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

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Citations Last Updated: Dec. 16, 2022

2023





2022




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.
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Identification and mechanism of G protein-biased ligands for chemokine receptor CCR1
Nature chemical biology. 2022. Shao, ZH et al. Zhejiang Univ, MOE Frontier Sci Ctr Brain Res & Brain Machine In, Sch Med, Hangzhou, Peoples R China; Zhejiang Univ, Affiliated Hosp 2, Dept Pharmacol, Key Lab Resp Dis Zhejiang Prov,Sch Med, Hangzhou, Peoples R China; Zhejiang Univ, Dept Pathol, Sir Run Run Shaw Hosp, Sch Med, Hangzhou, Peoples R China; Zhejiang Univ, Affiliated Hosp 2, Dept Resp & Crit Care Med, Key Lab Resp Dis Zhejiang Prov,Sch Med, Hangzhou, Peoples R China; Zhejiang Prov Key Lab Immun & Inflammatory Dis, Hangzhou, Peoples R China; Zhejiang Univ, Key Lab Resp Dis Zhejiang Prov, Dept Resp & Crit Care Med, Affiliated Hosp 2,Sch Med, Hangzhou, Peoples R China; State Key Lab Resp Dis, Guangzhou, Peoples R China; Zhejiang Univ, Liangzhu Lab, Med Ctr, Hangzhou, Peoples R China; Zhejiang Univ, Int Inst Med, Sch Med, Affiliated Hosp 4, Yiwu, Peoples R China; Zhejiang Univ, Dept Biophys, Sir Run Run Shaw Hosp, Sch Med, Hangzhou, Peoples R China
ABSTRACT: Biased signaling of G protein-coupled receptors describes an ability of different ligands that preferentially activate an alternative downstream signaling pathway. In this work, we identified and characterized different N-terminal truncations of endogenous chemokine CCL15 as balanced or biased agonists targeting CCR1, and presented three cryogenic-electron microscopy structures of the CCR1-G(i) complex in the ligand-free form or bound to different CCL15 truncations with a resolution of 2.6-2.9 angstrom, illustrating the structural basis of natural biased signaling that initiates an inflammation response.
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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.
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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.
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Probing strigolactone perception mechanisms with rationally designed small-molecule agonists stimulating germination of root parasitic weeds
Nature communications. 2022. Wang, DW et al. Hunan Univ, State Key Lab Chemo Biosensing & Chemometr, Hunan Prov Key Lab Plant Funct Genom & Dev Regula, Coll Biol, Changsha 410082, Peoples R China; Nankai Univ, Collaborat Innovat Ctr Chem Sci & Engn, Natl Pesticide Engn Res Ctr, Coll Chem,Dept Chem Biol, Tianjin 300071, Peoples R China; Univ Amsterdam, Swammerdam Inst Life Sci SILS, Sci Pk 904, NL-1098 XH Amsterdam, Netherlands; Nankai Univ, State Key Lab Elementoorgan Chem, Collaborat Innovat Ctr Chem Sci & Engn, Coll Chem,Natl Pesticide Engn Res Ctr, Tianjin 300071, Peoples R China
ABSTRACT: The development of potent strigolactone (SL) agonists as suicidal germination inducers could be a useful strategy for controlling root parasitic weeds, but uncertainty about the SL perception mechanism impedes real progress. Here we describe small-molecule agonists that efficiently stimulate Phelipanchce aegyptiaca, and Striga hermonthica, germination in concentrations as low as 10(-8) to 10(-17) M. We show that full efficiency of synthetic SL agonists in triggering signaling through the Striga SL receptor, ShHTL7, depends on the receptor-catalyzed hydrolytic reaction of the agonists.
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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.
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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.
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Comprehensive identification of protein orthologs in the family Ascoviridae facilitates an understanding of phylogenomics, protein conservation, and phosphorylation
Archives of Virology. 2022. Shi, YH et al. Weifang Med Univ, Sch Life Sci & Technol, Weifang 261053, Peoples R China; Hunan Agr Univ, Hunan Prov Key Lab Biol & Control Plant Dis & Ins, Changsha 410128, Hunan, Peoples R China
ABSTRACT: Analysis of orthology is important for understanding protein conservation, function, and phylogenomics. In this study, we performed a comprehensive analysis of gene orthology in the family Ascoviridae based on identification of 366 protein homologue groups and phylogenetic analysis of 34 non-single-copy proteins. Our findings revealed 90 newly annotated proteins, five newly identified core proteins for the family Ascoviridae, and 14 core proteins for the genus Ascovirus. A phylogenomic tree of 11 Ascoviridae members was constructed based on a concatenation of 35 of the 45 ortholog groups.
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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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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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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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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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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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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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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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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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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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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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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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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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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.
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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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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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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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Shelterin is a Dimeric Complex with Extensive Structural Heterogeneity
PNAS. 2022. Zinder, JC 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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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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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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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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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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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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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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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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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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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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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.
[more...]
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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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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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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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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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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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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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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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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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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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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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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.
[more...]
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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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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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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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Neuronal Rna Granules are Ribosome Complexes Stalled at the Pre-Translocation State
JOURNAL OF MOLECULAR BIOLOGY. 2022. Kipper, K et al. Univ Tartu, Inst Technol, Struct Biol Unit, EE-50411 Tartu, Estonia
ABSTRACT: The polarized cell morphology of neurons dictates many neuronal processes, including the axodendridic transport of specific mRNAs and subsequent translation. mRNAs together with ribosomes and RNA -binding proteins form RNA granules that are targeted to axodendrites for localized translation in neurons. It has been established that localized protein synthesis in neurons is essential for long-term memory for-mation, synaptic plasticity, and neurodegeneration. We have used proteomics and electron microscopy to characterize neuronal RNA granules (nRNAg) isolated from rat brain tissues or human neuroblastoma.
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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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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.
[more...]
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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.
[more...]
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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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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.
[more...]
Use: pNovo



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.
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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.
[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.
[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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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.
[more...]
Use: pGlyco



Exploration of quantitative site-specific serum O-glycoproteomics with isobaric labelling for the discovery of putative O-glycoprotein biomarkers
PROTEOMICS--Clinical Applications. 2022. Zhang, ZH et al. Tongji Univ, Shanghai Key Lab Chem Assessment & Sustainabil, Sch Chem Sci & Engn, Shanghai 200092, Peoples R China; Shanghai Jiao Tong Univ, Sch Med, Shanghai Peoples Hosp 9, Dept Plast & Reconstruct Surg, Shanghai 200011, Peoples R China
ABSTRACT: Purpose Exploration study of site-specific isobaric-TMT-labeling quantitative serum O-glycoproteomics for the discovery of putative O-glycoprotein cancer biomarkers. Experimental design Sera of 10 breast cancer patients was used as the exploration cohort. More abundant N-glycosylation was first removed with PNGase F. After tryptic digestion of de-N-glycosylated serum proteome, the TMT-labeled O-glycopeptides mixture was prepared and analyzed with RPLC-MS/MS. Site-specific qualitative and quantitative database search of O-glycopeptides was carried out with pGlyco 3.0.
[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.
[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



Comprehensive site-and structure-specific characterization of N-glycosylation in model plant Arabidopsis using mass-spectrometry-based N-glycoproteomics
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES. 2022. Qin, SS et al. Tongji Univ, Sch Chem Sci & Engn, Shanghai Key Lab Chem Assessment & Sustainabil, Shanghai 200092, Peoples R China
ABSTRACT: The N-glycosylation is an important bioprocess in plant. Monosaccharide composition-level characterization at the intact N-glycopeptides has been extensively reported, yet structure-specific study to resolve multiple sequence structures of a single composition is still lacking. Here, we present a comprehensive structure-specific identification of intact N-glycopeptides of Arabidopsis with both HILIC and RAX enrichment, as well as GPSeeker and pGlyco database search. With target-decoy searches and spectrum-level FDR <=1 %, 5,687 N-glycopeptides from 3,713 N-glycosites of 3,140 N-glycoproteins were identified, which represents the currently most comprohensive profilling to our best knowledge.
[more...]
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.
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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.
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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.
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2021




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.
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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...]
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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.
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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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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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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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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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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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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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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.
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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.
[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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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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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...]
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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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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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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...]
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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.
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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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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).
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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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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.
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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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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.
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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.
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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.
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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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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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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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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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Mimicked synthetic ribosomal protein complex for benchmarking cross-linking mass spectrometry workflows
Nature communications. 2021. Matzinger, M et al. Vienna BioCtr VBC, Inst Mol Pathol IMP, Vienna, Austria; Austrian Acad Sci, Vienna BioCtr VBC, Inst Mol Biotechnol, 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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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.
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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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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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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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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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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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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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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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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.
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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.
[more...]
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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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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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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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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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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.
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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.
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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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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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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.
[more...]
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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 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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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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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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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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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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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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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 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).
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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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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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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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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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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. Gonzalez, LJ et al. Ctr Genet Engn & Biotechnol CIGB, Dept Prote, 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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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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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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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.
[more...]
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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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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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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 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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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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Ribosome-Amplified Metabolism, RAMBO, Measured by NMR Spectroscopy
Biochemistry . 2021. Yu, JC 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.
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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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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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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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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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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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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.
[more...]
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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.
[more...]
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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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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.
[more...]
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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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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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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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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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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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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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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.
[more...]
Use: pNovo



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.
[more...]
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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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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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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.
[more...]
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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.
[more...]
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Membrane Interactions of $\alpha$-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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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.
[more...]
Use: pGlyco



Precision N-Glycoproteomic Profiling of Murine Peritoneal Macrophages After Different Stimulations
Frontiers in Immunology. 2021. Yang, LJ et al. Fudan Univ, Inst Biomed Sci, Shanghai, Peoples R China.
ABSTRACT: Macrophages are important immune cells that participate in both innate and adaptive immune responses, such as phagocytosis, recognition of molecular patterns, and activation of the immune response. In this study, murine peritoneal macrophages were isolated and then activated by LPS, HSV and VSV. Integrative proteomic and precision N-glycoproteomic profiling were conducted to assess the underlying macrophage activation. We identified a total of 587 glycoproteins, including 1239 glycopeptides, 526 monosaccharide components, and 8326 intact glycopeptides in glycoproteomics, as well as a total of 4496 proteins identified in proteomic analysis.
[more...]
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.
[more...]
Use: pGlyco



Mass Spectrometry Analysis of SARS-CoV-2 Nucleocapsid Protein Reveals Camouflaging Glycans and Unique Post-Translational Modifications
Infectious Microbes & Diseases. 2021. Sun, ZY et al. State Key Laboratory for the Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, No.79 Qingchun Road, Shangcheng District, Hangzhou 310003, China
ABSTRACT: The devastating coronavirus disease 2019 (COVID-19) pandemic has prompted worldwide efforts to study structural biological traits of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its viral components. Compared to the Spike protein, which is the primary target for currently available vaccines or antibodies, knowledge about other virion structural components is incomplete. Using high-resolution mass spectrometry, we report a comprehensive post-translational modification (PTM) analysis of nucleocapsid phosphoprotein (NCP), the most abundant structural component of the SARS-CoV-2 virion.
[more...]
Use: pGlyco



Extensive heterogeneity of glycopeptides in plasma revealed by deep glycoproteomic analysis using size-exclusion chromatography
Molecular Omics. 2021. Saraswat, M et al. Mayo Clin, Dept Lab Med & Pathol, Rochester, MN 55905 USA.
ABSTRACT: Several plasma glycoproteins are clinically useful as biomarkers in a variety of diseases. Although thousands of proteins are present in plasma, >95% of the plasma proteome by mass is represented by only 22 proteins. This necessitates strategies to deplete the abundant proteins and enrich other subsets of proteins. Although glycoproteins are abundant in plasma, in routine proteomic analyses, glycopeptides are not often investigated. Traditional methods such as lectin-based enrichment of glycopeptides followed by deglycosylation have helped understand the glycoproteome, but they lack any information about the attached glycans.
[more...]
Use: pGlyco



