACT: Polymer chemistry, rheology and cytotoxicity of cysteine initiated S-S redistribution in cold-extruded whey protein (TWPI) molecules were investigated. The locations of disulfide bonds in whey protein isolate (WPI), WPI dried without being extruded (OWPI) and cold-extruded WPI (TWPI), Cysteine (Cys)-treated WPI (WPI-Cys), OWPI (OWPI-Cys) and TWPI (TWPI-Cys) were precisely analyzed using liquid chromatography electrospray ionization tandem mass spectrometry (LC/MS/MS) combined with pLink software approaches.
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MS Annika: A New Cross-Linking Search Engine
Journal of Proteome Research. 2021. Pirklbauer, GJ et al. Univ Appl Sci Upper Austria, Bioinformat Res Grp, A-4232 Hagenberg, Austria.
ABSTRACT: Cross-linking mass spectrometry (XL-MS) has become a powerful technique that enables insights into protein structures and protein interactions. The development of cleavable cross-linkers has further promoted XL-MS through search space reduction, thereby allowing for proteome-wide studies. These new analysis possibilities foster the development of new cross-linkers, which not every search engine can deal with out of the box. In addition, some search engines for XL-MS data also struggle with the validation of identified cross-linked peptides, that is, false discovery rate (FDR) estimation, as FDR calculation is hampered by the fact that not only one but two peptides in a single spectrum have to be correct.
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Structure of cyanobacterial phycobilisome core revealed by structural modeling and chemical cross-linking
Science Advances. 2021. Liu, HJ et al. Washington Univ, Dept Chem, St Louis, MO 63130 USA.
ABSTRACT: In cyanobacteria and red algae, the structural basis dictating efficient excitation energy transfer from the phycobilisome (PBS) antenna complex to the reaction centers remains unclear. The PBS has several peripheral rods and a central core that binds to the thylakoid membrane, allowing energy coupling with photosystem II (PSII) and PSI. Here, we have combined chemical cross-linking mass spectrometry with homology modeling to propose a tricylindrical cyanobacterial PBS core structure. Our model reveals a side-view crossover configuration of the two basal cylinders, consolidating the essential roles of the anchoring domains composed of the ApcE PB loop and ApcD, which facilitate the energy transfer to PSII and PSI, respectively.
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Clostridium perfringens suppressing activity in black soldier fly protein preparations
LWT - Food Science and Technology. 2021. Dong, LY et al. Wageningen Res, Food & Biobased Res, Bornse Weilanden 9, NL-6708 WG Wageningen, Netherlands.
ABSTRACT: Clostridium perfringens is a commensal, but also an opportunistic pathogen that can lead to lethal diseases as a result of overgrowth when homeostasis is disrupted. The current course of treatment is antibiotics. However, with increasing antibiotic resistance alternatives are required. We investigated the antimicrobial capacity of digest from different black soldier fly- and mealworm-derived fractions towards C. perfringens by using in vitro models. Culturing C. perfringens with digest of insect-derived fractions showed that fractions containing black soldier fly larvae protein significantly (p < 0.05) inhibited the growth of C.
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Use: pNovo



Peptide presentations of marsupial MHC class I visualize immune features of lower mammals paralleled with bats
JOURNAL OF IMMUNOLOGY. 2021. Wang, PY et al. Wenzhou Med Univ, Sch Lab Med & Life Sci, Wenzhou, Peoples R China; Zhejiang Univ, Affiliated Hosp 1, Sch Med, State Key Lab Diag & Treatment Infect Dis, Hangzhou, Zhejiang, Peoples R China; Chinese Ctr Dis Control & Prevent, Natl Inst Viral Dis Control & Prevent, 155 Changbai Rd, Beijing 100101, Peoples R China
ABSTRACT: Marsupials are one of three major mammalian lineages that include the placental eutherians and the egg-laying monotremes. The marsupial brushtail possum is an important protected species in the Australian forest ecosystem. Molecules encoded by the MHC genes are essential mediators of adaptive immune responses in virus -host interactions. Yet, nothing is known about the peptide presentation features of any marsupial MHC class I (MHC I). This study identified a series of possum MHC I Trvu-UB*01:01 binding peptides derived from wobbly possum disease virus (WPDV), a lethal virus of both captive and feral possum populations, and unveiled the structure of marsupial peptide/MHC I complex.
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Use: pNovo