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.
[more...]
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Quantification of intact O-glycopeptides on haptoglobin in sera of patients with hepatocellular carcinoma and liver cirrhosis
frontiers in Chemistry. 2021. Shu, H et al. Fudan Univ, Zhongshan Hosp, Liver Canc Inst, Shanghai, Peoples R China.
ABSTRACT: Haptoglobin (Hp) is one of the acute-phase response proteins secreted by the liver, and its aberrant N-glycosylation was previously reported in hepatocellular carcinoma (HCC). Limited studies on Hp O-glycosylation have been previously reported. In this study, we aimed to discover and confirm its O-glycosylation in HCC based on lectin binding and mass spectrometry (MS) detection. First, serum Hp was purified from patients with liver cirrhosis (LC) and HCC, respectively. Then, five lectins with Gal or GalNAc monosaccharide specificity were chosen to perform lectin blot, and the results showed that Hp in HCC bound to these lectins in a much stronger manner than that in LC.
[more...]
Use: pGlyco; pQuant



O-GlcNAcylation of MEK2 promotes the proliferation and migration of breast cancer cells
Glycobiology. 2021. Xu, YY et al. Fudan Univ, Shanghai Canc Ctr, Shanghai 200032, Peoples R China.
ABSTRACT: Mitogen-activated protein kinase kinases are an important part of evolutionary conserved signaling modules that are involved in a variety of cellular processes in response to environmental stimuli. Among them, mitogen-activated protein kinase kinase 2 (MEK2) is the most crucial upstream signaling pathway of ERK1/2 cascade as a therapeutic target for overcoming Ras-driven cancers. However, the mechanisms of MEK2 regulation during tumor progression remain not fully elucidated. Herein, we identified that MEK2 was post-translationally regulated by O-GlcNAcylation.
[more...]
Use: pGlyco



GproDIA enables data-independent acquisition glycoproteomics with comprehensive statistical control
Nature Communications. 2021. Yang, Y et al. Fudan Univ, Dept Chem, Shanghai 200000, Peoples R China.
ABSTRACT: Data independent acquisition (DIA) proteomics provides deep coverage and high quantitative accuracy, but is not yet well established in glycoproteomics. Here, the authors develop a DIA-based glycoproteomics workflow with stringent statistical controls to enable accurate glycopeptide identification. Large-scale profiling of intact glycopeptides is critical but challenging in glycoproteomics. Data independent acquisition (DIA) is an emerging technology with deep proteome coverage and accurate quantitative capability in proteomics studies, but is still in the early stage of development in the field of glycoproteomics.
[more...]
Use: pGlyco



The need for community standards to enable accurate comparison of glycoproteomics algorithm performance
molecules. 2021. Hackett, WE et al. Boston Univ, Bioinformat Program, Boston, MA 02215 USA.
ABSTRACT: Protein glycosylation that mediates interactions among viral proteins, host receptors, and immune molecules is an important consideration for predicting viral antigenicity. Viral spike proteins, the proteins responsible for host cell invasion, are especially important to be examined. However, there is a lack of consensus within the field of glycoproteomics regarding identification strategy and false discovery rate (FDR) calculation that impedes our examinations. As a case study in the overlap between software, here as a case study, we examine recently published SARS-CoV-2 glycoprotein datasets with four glycoproteomics identification software with their recommended protocols: GlycReSoft, Byonic, pGlyco2, and MSFragger-Glyco.
[more...]
Use: pGlyco



Dissection of the Glycosylation in the Biosynthesis of the Heptadecaglycoside Antibiotic Saccharomicin A
JOURNAL OF ORGANIC CHEMISTRY. 2021. Zhao, JF et al. Fudan Univ, Dept Chem, Shanghai 200433, Peoples R China.
ABSTRACT: Oligosaccharide natural products have diverse biological activities and represent a potentially important source for drug development. In this study, we focus on the glycosylation pathway in the biosynthesis of saccharomicin A (SA-A), an oligosaccharide antibiotic containing 17 sugar moieties. By extensive gene-knockout studies with comparative metabolic profile analysis, we established a complete pathway in assembling the heptadecasaccharide chain of SA-A, the longest saccharide chain found in natural products.
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.
[more...]
Use: pGlyco



Identification of Dysregulated Complement Activation Pathways Driven by N-Glycosylation Alterations in T2D Patients
frontiers in Chemistry. 2021. Zhao, Y et al. Natl Inst Metrol, Ctr Adv Measurement Sci, Beijing, Peoples R China.
ABSTRACT: Diabetes has become a major public health concern worldwide, most of which are type 2 diabetes (T2D). The diagnosis of T2D is commonly based on plasma glucose levels, and there are no reliable clinical biomarkers available for early detection. Recent advances in proteome technologies offer new opportunity for the understanding of T2D; however, the underlying proteomic characteristics of T2D have not been thoroughly investigated yet. Here, using proteomic and glycoproteomic profiling, we provided a comprehensive landscape of molecular alterations in the fasting plasma of the 24 Chinese participants, including eight T2D patients, eight prediabetic (PDB) subjects, and eight healthy control (HC) individuals.
[more...]
Use: pGlyco



Evaluation and Optimization of High-Field Asymmetric Waveform Ion-Mobility Spectrometry for Multiplexed Quantitative Site-Specific N-Glycoproteomics
Analytical Chemistry. 2021. Fang, P et al. Max Planck Inst Biophys Chem, Bioanalyt Mass Spectrometry Grp, D-37077 Gottingen, Germany.
ABSTRACT: The heterogeneity and complexity of glycosylation hinder the depth of site-specific glycoproteomics analysis. High-field asymmetric-waveform ion-mobility spectrometry (FAIMS) has been shown to improve the scope of bottom-up proteomics. The benefits of FAIMS for quantitative N-glycoproteomics have not been investigated yet. In this work, we optimized FAIMS settings for N-glycopeptide identification, with or without the tandem mass tag (TMT) label. The optimized FAIMS approach significantly increased the identification of site-specific N-glycopeptides derived from the purified immunoglobulin M (IgM) protein or human lymphoma cells.
[more...]
Use: pGlyco; pQuant



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.
[more...]
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.
[more...]
Use: pQuant; pGlyco



Analysis of Serum Paraoxonase 1 Using Mass Spectrometry and Lectin Immunoassay in Patients With Alpha-Fetoprotein Negative Hepatocellular Carcinoma
FRONTIERS IN ONCOLOGY. 2021. Cao, XY et al. Fudan Univ, Inst Biomed Sci, Shanghai, Peoples R China.
ABSTRACT: The diagnosis of AFP (alpha-fetoprotein)-negative HCC (hepatocellular carcinoma) mostly relies on imaging and pathological examinations, and it lacks valuable and practical markers. Protein N-glycosylation is a crucial post-translation modifying process related to many biological functions in an organism. Alteration of N-glycosylation correlates with inflammatory diseases and infectious diseases including hepatocellular carcinoma. Here, serum N-linked intact glycopeptides with molecular weight (MW) of 40-55 kDa were analyzed in a discovery set (n = 40) including AFP-negative HCC and liver cirrhosis (LC) patients using label-free quantification methodology.
[more...]
Use: pGlyco; pQuant



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.
[more...]
Use: pGlyco



Effective Enrichment Strategy Using Boronic Acid-Functionalized Mesoporous Graphene--Silica Composites for Intact N-and O-Linked Glycopeptide Analysis in Human Serum
Analytical Chemistry. 2021. Kong, SY et al. Fudan Univ, Peoples Hosp 5, Shanghai 200032, Peoples R China.
ABSTRACT: The heterogeneity and low abundance of protein glycosylation present challenging barriers to the analysis of intact glycopeptides, which is key to comprehensively understanding the role of glycosylation in an organism. Efficient and specific enrichment of intact glycopeptides could help greatly with this problem. Here, we propose a new enrichment strategy using a boronic acid (BA)-functionalized mesoporous graphene-silica composite (denoted as GO@mSiO(2)-GLYMO-APB) for isolating intact glycopeptides from complex biological samples.
[more...]
Use: pGlyco



Enhancing Open Modification Searches via a Combined Approach Facilitated by Ursgal
Journal of Proteome Research. 2021. Schulze, S et al. Univ Penn, Dept Biol, Philadelphia, PA 19104 USA.
ABSTRACT: The identification of peptide sequences and their posttranslational modifications (PTMs) is a crucial step in the analysis of bottom-up proteomics data. The recent development of open modification search (OMS) engines allows virtually all PTMs to be searched for. This not only increases the number of spectra that can be matched to peptides but also greatly advances the understanding of the biological roles of PTMs through the identification, and the thereby facilitated quantification, of peptidoforms (peptide sequences and their potential PTMs).
[more...]
Use: pGlyco



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



DeepPhospho accelerates DIA phosphoproteome profiling through in silico library generation
Nature Communications. 2021. Lou, RH et al. ShanghaiTech Univ, iHuman Inst, Shanghai 201210, Peoples R China; ShanghaiTech Univ, Sch Life Sci & Technol, Shanghai 201210, Peoples R China; ShanghaiTech Univ, Sch Informat Sci & Technol, Shanghai 201210, Peoples R China; Shanghai Engn Res Ctr Intelligent Vis & Imaging, Shanghai 201210, Peoples R China
ABSTRACT: The coverage and throughput of data-independent acquisition (DIA)-based phosphoproteomics is limited by its dependence on experimental spectral libraries. Here the authors develop a DIA workflow based on in silico spectral libraries generated by a novel deep neural network to expand phosphoproteome coverage.Phosphoproteomics integrating data-independent acquisition (DIA) enables deep phosphoproteome profiling with improved quantification reproducibility and accuracy compared to data-dependent acquisition (DDA)-based phosphoproteomics.
[more...]
Use: pDeep



Multiregional profiling of the brain transmembrane proteome uncovers novel regulators of depression
Science Advances. 2021. Li, SS et al. ShanghaiTech Univ, iHuman Inst, Shanghai 201210, Peoples R China.
ABSTRACT: Transmembrane proteins play vital roles in mediating synaptic transmission, plasticity, and homeostasis in the brain. However, these proteins, especially the G protein-coupled receptors (GPCRs), are underrepresented in most large-scale proteomic surveys. Here, we present a new proteomic approach aided by deep learning models for comprehensive profiling of transmembrane protein families in multiple mouse brain regions. Our multiregional proteome profiling highlights the considerable discrepancy between messenger RNA and protein distribution, especially for region-enriched GPCRs, and predicts an endogenous GPCR interaction network in the brain.
[more...]
Use: pDeep




2020




Comprehensive Identification of Native Medium-Sized and Short Bioactive Peptides in Sea Bass Muscle
Food Chemistry. 2020. Cerrato, A et al. Sapienza Univ Roma, Dept Chem, Piazzale Aldo Moro 5, I-00185 Rome, Italy.
ABSTRACT: Native peptides from sea bass muscle were analyzed by two different approaches: medium-sized peptides by peptidomics analysis, whereas short peptides by suspect screening analysis employing an inclusion list of exact m/z values of all possible amino acid combinations (from 2 up to 4). The method was also extended to common post-translational modifications potentially interesting in food analysis, as well as non-proteolytic aminoacyl derivatives, which are well-known taste-active building blocks in pseudo-peptides.
[more...]
Use: pNovo; pFind



Persulfidation-based modification of cysteine desulfhydrase and the NADPH oxidase RBOHD controls guard cell abscisic acid signaling
The Plant Cell. 2020. Shen, J et al. Nanjing Agr Univ, Coll Life Sci, Lab Ctr Life Sci, Nanjing 210095, Peoples R China.
ABSTRACT: A persulfidation-based reversible post-translational modification of Cys desulfhydrase and NADPH oxidase RBOHD fine-tunes guard cell ABA signaling. Hydrogen sulfide (H2S) is a gaseous signaling molecule that regulates diverse cellular signaling pathways through persulfidation, which involves the post-translational modification of specific Cys residues to form persulfides. However, the mechanisms that underlie this important redox-based modification remain poorly understood in higher plants. We have, therefore, analyzed how protein persulfidation acts as a specific and reversible signaling mechanism during the abscisic acid (ABA) response in Arabidopsis (Arabidopsis thaliana).
[more...]
Use: pFind



Accurate annotation of human protein-coding small open reading frames
Nature chemical biology. 2020. Martinez, TF et al. Salk Inst Biol Studies, Clayton Fdn Labs Peptide Biol, 10010 N Torrey Pines Rd, La Jolla, CA 92037 USA.
ABSTRACT: Functional protein-coding small open reading frames (smORFs) are emerging as an important class of genes. However, the number of translated smORFs in the human genome is unclear because proteogenomic methods are not sensitive enough, and, as we show, Ribo-seq strategies require additional measures to ensure comprehensive and accurate smORF annotation. Here, we integrate de novo transcriptome assembly and Ribo-seq into an improved workflow that overcomes obstacles with previous methods, to more confidently annotate thousands of smORFs.
[more...]
Use: pFind



A streamlined mass spectrometry--based proteomics workflow for large-scale FFPE tissue analysis
The Journal of pathology. 2020. Coscia, F et al. Max Planck Inst Biochem, D-82152 Martinsried, Germany.
ABSTRACT: Formalin fixation and paraffin-embedding (FFPE) is the most common method to preserve human tissue for clinical diagnosis, and FFPE archives represent an invaluable resource for biomedical research. Proteins in FFPE material are stable over decades but their efficient extraction and streamlined analysis by mass spectrometry (MS)-based proteomics has so far proven challenging. Herein we describe a MS-based proteomic workflow for quantitative profiling of large FFPE tissue cohorts directly from histopathology glass slides.
[more...]
Use: pFind



Pervasive translation of circular RNAs driven by short IRES-like elements
Nature communications. 2020. Fan, XJ et al. Shanghai Inst Nutr & Hlth, Biomed Big Data Ctr, CAS Ctr Excellence Mol Cell Sci, CAS Key Lab Computat Biol, Shanghai, Peoples R China; Chinese Acad Sci, Univ Chinese Acad Sci, Beijing, Peoples R China
ABSTRACT: Some circular RNAs (circRNAs) were found to be translated through IRES-driven mechanism, however the scope and functions of circRNA translation are unclear because endogenous IRESs are rare. To determine the prevalence and mechanism of circRNA translation, we develop a cell-based system to screen random sequences and identify 97 overrepresented hexamers that drive cap-independent circRNA translation. These IRES-like short elements are significantly enriched in endogenous circRNAs and sufficient to drive circRNA translation.
[more...]
Use: pFind



Cancer neoantigen prioritization through sensitive and reliable proteogenomics analysis
Nature communications. 2020. Wen, B et al. Baylor Coll Med, Lester & Sue Smith Breast Ctr, Houston, TX 77030 USA.
ABSTRACT: Genomics-based neoantigen discovery can be enhanced by proteomic evidence, but there remains a lack of consensus on the performance of different quality control methods for variant peptide identification in proteogenomics. We propose to use the difference between accurately predicted and observed retention times for each peptide as a metric to evaluate different quality control methods. To this end, we develop AutoRT, a deep learning algorithm with high accuracy in retention time prediction. Analysis of three cancer data sets with a total of 287 tumor samples using different quality control strategies results in substantially different numbers of identified variant peptides and putative neoantigens.
[more...]
Use: pFind



Structural snapshots of human pre-60S ribosomal particles before and after nuclear export
Nature communications. 2020. Liang, XM et al. Peking Univ, Peking Tsinghua Joint Ctr Life Sci, Sch Life Sci, State Key Lab Membrane Biol, Beijing 100871, Peoples R China.
ABSTRACT: Ribosome biogenesis is an elaborate and energetically expensive program that involve two hundred protein factors in eukaryotes. Nuclear export of pre-ribosomal particles is one central step which also serves as an internal structural checkpoint to ensure the proper completion of nuclear assembly events. Here we present four structures of human pre-60S particles isolated through a nuclear export factor NMD3, representing assembly stages immediately before and after nuclear export. These structures reveal locations of a dozen of human factors, including an uncharacterized factor TMA16 localized between the 5S RNA and the P0 stalk.
[more...]
Use: pFind; pLink



Improvements on the quantitative analysis of Trypanosoma cruzi histone post translational modifications: Study of changes in epigenetic marks through the parasite's metacyclogenesis and life cycle
Journal of proteomics. 2020. de Lima, LP et al. Inst Butantan, Lab Especial Ciclo Celular, Ave Vital Brasil 1500, BR-05503900 Sao Paulo, Brazil.
ABSTRACT: Trypanosome histone N-terminal sequences are very divergent from the other eukaryotes, although they are still decorated by post-translational modifications (PTMs). Here, we used a highly robust workflow to analyze histone PTMs in the parasite Trypanosoma cruzi using mass spectrometry-based (MS-based) data-independent acquisition (DIA). We adapted the workflow for the analysis of the parasite's histone sequences by modifying the software EpiProfile 2.0, improving peptide and PTM quantification accuracy.
[more...]
Use: pFind



Atg1 kinase in fission yeast is activated by Atg11-mediated dimerization and cis-autophosphorylation
Elife. 2020. Pan, ZQ et al. Natl Inst Biol Sci, Beijing, Peoples R China.
ABSTRACT: Autophagy is a proteolytic pathway that is conserved from yeasts to mammals. Atg1 kinase is essential for autophagy, but how its activity is controlled remains insufficiently understood. Here, we show that, in the fission yeast Schizosaccharomyces pombe, Atg1 kinase activity requires Atg11, the ortholog of mammalian FIP200/RB1CC1, but does not require Atg13, Atg17, or Atg101. Remarkably, a 62 amino acid region of Atg11 is sufficient for the autophagy function of Atg11 and for supporting the Atg1 kinase activity.
[more...]
Use: pFind



Global lysine crotonylation profiling of mouse liver
Proteomics. 2020. Liu, JF et al. Chinese Acad Med Sci & Peking Union Med Coll, Inst Basic Med Sci, Dept Biochem & Mol Biol, State Key Lab Med Mol Biol, Beijing 100005, Peoples R China.
ABSTRACT: Lysine crotonylation (Kcr) is a recently discovered post-translational modification that potentially regulates multiple biological processes. With an objective to expand the available crotonylation datasets, LC-MS/MS is performed using mouse liver samples under normal physiological conditions to obtain in vivo crotonylome. A label-free strategy is used and 10 034 Class I (localization probabilities > 0.75) crotonylated sites are identified in 2245 proteins. The KcrE, KcrD, and EKcr motifs are significantly enriched in the crotonylated peptides.
[more...]
Use: pFind



Orally efficacious broad-spectrum allosteric inhibitor of paramyxovirus polymerase
Nature microbiology. 2020. Cox, RM et al. Georgia State Univ, Inst Biomed Sci, Atlanta, GA 30303 USA.
ABSTRACT: Paramyxoviruses such as human parainfluenza virus type-3 (HPIV3) and measles virus (MeV) are a substantial health threat. In a high-throughput screen for inhibitors of HPIV3 (a major cause of acute respiratory infection), we identified GHP-88309-a non-nucleoside inhibitor of viral polymerase activity that possesses unusual broad-spectrum activity against diverse paramyxoviruses including respiroviruses (that is, HPIV1 and HPIV3) and morbilliviruses (that is, MeV). Resistance profiles of distinct target viruses overlapped spatially, revealing a conserved binding site in the central cavity of the viral polymerase (L) protein that was validated by photoaffinity labelling-based target mapping.
[more...]
Use: pFind



A protein identification algorithm optimization for mass spectrometry data using deep learning
2020 3rd International Conference on Advanced Electronic Materials, Computers and Software Engineering (AEMCSE). 2020. Xu, Rui et al. State Key Laboratory of Proteomics, Beijing Proteome Research Center National Center for Protein Sciences (Beijing), Beijing
ABSTRACT: Protein sequence database search is one of the most commonly used methods for protein identification in shotgun proteomics. In tradition, searching a protein sequence database is usually required to construct the theoretical spectrum for each peptide at first, which only considers the information of mass-to-charge ratio at present. However, the information related to isotope peak intensity is neglected. Thanks to the rapid development of artificial intelligence technique in recent years, deep learning-based MS/MS spectrum prediction tools have showed a high accuracy and great potentials to improve the sensitivity and accuracy of protein sequence database searching.
[more...]
Use: pFind; pDeep



An Algorithm to Improve the Speed of Semi and Non-specific Enzyme Searches in Proteomics
Current bioinformatics. 2020. Rolfs, Z et al. Univ Wisconsin, Dept Chem, 1101 Univ Ave, Madison, WI 53706 USA.
ABSTRACT: Background: The identification of non-specifically cleaved peptides in proteomics and peptidomics poses a significant computational challenge. Current strategies for the identification of such peptides are typically time-consuming and hinder routine data analysis. Objective: We aimed to design an algorithm that would improve the speed of semi- and nonspecific enzyme searches and could be applied to existing search programs. Methods: We developed a novel search algorithm that leverages fragment-ion redundancy to simultaneously search multiple non-specifically cleaved peptides at once.
[more...]
Use: pFind



Characterization of Lysine Monomethylome and Methyltransferase in Model Cyanobacterium Synechocystis sp. PCC 6803
Genomics, proteomics & bioinformatics. 2020. Lin, XH et al. Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan 430072, Peoples R China.
ABSTRACT: Protein lysine methylation is a prevalent post-translational modification (PTM) and plays critical roles in all domains of life. However, its extent and function in photosynthetic organisms are still largely unknown. Cyanobacteria are a large group of prokaryotes that carry out oxygenic photosynthesis and are applied extensively in studies of photosynthetic mechanisms and environmental adaptation. Here we integrated propionylation of monomethylated proteins, enrichment of the modified peptides, and mass spectrometry (MS) analysis to identify monomethylated proteins in Synechocystis sp.
[more...]
Use: pFind



SlFERL interacts with S-adenosylmethionine synthetase to regulate fruit ripening
Plant Physiology. 2020. Ji, DC et al. Chinese Acad Sci, Innovat Acad Seed Design, Inst Bot, Key Lab Plant Resources, Beijing 100093, Peoples R China.
ABSTRACT: The tomato membrane protein SlFERL regulates fruit ripening via modulating ethylene production. Fruit ripening is a complex and genetically programmed process modulated by transcription factors, hormones, and other regulators. However, the mechanism underlying the regulatory loop involving the membrane-protein targets of RIPENING-INHIBITOR (RIN) remains poorly understood. To unravel the function of tomato (Solanum lycopersicum) FERONIA Like (SlFERL), a putative MADS-box transcription factor target gene, we investigated and addressed the significance of SlFERL in fruit ripening by combining reverse genetics, biochemical, and cytological analyses.
[more...]
Use: pFind



Unambiguous Phosphosite Localization through the Combination of Trypsin and LysargiNase Mirror Spectra in a Large-Scale Phosphoproteome Study
Journal of proteome research. 2020. Xu, F et al. Beijing Inst Life, Natl Ctr Prot Sci Beijing, Beijing Proteome Res Ctr, State Key Lab Prote, Beijing 102206, Peoples R China.
ABSTRACT: Understanding of the kinase-guided signaling pathways requires the identification and analysis of phosphosites. Mass spectrometry (MS)-based phosphoproteomics is a rapid and highly sensitive approach for high-throughput identification of phosphosites. However, phosphosite determination from MS data with a single protease is more likely to be ambiguous, regardless of the strategy used for phosphopeptide detection. Here, we explored the application of LysargiNase, which was recently reported to mirror trypsin in specificity to cleave arginine and lysine residues exclusively at the N-terminal side.
[more...]
Use: pFind



Global characterization of modifications to the charge isomers of IgG antibody
Journal of Pharmaceutical Analysis. 2020. Cui, XL et al. State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing
ABSTRACT: Posttranslational modifications of antibody products affect their stability, charge distribution, and drug activity and are thus a critical quality attribute. The comprehensive mapping of antibody modifications and different charge isomers (CIs) is of utmost importance but is challenging. We intended to quantitatively characterize the posttranslational modification status of CIs of antibody drugs and explore the impact of posttranslational modifications on charge heterogeneity. The CIs of antibodies were fractionated by strong cation exchange chromatography and verified by capillary isoelectric focusing-whole column imaging detection, followed by stepwise structural characterization at three levels.
[more...]
Use: pFind