Improved Identification of Small Open Reading Frames Encoded Peptides by Top-Down Proteomic Approaches and De Novo Sequencing
International journal of molecular sciences. 2021. Wang, B et al. Cent China Normal Univ, Sch Life Sci, Hubei Key Lab Genet Regulat & Integrat Biol, 152 Luoyu Rd, Wuhan 430079, Peoples R China.
ABSTRACT: Small open reading frames (sORFs) have translational potential to produce peptides that play essential roles in various biological processes. Nevertheless, many sORF-encoded peptides (SEPs) are still on the prediction level. Here, we construct a strategy to analyze SEPs by combining top-down and de novo sequencing to improve SEP identification and sequence coverage. With de novo sequencing, we identified 1682 peptides mapping to 2544 human sORFs, which were all first characterized in this work. Two-thirds of these new sORFs have reading frame shifts and use a non-ATG start codon.
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Mapping Microproteins and ncRNA-Encoded Polypeptides in Different Mouse Tissues
Frontiers in cell and developmental biology. 2021. Pan, N et al. Cent China Normal Univ, Sch Life Sci, Hubei Key Lab Genet Regulat & Integrat Biol, Wuhan, Peoples R China.
ABSTRACT: Small open reading frame encoded peptides (SEPs), also called microproteins, play a vital role in biological processes. Plenty of their open reading frames are located within the non-coding RNA (ncRNA) range. Recent research has demonstrated that ncRNA-encoded polypeptides have essential functions and exist ubiquitously in various tissues. To better understand the role of microproteins, especially ncRNA-encoded proteins, expressed in different tissues, we profiled the proteomic characterization of five mouse tissues by mass spectrometry, including bottom-up, top-down, and de novo sequencing strategies.
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Use: pNovo



Peptide Presentations of Marsupial MHC Class I Visualize Immune Features of Lower Mammals Paralleled with Bats
JOURNAL OF IMMUNOLOGY. 2021. Wang, PY et al. Wenzhou Med Univ, Sch Lab Med & Life Sci, Wenzhou, Peoples R China; Zhejiang Univ, Affiliated Hosp 1, Sch Med, State Key Lab Diag & Treatment Infect Dis, Hangzhou, Zhejiang, Peoples R China; Chinese Ctr Dis Control & Prevent, Natl Inst Viral Dis Control & Prevent, 155 Changbai Rd, Beijing 100101, Peoples R China
ABSTRACT: Marsupials are one of three major mammalian lineages that include the placental eutherians and the egg-laying monotremes. The marsupial brushtail possum is an important protected species in the Australian forest ecosystem. Molecules encoded by the MHC genes are essential mediators of adaptive immune responses in virus -host interactions. Yet, nothing is known about the peptide presentation features of any marsupial MHC class I (MHC I). This study identified a series of possum MHC I Trvu-UB*01:01 binding peptides derived from wobbly possum disease virus (WPDV), a lethal virus of both captive and feral possum populations, and unveiled the structure of marsupial peptide/MHC I complex.
[more...]
Use: pNovo



Computationally instrument-resolution-independent de novo peptide sequencing for high-resolution devices
Nature Machine Intelligence. 2021. Qiao, R et al. Univ Waterloo, Dept Stat & Actuarial Sci, Waterloo, ON, Canada.
ABSTRACT: De novo peptide sequencing is the key technology for finding novel peptides from mass spectra. The overall quality of sequencing results depends on the de novo peptide sequencing algorithm as well as the quality of mass spectra. Over the past decade, the resolution and accuracy of mass spectrometers have improved by orders of magnitude and higher-resolution mass spectra have been generated. How to effectively take advantage of those high-resolution data without substantially increasing the computational complexity remains a challenge for de novo peptide sequencing tools.
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Use: pNovo



Reaction Tracking and High-Throughput Screening of Active Compounds in Combinatorial Chemistry by Tandem Mass Spectrometry Molecular Networking
Analytical chemistry. 2021. Chung, HH et al. Natl Taiwan Univ, Dept Chem, Taipei 10617, Taiwan.
ABSTRACT: Combinatorial synthesis has been widely used as an efficient strategy to screen for active compounds. Mass spectrometry is the method of choice in the identification of hits resulting from high-throughput screenings due to its high sensitivity, specificity, and speed. However, manual data processing of mass spectrometry data, especially for structurally diverse products in combinatorial chemistry, is extremely time-consuming and one of the bottlenecks in this process. In this study, we demonstrated the effectiveness of a tandem mass spectrometry molecular networking-based strategy for product identification, reaction dynamics monitoring, and active compound targeting in combinatorial synthesis.
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Use: pNovo



StrucGP: de novo structural sequencing of site-specific N-glycan on glycoproteins using a modularization strategy
Nature Methods. 2021. Shen, JC et al. Northwest Univ, Coll Life Sci, Xian, Peoples R China.
ABSTRACT: Precision mapping of glycans at structural and site-specific level is still one of the most challenging tasks in the glycobiology field. Here, we describe a modularization strategy for de novo interpretation of N-glycan structures on intact glycopeptides using tandem mass spectrometry. An algorithm named StrucGP is also developed to automate the interpretation process for large-scale analysis. By dividing an N-glycan into three modules and identifying each module using distinct patterns of Y ions or a combination of distinguishable B/Y ions, the method enables determination of detailed glycan structures on thousands of glycosites in mouse brain, which comprise four types of core structure and 17 branch structures with three glycan subtypes.
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Use: pGlyco



In-depth site-specific analysis of N-glycoproteome in human cerebrospinal fluid and glycosylation landscape changes in Alzheimer's disease
Molecular & Cellular Proteomics. 2021. Chen, ZW et al. Univ Wisconsin, Dept Chem, 1101 Univ Ave, Madison, WI 53706 USA.
ABSTRACT: As the body fluid that directly interchanges with the extracellular fluid of the central nervous system (CNS), cerebrospinal fluid (CSF) serves as a rich source for CNS-related disease biomarker discovery. Extensive proteome profiling has been conducted for CSF, but studies aimed at unraveling site-specific CSF N-glycoproteome are lacking. Initial efforts into site-specific N-glycoproteomics study in CSF yield limited coverage, hindering further experimental design of glycosylation-based disease biomarker discovery in CSF.
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Use: pGlyco



Identification of 22 N-glycosites on spike glycoprotein of SARS-CoV-2 and accessible surface glycopeptide motifs: Implications for vaccination and antibody therapeutics
Glycobiology. 2021. Zhou, DP et al. Tongji Univ, Sch Med, 1239 Siping Rd, Shanghai 200092, Peoples R China.
ABSTRACT: Coronaviruses hijack human enzymes to assemble the sugar coat on their spike glycoproteins. The mechanisms by which human antibodies may recognize the antigenic viral peptide epitopes hidden by the sugar coat are unknown. Glycosylation by insect cells differs from the native form produced in human cells, but insect cell-derived influenza vaccines have been approved by the US Food and Drug Administration. In this study, we analyzed recombinant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein secreted from BTI-Tn-5B1-4 insect cells, by trypsin and chymotrypsin digestion followed by mass spectrometry analysis.
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Use: pGlyco



Fecal multi-omics analysis reveals diverse molecular alterations of gut ecosystem in COVID-19 patients
ANALYTICA CHIMICA ACTA. 2021. He, FX et al. Sun Yat Sen Univ, Guangdong Prov Key Lab Biomed Imaging, Affiliated Hosp 5, Zhuhai 519000, Peoples R China.
ABSTRACT: Gut ecosystem has profound effects on host physiology and health. Gastrointestinal (GI) symptoms were frequently observed in patients with COVID-19. Compared with other organs, gut antiviral response can result in more complicated immune responses because of the interactions between the gut microbiota and host immunity. However, there are still large knowledge gaps in the impact of COVID-19 on gut molecular profiles and commensal microbiome, hindering our comprehensive understanding of the pathogenesis of SARS-CoV-2 and the treatment of COVID-19.
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Use: pGlyco



Sorbitol is a severity biomarker for PMM2-CDG with therapeutic implications
Annals of Neurology. 2021. Ligezka, AN et al. Mayo Clin, Dept Clin Genom, 200 First St SW, Rochester, MN 55905 USA.
ABSTRACT: Objective Epalrestat, an aldose reductase inhibitor increases phosphomannomutase (PMM) enzyme activity in a PMM2-congenital disorders of glycosylation (CDG) worm model. Epalrestat also decreases sorbitol level in diabetic neuropathy. We evaluated the genetic, biochemical, and clinical characteristics, including the Nijmegen Progression CDG Rating Scale (NPCRS), urine polyol levels and fibroblast glycoproteomics in patients with PMM2-CDG. Methods We performed PMM enzyme measurements, multiplexed proteomics, and glycoproteomics in PMM2-deficient fibroblasts before and after epalrestat treatment.
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Use: pGlyco



Glycoproteomics analysis reveals differential expression of site-specific glycosylation in human milk whey during lactation
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY. 2021. Wang, ZY et al. Chinese Acad Sci, Dalian Inst Chem Phys, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Peoples R China.
ABSTRACT: Protein N-glycosylation in human milk whey plays a substantial role in infant health during postnatal development. Changes in site-specific glycans in milk whey reflect the needs of infants under different circumstances. However, the conventional glycoproteomics analysis of milk whey cannot reveal the changes in site-specific glycans because the attached glycans are typically enzymatically removed from the glycoproteins prior to analysis. In this study, N-glycoproteomics analysis of milk whey was performed without removing the attached glycans, and 330 and 327 intact glycopeptides were identified in colostrum and mature milk whey, respectively.
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Use: pQuant; pGlyco



Automated intact glycopeptide enrichment method facilitating highly reproducible analysis of serum site-specific N-glycoproteome
Analytical Chemistry. 2021. Liu, LY et al. Chinese Acad Sci, Dalian Inst Chem Phys, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Peoples R China.
ABSTRACT: Bottom-up proteomics has been increasingly applied in clinical research to study the disease pathophysiology and to discover disease biomarkers. However, glycoproteomic analysis always requires tedious experimental steps for intact glycopeptide enrichment, which has been the technique bottleneck for large-scale analysis of clinical samples. Herein, we developed an automated glycopeptide enrichment method for the analysis of serum site-specific N-glycoproteome. This automated method allowed for processing one sample within 20 min.