UPEFinder: A Bioinformatic Tool for the Study of Uncharacterized Proteins Based on Gene Expression Correlation and the PageRank Algorithm
Journal of proteome research. 2020. Gonzalez-Gomariz, J et al. Navarra Inst Hlth Res, IdiSNA, E-31008 Pamplona, Spain.
ABSTRACT: The Human Proteome Project (HPP) is leading the international effort to characterize the human proteome. Although the main goal of this project was first focused on the detection of missing proteins, a new challenge arose from the need to assign biological functions to the uncharacterized human proteins and describe their implications in human diseases. Not only the proteins with experimental evidence (uPE1 proteins) but also the uncharacterized missing proteins (uMPs) were the objects of study in this challenge, neXt-CP50.
[more...]
Use: pFind



Quantitative Perspective on Online Flow Reaction Profiling Using a Miniature Mass Spectrometer
ORGANIC PROCESS RESEARCH & DEVELOPMENT. 2020. Sheng, HM et al. Merck & Co Inc, Analyt Sci, Rahway, NJ 07065 USA.
ABSTRACT: Online mass spectrometry has proven to be a useful tool for characterizing many aspects of chemical reactions. However, to the best of the authors' knowledge, no reference standard (RS) quantitation approach has been applied in online MS profiling work to date. In this study, we present a RS approach for online quantitation of an aerobic oxidation reaction in flow using a miniature mass spectrometer, with both internal RS and external RS quantitation approaches being evaluated. Quinoline, a structurally similar and chemically inert compound under these reaction conditions, was chosen as the RS to quantify the pyridine aldehyde product.
[more...]
Use: pFind



Open-pFind Verified Four Missing Proteins from Multi-Tissues
Journal of proteome research. 2020. Wu, SJ et al. Wuhan Univ, Sch Basic Med Sci, Minist Educ, Key Lab Combinatl Biosynth & Drug Discovery, Wuhan 430072, Peoples R China.
ABSTRACT: The Chromosome-Centric Human Proteome Project (C-HPP) was launched in 2012 to perfect the annotation of human protein existence by identifying stronger evidence of the expression of missing proteins (MPs) at the protein level. After an 8 year effort all over the world, the number of MPs in the neXtProt database significantly decreased from 5511 (2012-02-24) to 1899 (2020-01-17). It is now more difficult to provide confident evidence of the remaining MPs because of their specific characteristics, including low abundance, low molecular weight, unexpected modifications, transmembrane structure, tissue-expression specificity, and so on.
[more...]
Use: pFind



D283 Med, a Cell Line Derived from Peritoneal Metastatic Medulloblastoma: A Good Choice for Missing Protein Discovery
Journal of proteome research. 2020. Zhang, YL et al. BGI Shenzhen, BGI Genom, Shenzhen 518083, Guangdong, Peoples R China.
ABSTRACT: Since the Chromosome-Centric Human Proteome Project (C-HPP) was launched in 2010, many techniques have been adopted for the discovery of missing proteins (MPs). Because of these efforts, only 1481 MPs remained as of July 2020; however, by relying only on technique optimization, researchers have reached a bottleneck in MP discovery. Protein expression is tissue- or cell-type-dependent. The tissues of the human testis and brain have been reported to harbor a large number of tissue-specific genes and proteins; however, few studies have been performed on human brain tissue or cells to identify MPs.
[more...]
Use: pFind



Open Search Strategy for Inferring the Masses of Cross-Link Adducts on Proteins
Analytical Chemistry. 2020. Slavin, M et al. Hebrew Univ Jerusalem, Inst Life Sci, IL-9190401 Jerusalem, Israel.
ABSTRACT: Development of new reagents for protein cross-linking is constantly ongoing. The chemical formulas for the linker adducts formed by these reagents are usually deduced from expert knowledge and then validated by mass spectrometry. Clearly, it would be more rigorous to infer the chemical compositions of the adducts directly from the data without any prior assumptions on their chemistries. Unfortunately, the analysis tools that are currently available to detect chemical modifications on linear peptides are not applicable to the case of two cross-linked peptides.
[more...]
Use: pFind



Pre-termination transcription complex: structure and function
Molecular cell. 2020. Hao, ZT et al. NYU, Sch Med, Dept Biochem & Mol Pharmacol, New York, NY 10016 USA.
ABSTRACT: Rho is a general transcription termination factor playing essential roles in RNA polymerase (RNAP) recycling, gene regulation, and genomic stability in most bacteria. Traditional models of transcription termination postulate that hexameric Rho loads onto RNA prior to contacting RNAP and then translocates along the transcript in pursuit of the moving RNAP to pull RNA from it. Here, we report the cryoelectron microscopy (cryo-EM) structures of two termination process intermediates. Prior to interacting with RNA, Rho forms a specific "pre-termination complex'' (PTC) with RNAP and elongation factors NusA and NusG, which stabilize the PTC.
[more...]
Use: pFind; pLink



Synergistic optimization of Liquid Chromatography and Mass Spectrometry parameters on Orbitrap Tribrid mass spectrometer for high efficient data-dependent proteomics
Journal of Mass Spectrometry. 2020. Huang, PW et al. Southern Univ Sci & Technol, Dept Chem, Shenzhen 518055, Peoples R China.
ABSTRACT: Steady improvement in Orbitrap-based mass spectrometry (MS) technologies has greatly advanced the peptide sequencing speed and depth. In-depth analysis of the performance of state-of-the-art MS and optimization of key parameters can improve sequencing efficiency. In this study, we first systematically compared the performance of two popular data-dependent acquisition approaches, with Orbitrap as the first-stage (MS1) mass analyzer and the same Orbitrap (high-high approach) or ion trap (high-low approach) as the second-stage (MS2) mass analyzer, on the Orbitrap Fusion mass spectrometer.
[more...]
Use: pFind; pParse



Activation of the CARD8 inflammasome requires a disordered region
Cell reports. 2020. Chui, AJ et al. Mem Sloan Kettering Canc Ctr, Triinst PhD Program Chem Biol, New York, NY 10065 USA.
ABSTRACT: Several cytosolic pattern-recognition receptors (PRRs) form multiprotein complexes called canonical inflammasomes in response to intracellular danger signals. Canonical inflammasomes recruit and activate caspase-1 (CASP1), which in turn cleaves and activates inflammatory cytokines and gasdermin D (GSDMD), inducing pyroptotic cell death. Inhibitors of the dipeptidyl peptidases DPP8 and DPP9 (DPP8/9) activate both the human NLRP1 and CARD8 inflammasomes. NLRP1 and CARD8 have different N-terminal regions but have similar C-terminal regions that undergo autoproteolysis to generate two non-covalently associated fragments.
[more...]
Use: pFind



TransCirc: an interactive database for translatable circular RNAs based on multi-omics evidence
Nucleic Acids Research. 2020. Huang, WD et al. Chinese Acad Sci, Biomed Big Data Ctr, CAS MPG Partner Inst Computat Biol, CAS Key Lab Computat Biol,Shanghai Inst Nutr & Hl, Shanghai 200031, Peoples R China.
ABSTRACT: TransCirc (https://www.biosino.org/transcirc/) is a specialized database that provide comprehensive evidences supporting the translation potential of circular RNAs (circRNAs). This database was generated by integrating various direct and indirect evidences to predict coding potential of each human circRNA and the putative translation products. Seven types of evidences for circRNA translation were included: (i) ribosome/polysome binding evidences supporting the occupancy of ribosomes onto circRNAs; (ii) experimentally mapped translation initiation sites on circRNAs; (iii) internal ribosome entry site on circRNAs; (iv) published N-6-methyladenosine modification data in circRNA that promote translation initiation; (v) lengths of the circRNA specific open reading frames; (vi) sequence composition scores froma machine learning prediction of all potential open reading frames; (vii) mass spectrometry data that directly support the circRNA encoded peptides across back-splice junctions.
[more...]
Use: pFind



Protocol for Proximity-Dependent Proteomic Profiling in Yeast Cells by APEX and Alk-Ph Probe
STAR Protocols. 2020. Li, Yi et al. Peking University
ABSTRACT: Alk-Ph is a clickable APEX2 substrate developed for spatially restricted protein/RNA labeling in intact yeast cells. Alk-Ph is more water soluble and cell wall permeable than biotin-phenol substrate, allowing more efficient profiling of the subcellular proteome in microorganisms. We describe the protocol for Alk-Ph probe synthesis, APEX2 expression, and protein/RNA labeling in yeast and the workflow for quantitative proteomic experiments and data analysis. Using the yeast mitochondria as an example, we provide guidelines to achieve high-resolution mapping of subcellular yeast proteome and transcriptome.
[more...]
Use: pQuant; pFind



Comprehensive identification of native medium-sized and short bioactive peptides in sea bass muscle
Food Chemistry. 2020. Cerrato, A et al. Sapienza Univ Roma, Dept Chem, Piazzale Aldo Moro 5, I-00185 Rome, Italy.
ABSTRACT: Native peptides from sea bass muscle were analyzed by two different approaches: medium-sized peptides by peptidomics analysis, whereas short peptides by suspect screening analysis employing an inclusion list of exact m/z values of all possible amino acid combinations (from 2 up to 4). The method was also extended to common post-translational modifications potentially interesting in food analysis, as well as non-proteolytic aminoacyl derivatives, which are well-known taste-active building blocks in pseudo-peptides.
[more...]
Use: pNovo; pFind



Comprehensive structural glycomic characterization of the glycocalyxes of cells and tissues
NATURE PROTOCOLS. 2020. Li, QY et al. Univ Calif Davis, Dept Chem, Davis, CA 95616 USA.
ABSTRACT: The glycocalyx comprises glycosylated proteins and lipids and fcorms the outermost layer of cells. It is involved in fundamental inter- and intracellular processes, including non-self-cell and self-cell recognition, cell signaling, cellular structure maintenance, and immune protection. Characterization of the glycocalyx is thus essential to understanding cell physiology and elucidating its role in promoting health and disease. This protocol describes how to comprehensively characterize the glycocalyx N-glycans and O-glycans of glycoproteins, as well as intact glycolipids in parallel, using the same enriched membrane fraction.
[more...]
Use: pFind; pGlyco



A quantitative thiol reactivity profiling platform to analyze redox and electrophile reactive cysteine proteomes
NATURE PROTOCOLS. 2020. Fu, L et al. Beijing Inst Life, State Key Lab Prote, Beijing Proteome Res Ctr, Natl Ctr Prot Sci Beijing, Beijing, Peoples R China.
ABSTRACT: Cysteine is unique among all protein-coding amino acids, owing to its intrinsically high nucleophilicity. The cysteinyl thiol group can be covalently modified by a broad range of redox mechanisms or by various electrophiles derived from exogenous or endogenous sources. Measuring the response of protein cysteines to redox perturbation or electrophiles is critical for understanding the underlying mechanisms involved. Activity-based protein profiling based on thiol-reactive probes has been the method of choice for such analyses.
[more...]
Use: pFind; pQuant



鸽毛滴虫外泌体的分离鉴定及蛋白质谱分析
Acta Veterinaria et Zootechnica Sinica (畜牧兽医学报). 2020. Ni, xinai et al. 中国农业科学院北京畜牧兽医研究所
ABSTRACT: The aim of this study was to determine whether Trichomonas gallinae(T.gallinae)secrete exosomes and investigate the protein composition of exosomes derived fromT.gallinae.T.gallinae was isolated from upper digestive tract of infected pigeons and identified by microscopy and sequencing.Exosomes were extracted from serum absent culture medium,and further verified by transmission electron microscopy,Western blot and nanoparticle tracking analysis(NTA).Label-free was used to identify the proteins of exosomes.Results showed that the parasite had T.gallinae morphology,and sequence alignment of ITS1/5.8S/ITS2gene showed an identity of 98%.The isolated vesicles presented a typical cup-shaped morphology;NTA results showed that particle size concentrated on 125.1nm,percentage of peak diameter at 132.3nm was 99.3%;proteins such as CD63and TSG101were conspicuously detected,suggesting that the extracts were exosomes.Mass spectrometry results showed that enolase,glyceraldehyde-3-phosphate dehydrogenase,phosphoglycerate mutase and elongation factor were the highly expressed proteins.GO pathways showed that proteins of exosomes mainly enriched into cellular components such as intracellular and cytoplasmic,played GTP binding and GTPase activity function, and participated in biological process of small GTPase mediated signal transduction and glycolytic process.KEGG enrichment analysis showed that proteins of exosomes were enriched in pathways as metabolic pathways,glycolysis/gluconeogenesis,biosynthesis of antibiotics and secondary metabolites.These results indicated that T.gallinaecan secrete exosomes and proteins of exosomes might play roles in energy metabolism,signal transduction and biosynthesis.
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Combination of continuous digestion by peptidase and spectral similarity comparisons for peptide sequencing
JOURNAL OF SEPARATION SCIENCE. 2020. Yang, C 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.
ABSTRACT: Peptide sequencing is critical to the quality control of peptide drugs and functional studies of active peptides. A combination of peptidase digestion and mass spectrometry technology is common for peptide sequencing. However, such methods often cannot obtain the complete sequence of a peptide due to insufficient amino acid sequence information. Here, we developed a method of generating full peptide ladders and comparing their MS(2)spectral similarities. The peptide ladders, of which each component was different from the next component with one residue, were generated by continuous digestion by peptidase (carboxypeptidase Y and aminopeptidase).
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Identification of modified peptides using localization-aware open search
Nature communications. 2020. Yu, FC et al. Univ Michigan, Dept Pathol, Ann Arbor, MI 48109 USA.
ABSTRACT: Identification of post-translationally or chemically modified peptides in mass spectrometry-based proteomics experiments is a crucial yet challenging task. We have recently introduced a fragment ion indexing method and the MSFragger search engine to empower an open search strategy for comprehensive analysis of modified peptides. However, this strategy does not consider fragment ions shifted by unknown modifications, preventing modification localization and limiting the sensitivity of the search. Here we present a localization-aware open search method, in which both modification-containing (shifted) and regular fragment ions are indexed and used in scoring.
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Rapid and easy enrichment strategy for naturally acetylated N termini based on LysN digestion and amine-reactive resin capture
Analytical chemistry. 2020. Du, XX et al. Fudan Univ, Shanghai Canc Ctr, Shanghai 200032, Peoples R China.
ABSTRACT: Protein N-terminal acetylation (N-alpha-acetylation) is one of the most common modifications in both eukaryotes and prokaryotes. Although studies have shown that N-alpha-acetylation plays important roles in protein assembly, stability, and location, the physiological role has not been fully elucidated. Therefore, a robust and large-scale analytical method is important for a better understanding of N-alpha-acetylation. Here, an enrichment strategy was presented based on LysN digestion and amine-reactive resin capture to study naturally acetylated protein N termini.
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Investigation of indigoidine synthetase reveals a conserved active-site base residue of nonribosomal peptide synthetase oxidases
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY. 2020. Pang, B et al. Univ Calif Berkeley, QB3 Inst, Berkeley, CA 94720 USA.
ABSTRACT: Nonribosomal peptide synthetase (NRPS) oxidase (Ox) domains oxidize protein-bound intermediates to install crucial structural motifs in bioactive natural products. The mechanism of this domain remains elusive. Here, by studying indigoidine synthetase, a single-module NRPS involved in the biosynthesis of indigoidine and several other bacterial secondary metabolites, we demonstrate that its Ox domain utilizes an active-site base residue, tyrosine 665, to deprotonate a protein-bound L-glutaminyl residue.
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The proteome landscape of the kingdoms of life
Nature. 2020. Muller, JB et al. Max Planck Inst Biochem, Dept Prote & Signal Transduct, Martinsried, Germany.
ABSTRACT: Proteins carry out the vast majority of functions in all biological domains, but for technological reasons their large-scale investigation has lagged behind the study of genomes. Since the first essentially complete eukaryotic proteome was reported(1), advances in mass-spectrometry-based proteomics(2)have enabled increasingly comprehensive identification and quantification of the human proteome(3-6). However, there have been few comparisons across species(7,8), in stark contrast with genomics initiatives(9).
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A comprehensive evaluation of MS/MS spectrum prediction tools for shotgun proteomics
Proteomics. 2020. Xu, R et al. Beijing Inst Life, Natl Ctr Prot Sci Beijing, Beijing Proteome Res Ctr, State Key Lab Prote, Beijing 102206, Peoples R China.
ABSTRACT: Spectrum prediction using machine learning or deep learning models is an emerging method in computational proteomics. Several deep learning-based MS/MS spectrum prediction tools have been developed and showed their potentials not only for increasing the sensitivity and accuracy of data-dependent acquisition search engines, but also for building spectral libraries for data-independent acquisition analysis. Different tools with their unique algorithms and implementations may result in different performances.
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A clickable APEX probe for proximity-dependent proteomic profiling in yeast
Cell Chemical Biology. 2020. Li, Y et al. Peking Univ, Coll Chem & Mol Engn, Key Lab Bioorgan Chem & Mol Engn,Minist Educ, Beijing Natl Lab Mol Sci,Synthet & Funct Biomol C, Beijing 100871, Peoples R China.
ABSTRACT: The engineered ascorbate peroxidase (APEX) is a powerful tool for the proximity-dependent labeling of proteins and RNAs in live cells. Although widely use in mammalian cells, APEX applications in microorganisms have been hampered by the poor labeling efficiency of its biotin-phenol (BP) substrate. In this study, we sought to address this challenge by designing and screening a panel of alkyne-functionalized substrates. Our best probe, Alk-Ph, substantially improves APEX-labeling efficiency in intact yeast cells, as it is more cell wall-permeant than BP.
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Proteomic Analysis Reveals that EPHX1 Contributes to 5-Fluorouracil Resistance in a Human Hepatocellular Carcinoma Cell Line
Proteomics Clinical Applications. 2020. Sun, R et al. Dalian Med Univ, Dept Gen Surg, Div Hepatobiliary & Pancreat Surg, Affiliated Hosp 2, Dalian 116023, Peoples R China.
ABSTRACT: Purpose The extensive drug resistance of hepatocellular carcinoma (HCC) has become a major cause of chemotherapy failure. A deeper understanding of the drug resistance mechanism of tumor cells is very significant for improving the clinical prognosis of patients with HCC. Experimental Design In this study, proteomic studies on the composition of 5-fluorouracil (5-Fu) resistant Bel/5Fu cell line and its parent Bel7402 cell line by using an ionic liquid assisted proteins extraction method with the advantage of extracting plasma membrane proteins to a wider extent are performed.
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Identification of novel DPP--IV inhibitory peptides from Atlantic salmon (Salmo salar) skin
Food Research International. 2020. Jin, RT et al. Northeast Agr Univ, Coll Food Sci, 600 Changjiang Rd, Harbin 150030, Peoples R China.
ABSTRACT: The aim of this study was to identify dipeptidyl peptidase IV (DPP-IV) inhibitory peptides from salmon skin collagen hydrolysate, and to evaluate the possible inhibition mechanism of DPP-IV and peptide. Salmon skin collagen was hydrolyzed by pepsin, trypsin, papain, or Alcalase 2.4 L, separately. Trypsin hydrolysate (10 mg/mL) showed the highest inhibitory activity of 66.12 +/- 0.68%. The hydrolysate was separated into three fractions by ultrafiltration, and the inhibitory IC50 of M1 (molecular weight < 3 kDa) was 1.54 +/- 0.06 mg/mL.
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Sequence analysis of digestion-resistant peptides may be an efficient strategy for studying the linear epitopes of Jug r 1, the major walnut allergen
Food Chemistry. 2020. Guo, XY et al. China Agr Univ, Coll Food Sci & Nutr Engn, Beijing Adv Innovat Ctr Food Nutr & Human Hlth, 17 Qinghua East Rd, Beijing, Peoples R China.
ABSTRACT: Jug r 1, the major allergen of walnut, triggers severe allergic reactions through epitopes. Hence, research on the efficient strategy for analyzing the linear epitopes of Jug r 1 are necessary. In this work, bioinformatics analysis was used to predict the linear epitopes of Jug r 1. Overlapping peptide synthesis was used to map linear epitopes. In vitro simulated gastrointestinal digestion and HPLC-MS/MS were used to identify digestion-resistant peptides. The results showed that six predicted linear epitopes were AA28-35, AA42-49, AA55-62, AA65-73, AA97-104, and AA109-121.
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Characterization of urinary exosomes purified with size exclusion chromatography and ultracentrifugation
Journal of Proteome Research. 2020. Guan, S et al. Fudan Univ, Dept Chem, Shanghai 200438, Peoples R China.
ABSTRACT: Exosomes, a subtype of extracellular vesicles secreted by mammalian cells with a typical size range of 30-150 nm, have been implicated in many biological processes as intercellular communication carriers. The isolation of exosomes is an essential and challenging step before subsequent analysis and functional studies, due to the complexity of body fluids, as well as the small size and low density of exosomes. Ultracentrifugation (UC) and size exclusion chromatography (SEC) are two methods that have been extensively used for exosomes isolation in biological studies in recent years.
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An Arabidopsis secondary metabolite directly targets expression of the bacterial type III secretion system to inhibit bacterial virulence
Cell host & microbe. 2020. Wang, W et al. Chinese Acad Sci, Innovat Acad Seed Design, Inst Genet & Dev Biol, State Key Lab Plant Genom, Beijing 100101, Peoples R China.
ABSTRACT: Plants deploy a variety of secondary metabolites to fend off pathogen attack. Although defense compounds are generally considered toxic to microbes, the exact mechanisms are often unknown. Here, we show that the Arabidopsis defense compound sulforaphane (SFN) functions primarily by inhibiting Pseudomonas syringae type III secretion system (TTSS) genes, which are essential for pathogenesis. Plants lacking the aliphatic glucosinolate pathway, which do not accumulate SFN, were unable to attenuate TTSS gene expression and exhibited increased susceptibility to P.
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Parallel channels-multidimensional protein identification technology
Journal of the American Society for Mass Spectrometry. 2020. Zhang, N et al. Peking Univ, Sch Pharmaceut Sci, Sch Basic Med Sci, Hlth Sci Ctr,Ctr Precis Med Multi Res, Beijing 100191, Peoples R China.
ABSTRACT: Multidimensional protein identification (MudPIT), developed in the Yates Laboratory 20 years ago, is regarded as a powerful tool for proteomics research. Due to its remarkable online separation advantages, MudPIT has been widely used to facilitate discoveries in the field of proteomics research. However, it has one major disadvantage: the process of eluting peptides during strong cation exchange introduces salts, of different concentrations, into the mass spectrometer. Considering the sensitivity of the new generation of high-resolution mass spectrometers, developing a new version of MudPIT that could eliminate the introduction of salts in the elute would be a significant advancement to current technology.
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Sequential amidation of peptide C-termini for improving fragmentation efficiency
Journal of Mass Spectrometry. 2020. Wu, Q et al. 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: Owing to the poor fragmentation efficiency caused by the lack of a positively charged basic group at the C-termini of peptides, the identification of nontryptic peptides in classical proteomics is known to be less efficient. Particularly, attaching positively charged basic groups to C-termini via chemical derivatizations is known to be able to enhance their fragmentation efficiency. In this study, we introduced a novel strategy, C-termini sequential amidation reaction (CSAR), to improve peptide fragmentation efficiency.
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The F1 loop of the talin head domain acts as a gatekeeper in integrin activation and clustering
Journal of cell science. 2020. Kukkurainen, S et al. Tampere Univ, Fac Med & Hlth Technol, Arvo Ylpon Katu 34, FI-33520 Tampere, Finland.
ABSTRACT: Integrin activation and clustering by talin are early steps of cell adhesion. Membrane-bound talin head domain and kindlin bind to the beta integrin cytoplasmic tail, cooperating to activate the heterodimeric integrin, and the talin head domain induces integrin clustering in the presence of Mn2+. Here we show that kindlin-1 can replace Mn2+ to mediate beta 3 integrin clustering induced by the talin head, but not that induced by the F2-F3 fragment of talin. Integrin clustering mediated by kindlin-1 and the talin head was lost upon deletion of the flexible loop within the talin head F1 subdomain.
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Structure of nucleosome-bound human BAF complex
SCIENCE. 2020. He, S et al. Fudan Univ, Shanghai Med Coll, State Key Lab Genet Engn, Shanghai Canc Ctr,Inst Biomed Sci, Shanghai 200032, Peoples R China.
ABSTRACT: Mammalian SWI/SNF family chromatin remodelers, BRG1/BRM-associated factor (BAF) and polybromoassociated BAF (PBAF), regulate chromatin structure and transcription, and their mutations are linked to cancers. The 3.7-angstrom-resolution cryo-electron microscopy structure of human BAF bound to the nucleosome reveals that the nucleosome is sandwiched by the base and the adenosine triphosphatase (ATPase) modules, which are bridged by the actin-related protein (ARP) module. The ATPase motor is positioned proximal to nucleosomal DNA and, upon ATP hydrolysis, engages with and pumps DNA along the nucleosome.
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High-density chemical cross-linking for modeling protein interactions
PNAS. 2020. Mintseris, J et al. Harvard Med Sch, Dept Cell Biol, Boston, MA 02115 USA.
ABSTRACT: Detailed mechanistic understanding of protein complex function is greatly enhanced by insights from its 3-dimensional structure. Traditional methods of protein structure elucidation remain expensive and labor-intensive and require highly purified starting material. Chemical cross-linking coupled with mass spectrometry offers an alternative that has seen increased use, especially in combination with other experimental approaches like cryo-electron microscopy. Here we report advances in method development, combining several orthogonal cross-linking chemistries as well as improvements in search algorithms, statistical analysis, and computational cost to achieve coverage of 1 unique cross-linked position pair for every 7 amino acids at a 1% false discovery rate.
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Nicotinamide mononucleotide adenylyltransferase uses its NAD+ substrate-binding site to chaperone phosphorylated Tau
eLife. 2020. Ma, XJ et al. Chinese Acad Sci, Interdisciplinary Res Ctr Biol & Chem, Shanghai Inst Organ Chem, Shanghai, Peoples R China.
ABSTRACT: Tau hyper-phosphorylation and deposition into neurofibrillary tangles have been found in brains of patients with Alzheimer's disease (AD) and other tauopathies. Molecular chaperones are involved in regulating the pathological aggregation of phosphorylated Tau (pTau) and modulating disease progression. Here, we report that nicotinamide mononucleotide adenylyltransferase (NMNAT), a well-known NAD(+) synthase, serves as a chaperone of pTau to prevent its amyloid aggregation in vitro as well as mitigate its pathology in a fly tauopathy model.
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A molecular switch regulating transcriptional repression and activation of PPARγ
Nature communications. 2020. Shang, JS et al. Scripps Res Inst, Dept Integrat Struct & Computat Biol, Jupiter, FL 33458 USA.
ABSTRACT: Nuclear receptor (NR) transcription factors use a conserved activation function-2 (AF-2) helix 12 mechanism for agonist-induced coactivator interaction and NR transcriptional activation. In contrast, ligand-induced corepressor-dependent NR repression appears to occur through structurally diverse mechanisms. We report two crystal structures of peroxisome proliferator-activated receptor gamma (PPAR gamma) in an inverse agonist/corepressor-bound transcriptionally repressive conformation. Helix 12 is displaced from the solvent-exposed active conformation and occupies the orthosteric ligand-binding pocket enabled by a conformational change that doubles the pocket volume.
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Structures of three MORN repeat proteins and a re-evaluation of the proposed lipid-binding properties of MORN repeats
PLOS ONE. 2020. Sajko, S et al. Univ Vienna, Dept Struct & Computat Biol, Max Perutz Labs, Vienna, Austria.
ABSTRACT: MORN (Membrane Occupation and Recognition Nexus) repeat proteins have a wide taxonomic distribution, being found in both prokaryotes and eukaryotes. Despite this ubiquity, they remain poorly characterised at both a structural and a functional level compared to other common repeats. In functional terms, they are often assumed to be lipid-binding modules that mediate membrane targeting. We addressed this putative activity by focusing on a protein composed solely of MORN repeats-Trypanosoma brucei MORN1.
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The Nucleosome Remodeling and Deacetylase complex has an asymmetric, dynamic, and modular architecture
Cell reports. 2020. Low, JKK et al. Univ Sydney, Sch Life & Environm Sci, Sydney, NSW, Australia.
ABSTRACT: The nucleosome remodeling and deacetylase (NuRD) complex is essential for metazoan development but has been refractory to biochemical analysis. We present an integrated analysis of the native mammalian NuRD complex, combining quantitative mass spectrometry, cross-linking, protein biochemistry, and electron microscopy to define the architecture of the complex. NuRD is built from a 2:2:4 (MTA, HDAC, and RBBP) deacetylase module and a 1:1:1 (MBD, GATAD2, and Chromodomain-Helicase-DNA-binding [CHD]) remodeling module, and the complex displays considerable structural dynamics.
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Exaptation of two ancient immune proteins into a new dimeric pore-forming toxin in snails
JOURNAL OF STRUCTURAL BIOLOGY. 2020. Giglio, ML et al. Consejo Nacl Invest Cient & Tecn, Fdn Inst Leloir, IIBBA, Av Patricias Argentinas 435,C1405BWE, Buenos Aires, DF, Argentina.
ABSTRACT: The Membrane Attack Complex-Perforin (MACPF) family is ubiquitously found in all kingdoms. They have diverse cellular roles, however MACPFs with pore-forming toxic function in venoms and poisons are very rare in animals. Here we present the structure of PmPV2, a MACPF toxin from the poisonous apple snail eggs, that can affect the digestive and nervous systems of potential predators. We report the three-dimensional structure of PmPV2, at 17.2 angstrom resolution determined by negative-stain electron microscopy and its solution structure by small angle X-ray scattering (SAXS).
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Methionine sulfoxide reductase B from Corynebacterium diphtheriae catalyzes sulfoxide reduction via an intramolecular disulfide cascade
Journal of Biological Chemistry. 2020. Tossounian, MA et al. VUB, VIB, Ctr Structural Biol, B-1050 Brussels, Belgium.
ABSTRACT: Corynebacterium diphtheriae is a human pathogen that causes diphtheria. In response to immune system-induced oxidative stress, C. diphtheriae expresses antioxidant enzymes, among which are methionine sulfoxide reductase (Msr) enzymes, which are critical for bacterial survival in the face of oxidative stress. Although some aspects of the catalytic mechanism of the Msr enzymes have been reported, several details still await full elucidation. Here, we solved the solution structure of C. diphtheriae MsrB (Cd-MsrB) and unraveled its catalytic and oxidation-protection mechanisms.
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crisscrosslinkeR: identification and visualization of protein--RNA and protein--protein interactions from crosslinking mass spectrometry
Bioinformatics. 2020. Gail, EH et al. Monash Univ, Dept Biochem & Mol Biol, Biomed Discovery Inst, Clayton, Vic 3800, Australia.
ABSTRACT: The Summary: Unbiased detection of protein-protein and protein-RNA interactions within ribonucleoprotein complexes are enabled through crosslinking followed by mass spectrometry. Yet, different methods detect different types of molecular interactions and therefore require the usage of different software packages with limited compatibility. We present crisscrosslinkeR, an R package that maps both protein-protein and protein-RNA interactions detected by different types of approaches for crosslinking with mass spectrometry.
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A basic motif anchoring ISWI to nucleosome acidic patch regulates nucleosome spacing
Nature chemical biology. 2020. Dao, HT et al. Princeton Univ, Dept Chem, Princeton, NJ USA.
ABSTRACT: A photocrosslinking-based nucleosome profiling approach is used to identify a conserved basic motif in the ISWI remodeler SNF2h that anchors it to the acidic patch of nucleosome and enables nucleosome sliding activity. Recent studies have implicated the nucleosome acidic patch in the activity of ATP-dependent chromatin remodeling machines. We used a photocrosslinking-based nucleosome profiling technology (photoscanning) to identify a conserved basic motif within the catalytic subunit of ISWI remodelers, SNF2h, which engages this nucleosomal epitope.
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ADAM17 cytoplasmic domain modulates Thioredoxin-1 conformation and activity
Redox biology. 2020. Costa, RAPE et al. CNPEM, Lab Nacl Biociencias, LNBio, Campinas, SP, Brazil.
ABSTRACT: The activity of Thioredoxin-1 (Trx-1) is adjusted by the balance of its monomeric, active and its dimeric, inactive state. The regulation of this balance is not completely understood. We have previously shown that the cytoplasmic domain of the transmembrane protein A Disintegrin And Metalloprotease 17 (ADAM17cyto) binds to Thioredoxin-1 (Trx-1) and the destabilization of this interaction favors the dimeric state of Trx-1. Here, we investigate whether ADAM17 plays a role in the conformation and activation of Trx-1.
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A structural model of the endogenous human BAF complex informs disease mechanisms
Cell. 2020. Mashtalir, N et al. Dana Farber Canc Inst, Dept Pediat Oncol, Boston, MA 02115 USA.
ABSTRACT: Mammalian SWI/SNF complexes are ATP-dependent chromatin remodeling complexes that regulate genomic architecture. Here, we present a structural model of the endogenously purified human canonical BAF complex bound to the nucleosome, generated using cryoelectron microscopy (cryo-EM), cross-linking mass spectrometry, and homology modeling. BAF complexes bilaterally engage the nucleosome H2A/H2B acidic patch regions through the SMARCB1 C-terminal alpha-helix and the SMARCA4/2 C-terminal SnAc/post-SnAc regions, with disease-associated mutations in either causing attenuated chromatin remodeling activities.
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Structure of SWI/SNF chromatin remodeller RSC bound to a nucleosome
Nature. 2020. Wagner, FR et al. Max Planck Inst Biophys Chem, Dept Mol Biol, Gottingen, Germany.
ABSTRACT: The cryo-electron microscopy structure of the 16-subunit yeast SWI/SNF complex RSC in complex with a nucleosome substrate provides insights into the chromatin-remodelling function of this family of protein complexes. Chromatin-remodelling complexes of the SWI/SNF family function in the formation of nucleosome-depleted, transcriptionally active promoter regions (NDRs)(1,2). In the yeast Saccharomyces cerevisiae, the essential SWI/SNF complex RSC3 contains 16 subunits, including the ATP-dependent DNA translocase Sth1(4,5).
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Discovery of a molecular glue promoting CDK12-DDB1 interaction to trigger cyclin K degradation
Elife. 2020. Lv, L et al. Natl Inst Biol Sci, Beijing, Peoples R China.
ABSTRACT: Molecular-glue degraders mediate interactions between target proteins and components of the ubiquitin-proteasome system to cause selective protein degradation. Here, we report a new molecular glue HQ461 discovered by high-throughput screening. Using loss-of-function and gain-of-function genetic screening in human cancer cells followed by biochemical reconstitution, we show that HQ461 acts by promoting an interaction between CDK12 and DDB1-CUL4-RBX1 E3 ubiquitin ligase, leading to polyubiquitination and degradation of CDK12-interacting protein Cyclin K (CCNK).
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Molecular architecture of the human 17S U2 snRNP
Nature. 2020. Zhang, ZW et al. MPI Biophys Chem, Dept Struct Dynam, Gottingen, Germany.
ABSTRACT: The U2 small nuclear ribonucleoprotein (snRNP) has an essential role in the selection of the precursor mRNA branch-site adenosine, the nucleophile for the first step of splicing'. Stable addition of U2 during early spliceosome formation requiresthe DEAD-box ATPase PRP5(2-7). Yeast U2 small nuclear RNA (snRNA) nucleotides that form base pairs with the branch site are initially sequestered in a branchpoint-interacting stem-loop (BSL)(8), but whether the human U2 snRNA folds in a similar manner is unknown.
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Epitope and Paratope Mapping of PD-1/Nivolumab by Mass Spectrometry-Based Hydrogen--Deuterium Exchange, Cross-linking, and Molecular Docking
Analytical Chemistry. 2020. Zhang, MM et al. Bristol Myers Squibb Co, Nonclin Res & Dev, Pharmaceut Candidate Optimizat, Princeton, NJ 08540 USA.
ABSTRACT: Programmed cell death-1 (PD-1), an antigen co-receptor on cell surfaces, is one of the conspicuous immune checkpoints. Nivolumab, a monoclonal antibody therapeutic approved by the FDA, binds to PD-1 and efficiently blocks its pathways. In this study, an integrated approach was developed to map the epitope/paratope of PD-1/nivolumab. The approach includes hydrogen -deuterium exchange mass spectrometry (HDX-MS) followed by electron-transfer dissociation (ETD), chemical cross-linking, and molecular docking.
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Identification of sulfenylated cysteines in Arabidopsis thaliana proteins using a disulfide-linked peptide reporter
Frontiers in plant science. 2020. Wei, B et al. Univ Ghent, Dept Plant Biotechnol & Bioinformat, Ghent, Belgium.
ABSTRACT: In proteins, hydrogen peroxide (H2O2) reacts with redox-sensitive cysteines to form cysteine sulfenic acid, also known asS-sulfenylation. These cysteine oxidation events can steer diverse cellular processes by altering protein interactions, trafficking, conformation, and function. Previously, we had identifiedS-sulfenylated proteins by using a tagged proteinaceous probe based on the yeast AP-1-like (Yap1) transcription factor that specifically reacts with sulfenic acids and traps them through a mixed disulfide bond.
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Expanding the Depth and Sensitivity of Cross-Link Identification by Differential Ion Mobility Using High-Field Asymmetric Waveform Ion Mobility Spectrometry
Analytical Chemistry. 2020. Schnirch, L et al. Leibniz Forschungsinst Mol Pharmakol FMP, Dept Chem Biol, D-13125 Berlin, Germany.
ABSTRACT: In cross-linking mass spectrometry (XL-MS), the depth and sensitivity of cross-link detection is often limited by the low abundance of cross-links compared to non-cross-linked peptides in the digestion mixture. To improve the identification efficiency of cross-links, here, we present a gas-phase separation strategy using high-field asymmetric waveform ion mobility spectrometry (FAIMS) coupled to the Orbitrap Tribrid mass spectrometers. By enabling an additional peptide separation step in the gas phase using the FAIMS device, we increase the number of cross-link identifications by 22% for a medium complex sample and 59% for strong cation exchange-fractionated HEK293T cell lysate in XL-MS experiments using disuccinimidyl sulfoxide (DSSO) cross-linker.
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Vaccinia Virus Immunomodulator A46: Destructive Interactions with MAL and MyD88 Shown by Negative-Stain Electron Microscopy
Structure. 2020. Azar, DF et al. Med Univ Vienna, Vienna Bioctr, Max Perutz Labs, Dr Bohr Gasse 9-3, A-1030 Vienna, Austria.
ABSTRACT: Vaccinia virus A46 is an anti-inflammatory and non-anti-apoptotic, two-domain member of the poxviral Bcl-2-like protein family that inhibits the cellular innate immune response at the level of the Toll/interleukin-1 receptor (TIR) domain-containing TLR adaptor proteins MAL, MyD88, TRAM, and TRIF. The mechanism of interaction of A46 with its targets has remained unclear. The TIR domains of MAL and MyD88 have been shown to signal by forming filamentous assemblies. We show a clear concentration-dependent destruction of both of these assemblies by A46 by means of negative-stain electron microscopy from molar ratios of 1:15 for MAL and 1:30 for MyD88.
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Integrated structural modeling of full-length LRH-1 reveals inter-domain interactions contribute to receptor structure and function
Structure. 2020. Seacrist, CD et al. Vanderbilt Univ, Dept Pharmacol, Nashville, TN 37235 USA.
ABSTRACT: Liver receptor homolog-1 (LRH-1; NR5A2) is a nuclear receptor that regulates a diverse array of biological processes. In contrast to dimeric nuclear receptors, LRH-1 is an obligate monomer and contains a subtype-specific helix at the C terminus of the DNA-binding domain (DBD), termed FTZ-F1. Although detailed structural information is available for individual domains of LRH-1, it is unknown how these domains exist in the intact nuclear receptor Here, we developed an integrated structural model of human full-length LRH-1 using a combination of HDX-MS, XL-MS, Rosetta computational docking, and SAXS, The model predicts the DBD FTZ-F1 helix directly interacts with ligand binding domain helix 2, We confirmed several other predicted inter-domain interactions via structural and functional analyses.
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DNA binding reorganizes the intrinsically disordered C-terminal region of PSC in drosophila PRC1
Journal of Molecular Biology. 2020. Kang, JJ et al. Inst Rech Clin Montreal, 110 Ave Pins Ouest, Montreal, PQ H2W 1R7, Canada.
ABSTRACT: Polycomb Group proteins regulate gene expression by modifying chromatin. Polycomb Repressive Complex 1 (PRC1) has two activities: a ubiquitin ligase activity for histone H2A and a chromatin compacting activity. In Drosophila, the Posterior Sex Combs (PSC) subunit of PRC1 is central to both activities. The N-terminal of PSC assembles into PRC1, including partnering with dRING to form the ubiquitin ligase. The intrinsically disordered C-terminal region of PSC compacts chromatin and inhibits chromatin remodeling and transcription in vitro.
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An experimentally generated peptide database increases the sensitivity of XL-MS with complex samples
Journal of proteomics. 2020. Parfentev, I et al. Max Planck Inst Biophys Chem, Res Grp Bioanalyt Mass Spectrometry, Gottingen, Germany.
ABSTRACT: Cross-linking mass spectrometry (XL-MS) is steadily expanding its range of applications from purified protein complexes to more complex samples like organelles and even entire cells. One main challenge using non-cleavable cross-linkers is the so-called n 2 problem: With linearly increasing database size, the search space for the identification of two covalently linked peptides per spectrum increases quadratically. Here, we report an alternative search strategy that focuses on only those peptides, which were demonstrated to cross-link under the applied experimental conditions.
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Structure of the human sodium leak channel NALCN in complex with FAM155A
Nature communications. 2020. Xie, JF et al. Westlake Univ, Sch Life Sci, Key Lab Struct Biol Zhejiang Prov, Hangzhou 310024, Zhejiang, Peoples R China.
ABSTRACT: NALCN, a sodium leak channel expressed mainly in the central nervous system, is responsible for the resting Na+ permeability that controls neuronal excitability. Dysfunctions of the NALCN channelosome, NALCN with several auxiliary subunits, are associated with a variety of human diseases. Here, we report the cryo-EM structure of human NALCN in complex with FAM155A at an overall resolution of 3.1 angstroms. FAM155A forms extensive interactions with the extracellular loops of NALCN that may help stabilize NALCN in the membrane.
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Structural insights into the roles of metazoan-specific splicing factors in the human step 1 spliceosome
Molecular Cell. 2020. Bertram, K et al. MPI Biophys Chem, Dept Struct Dynam, Fassberg 11, D-37077 Gottingen, Germany.
ABSTRACT: Human spliceosomes contain numerous proteins absent in yeast, whose functions remain largely unknown. Here we report a 3D cryo-EM structure of the human spliceosomal C complex at 3.4 angstrom core resolution and 4.5-5.7 angstrom at its periphery, and aided by protein crosslinking we determine its molecular architecture. Our structure provides additional insights into the spliceosome's architecture between the catalytic steps of splicing, and how proteins aid formation of the spliceosome's catalytically active RNP (ribonucleoprotein) conformation.
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Decision tree searching strategy to boost the identification of cross-linked peptides
Analytical Chemistry. 2020. Huang, R et al. ShanghaiTech Univ, Shanghai Inst Adv Immunochem Studies, Shanghai 201210, Peoples R China.
ABSTRACT: We describe an efficient decision tree searching strategy (DTSS) to boost the identification of cross-linked peptides. The DTSS approach allows the identification of a wealth of complementary information to facilitate the construction of more protein-protein interaction networks for human cell lysate, which was tested by the use of a recently reported cross-linking data set (ACS Cent. Sci. 2019, 5, 1514-1522). A variant of the PhoX-linker, named pDSPE, was synthesized and applied to cross-link Escherichia coli cell lysate to demonstrate that the acquisition of doubly charged ions can significantly improve identification results.
[more...]
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Use of Multiple Ion Fragmentation Methods to Identify Protein Cross-Links and Facilitate Comparison of Data Interpretation Algorithms
Journal of Proteome Research. 2020. Zhao, BQ et al. Indiana Univ, Dept Chem, Bloomington, IN 47405 USA.
ABSTRACT: Multiple ion fragmentation methods involving collision-induced dissociation (CID), higher-energy collisional dissociation (HCD) with regular and very high energy settings, and electron-transfer dissociation with supplementary HCD (EThcD) are implemented to improve the confidence of cross-link identifications. Three different S. cerevisiae proteasome samples cross-linked by diethyl suberthioimidate (DEST) or bis(sulfosuccinimidyl)suberate (BS3) are analyzed. Two approaches are introduced to combine interpretations from the above four methods.
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FGF23 contains two distinct high-affinity binding sites enabling bivalent interactions with $\alpha$-Klotho
PNAS. 2020. Suzuki, Y et al. Yale Univ, Dept Pharmacol, Sch Med, New Haven, CT 06510 USA.
ABSTRACT: The three members of the endocrine-fibroblast growth factor (FGF) family, FGF19, 21, and 23 are circulating hormones that regulate critical metabolic processes. FGF23 stimulates the assembly of a signaling complex composed of alpha-Klotho (KLA) and FGF receptor (FGFR) resulting in kinase activation, regulation of phosphate homeostasis, and vitamin D levels. Here we report that the C-terminal tail of FGF23, a region responsible for KLA binding, contains two tandem repeats, repeat 1 (R1) and repeat 2 (R2) that function as two distinct ligands for KLA.
[more...]
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OpenPepXL: An open-source tool for sensitive identification of cross-linked peptides in XL-MS
Molecular & Cellular Proteomics. 2020. Netz, E et al. Max Planck Inst Dev Biol, Biomol Interact, Tubingen, Germany.
ABSTRACT: XL-MS has been recognized as an effective source of information about protein structures and interactions. OpenPepXL is a sensitive XL-MS identification software that reports from 7% to 40% more structurally validated cross-links than other tools on data sets with available high-resolution structures for cross-link validation. It is open source and has been built as part of the OpenMS suite of tools. OpenPepXL supports all common operating systems and open data formats. Cross-linking MS (XL-MS) has been recognized as an effective source of information about protein structures and interactions.
[more...]
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Mechanism of protein-guided folding of the active site U2/U6 RNA during spliceosome activation
Science (New York, N.Y.). 2020. Townsend, Cole et al. Cellular Biochemistry, MPI for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany.
ABSTRACT: Spliceosome activation involves extensive protein and RNA rearrangements that lead to formation of a catalytically active U2/U6 RNA structure. At present, little is known about the assembly pathway of the latter and the mechanism whereby proteins aid its proper folding. Here, we report the cryo-electron microscopy structures of two human, activated spliceosome precursors (that is, pre-Bact complexes) at core resolutions of 3.9 and 4.2 angstroms. These structures elucidate the order of the numerous protein exchanges that occur during activation, the mutually exclusive interactions that ensure the correct order of ribonucleoprotein rearrangements needed to form the U2/U6 catalytic RNA, and the stepwise folding pathway of the latter.
[more...]
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Defining the architecture of the human TIM22 complex by chemical crosslinking
FEBS Letters. 2020. Valpadashi, A et al. Univ Med Ctr Gottingen, Dept Cellular Biochem, Humboldtallee 23, D-37073 Gottingen, Germany.
ABSTRACT: The majority of mitochondrial proteins are nuclear encoded and imported into mitochondria as precursor proteins via dedicated translocases. The translocase of the inner membrane 22 (TIM22) is a multisubunit molecular machine specialized for the translocation of hydrophobic, multi-transmembrane-spanning proteins with internal targeting signals into the inner mitochondrial membrane. Here, we undertook a crosslinking-mass spectrometry (XL-MS) approach to determine the molecular arrangement of subunits of the human TIM22 complex.
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Dynamic folding modulation generates FGF21 variant against diabetes
EMBO reports. 2020. Zhu, Lei et al. High Magnetic Field Laboratory, CAS Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy ofSciences, Hefei, China
ABSTRACT: Fibroblast growth factor 21 (FGF21) is a regulator of glucose and lipid metabolism. It has been widely considered as a promising candidate for the treatment of type 2 diabetes mellitus (T2DM) and other related metabolic disorders. However, lack of structural and dynamic information has limited FGF21-based drug development. Here, using nuclear magnetic resonance (NMR) spectroscopy, we determine the structure of FGF21 and find that its non-canonical flexible beta-trefoil conformation affects the folding of beta2-beta3 hairpin and further overall protein stability.
[more...]
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Dimerization regulates the human APC/C-associated ubiquitin-conjugating enzyme UBE2S
Science Signaling. 2020. Liess, AKL et al. Univ Wurzburg, Rudolf Virchow Ctr Integrat & Translat Bioimaging, D-97080 Wurzburg, Germany.
ABSTRACT: At the heart of protein ubiquitination cascades, ubiquitin-conjugating enzymes (E2s) form reactive ubiquitin-thioester intermediates to enable efficient transfer of ubiquitin to cellular substrates. The precise regulation of E2s is thus crucial for cellular homeostasis, and their deregulation is frequently associated with tumorigenesis. In addition to driving substrate ubiquitination together with ubiquitin ligases (E3s), many E2s can also autoubiquitinate, thereby promoting their own proteasomal turnover.
[more...]
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Switch from Polymorphic to Homogenous Self-Assembly of Virus-Like Particles of Simian Virus 40 through Double-Cysteine Substitution
Small. 2020. Xu, CC et al. Guangzhou Med Univ, Guangzhou Women & Childrens Med Ctr, Guangzhou 510623, Peoples R China.
ABSTRACT: Self-assembled virus-like particles (VLPs) hold great potential as natural nanomaterials for applications in many fields. For such purposes, monodisperse size distribution is a desirable property. However, the VLPs of simian virus 40 (SV40), a representative VLP platform, are characterized by polymorphism. In an attempt to eliminate the polymorphism, 15 mutants of the VLP subunit (VP1) are constructed through the substitution of double cysteines at the VP1 pentamer interfaces, generating a group of VLPs with altered size distributions.
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Mapping the native interaction surfaces of PREP1 with PBX1 by cross-linking mass-spectrometry and mutagenesis
Scientific Reports. 2020. Bruckmann, C et al. IFOM Fdn FIRC, Inst Mol Oncol, Via Adamello 16, I-20139 Milan, Italy.
ABSTRACT: Both onco-suppressor PREP1 and the oncogene MEIS1 bind to PBX1. This interaction stabilizes the two proteins and allows their translocation into the nucleus and thus their transcriptional activity. Here, we have combined cross-linking mass-spectrometry and systematic mutagenesis to detail the binding geometry of the PBX1-PREP1 (and PBX1-MEIS1) complexes, under native in vivo conditions. The data confirm the existence of two distinct interaction sites within the PBC domain of PBX1 and unravel differences among the highly similar binding sites of MEIS1 and PREP1.
[more...]
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Glucocorticoid receptor complexes form cooperatively with the Hsp90 co-chaperones Pp5 and FKBPs
Scientific Reports. 2020. Kaziales, A et al. Tech Univ Munich, Dept Chem, Ctr Integrated Prot Sci Munich, Lichtenbergstr 4, D-85748 Garching, Germany.
ABSTRACT: The function of steroid receptors in the cell depends on the chaperone machinery of Hsp90, as Hsp90 primes steroid receptors for hormone binding and transcriptional activation. Several conserved proteins are known to additionally participate in receptor chaperone assemblies, but the regulation of the process is not understood in detail. Also, it is unknown to what extent the contribution of these cofactors is conserved in other eukaryotes. We here examine the reconstituted C. elegans and human chaperone assemblies.
[more...]
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Structural basis of human full-length kindlin-3 homotrimer in an auto-inhibited state
PLOS Biology. 2020. Bu, WT et al. Nanyang Technol Univ, Sch Biol Sci, Singapore, Singapore.
ABSTRACT: Kindlin-1, -2, and -3 directly bind integrin beta cytoplasmic tails to regulate integrin activation and signaling. Despite their functional significance and links to several diseases, structural information on full-length kindlin proteins remains unknown. Here, we report the crystal structure of human full-length kindlin-3, which reveals a novel homotrimer state. Unlike kindlin-3 monomer, which is the major population in insect and mammalian cell expression systems, kindlin-3 trimer does not bind integrin beta cytoplasmic tail as the integrin-binding pocket in the F3 subdomain of 1 protomer is occluded by the pleckstrin homology (PH) domain of another protomer, suggesting that kindlin-3 is auto-inhibited upon trimer formation.
[more...]
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Generation of aggregates of $\alpha$-lactalbumin by UV-B light exposure
Journal of Agricultural and Food Chemistry. 2020. Zhao, ZC et al. Univ Copenhagen, Fac Sci, Dept Food Sci, DK-1958 Frederiksberg C, Denmark.
ABSTRACT: Whey proteins are widely used as ingredients in the form of aggregates to obtain certain functionalities in food applications. The aim of this study was to understand how UV illumination generates aggregates of alpha-lactalbumin (alpha-LA) as an alternative to heat treatments traditionally used for industrial production of protein aggregates. Absorption of UV light by alpha-LA caused cleavage of disulfide bonds and release of thiol groups, which resulted in primarily disulfide-mediated aggregation.
[more...]
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Structure of complete Pol II--DSIF--PAF--SPT6 transcription complex reveals RTF1 allosteric activation
Nature Structural & Molecular Biology. 2020. Vos, SM et al. Max Planck Inst Biophys Chem, Dept Mol Biol, Gottingen, Germany.
ABSTRACT: Transcription by RNA polymerase II (Pol II) is carried out by an elongation complex. We previously reported an activated porcine Pol II elongation complex, EC*, encompassing the human elongation factors DSIF, PAF1 complex (PAF) and SPT6. Here we report the cryo-EM structure of the complete EC* that contains RTF1, a dissociable PAF subunit critical for chromatin transcription. The RTF1 Plus3 domain associates with Pol II subunit RPB12 and the phosphorylated C-terminal region of DSIF subunit SPT5. RTF1 also forms four alpha-helices that extend from the Plus3 domain along the Pol II protrusion and RPB10 to the polymerase funnel.
[more...]
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Functional and mass spectrometric evaluation of an anti-tick antigen based on the P0 peptide conjugated to Bm86 protein
Pathogens. 2020. Mallon, AR et al. Ctr Genet Engn & Biotechnol CIGB, Anim Biotechnol Dept, Havana 10600, Cuba.
ABSTRACT: A synthetic 20 amino acid peptide of the ribosomal protein P0 from ticks, when conjugated to keyhole limpet hemocyanin fromMegathura crenulataand used as an immunogen againstRhipicephalus microplusandRhipicephalus sanguineuss.l. species, has shown efficacies of around 90%. There is also experimental evidence of a high efficacy of this conjugate againstAmblyomma mixtumandIxodes ricinusspecies, which suggest that this antigen could be a good broad-spectrum anti-tick vaccine candidate. In this study, the P0 peptide (pP0) was chemically conjugated to Bm86 as a carrier protein.
[more...]
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A cross-linking mass spectrometry approach defines protein interactions in yeast mitochondria
Molecular & Cellular Proteomics. 2020. Linden, A et al. Max Planck Inst Biophys Chem, Bioanalyt Mass Spectrometry Grp, Gottingen, Germany.
ABSTRACT: Protein cross-linking and the analysis of cross-linked peptides by mass spectrometry is currently receiving much attention. Not only is this approach applied to isolated complexes to provide information about spatial arrangements of proteins, but it is also increasingly applied to entire cells and their organelles. As in quantitative proteomics, the application of isotopic labeling further makes it possible to monitor quantitative changes in the protein-protein interactions between different states of a system.
[more...]
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Structure of the transcription coactivator SAGA
Nature. 2020. Haibo Wang et al. Max Planck Institute for Biophysical Chemistry, Department of Molecular Biology, Göttingen, Germany
ABSTRACT: Gene transcription by RNA polymerase II is regulated by activator proteins that recruit the coactivator complexes SAGA (Spt-Ada-Gcn5-acetyltransferase)(1,2) and transcription factor IID (TFIID)(2-4). SAGA is required for all regulated transcription(5) and is conserved among eukaryotes(6). SAGA contains four modules(7-9): the activator-binding Tra1 module, the core module, the histone acetyltransferase (HAT) module and the histone deubiquitination (DUB) module. Previous studies provided partial structures(10-14), but the structure of the central core module is unknown.
[more...]
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A synthetic peptide library for benchmarking crosslinking-mass spectrometry search engines for proteins and protein complexes
Nature Communications. 2020. Beveridge, R et al. Vienna Bioctr VBC, Res Inst Mol Pathol IMP, Campus Vienna Bioctr 1, A-1030 Vienna, Austria.
ABSTRACT: Crosslinking-mass spectrometry (XL-MS) serves to identify interaction sites between proteins. Numerous search engines for crosslink identification exist, but lack of ground truth samples containing known crosslinks has precluded their systematic validation. Here we report on XL-MS data arising from measuring synthetic peptide libraries that provide the unique benefit of knowing which identified crosslinks are true and which are false. The data are analysed with the most frequently used search engines and the results filtered to an estimated false discovery rate of 5%.
[more...]
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The acetyltransferase Eco1 elicits cohesin dimerization during S phase
Journal of Biological Chemistry. 2020. Shi, D et al. China Agr Univ, State Key Lab Agrobiotechnol, Beijing 100193, Peoples R China.
ABSTRACT: Cohesin is a DNA-associated protein complex that forms a tripartite ring controlling sister chromatid cohesion, chromosome segregation and organization, DNA replication, and gene expression. Sister chromatid cohesion is established by the protein acetyltransferase Eco1, which acetylates two conserved lysine residues on the cohesin subunit Smc3 and thereby ensures correct chromatid separation in yeast (Saccharomyces cerevisiae) and other eukaryotes. However, the consequence of Eco1-catalyzed cohesin acetylation is unknown, and the exact nature of the cohesive state of chromatids remains controversial.
[more...]
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Rett syndrome-causing mutations compromise MeCP2-mediated liquid--liquid phase separation of chromatin
Cell Research. 2020. Wang, L et al. Tsinghua Univ, Beijing Adv Innovat Ctr Struct Biol, Tsinghua Univ Peking Univ Joint Ctr Life Sci, Sch Life Sci,Beijing Frontier Res Ctr Biol Struct, Beijing, Peoples R China.
ABSTRACT: Rett syndrome (RTT), a severe postnatal neurodevelopmental disorder, is caused by mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MeCP2). MeCP2 is a chromatin organizer regulating gene expression. RTT-causing mutations have been shown to affect this function. However, the mechanism by which MeCP2 organizes chromatin is unclear. In this study, we found that MeCP2 can induce compaction and liquid-liquid phase separation of nucleosomal arrays in vitro, and DNA methylation further enhances formation of chromatin condensates by MeCP2.
[more...]
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Discovery of a regulatory subunit of the yeast fatty acid synthase
Cell. 2020. Singh, K et al. Max Planck Inst Biophys Chem, Dept Struct Dynam, D-37077 Gottingen, Germany.
ABSTRACT: Fatty acid synthases (FASs) are central to metabolism but are also of biotechnological interest for the production of fine chemicals and biofuels from renewable resources. During fatty acid synthesis, the growing fatty acid chain is thought to be shuttled by the dynamic acyl carrier protein domain to several enzyme active sites. Here, we report the discovery of a gamma subunit of the 2.6 megadalton alpha(6)-beta(6) S. cerevisiae FAS, which is shown by high-resolution structures to stabilize a rotated FAS conformation and rearrange ACP domains from equatorial to axial positions.
[more...]
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A receptor for the complement regulator factor H increases transmission of trypanosomes to tsetse flies
Nature Communications. 2020. Macleod, OJS et al. Univ Cambridge, Dept Biochem, Tennis Court Rd, Cambridge CB2 1QW, England.
ABSTRACT: Persistent pathogens have evolved to avoid elimination by the mammalian immune system including mechanisms to evade complement. Infections with African trypanosomes can persist for years and cause human and animal disease throughout sub-Saharan Africa. It is not known how trypanosomes limit the action of the alternative complement pathway. Here we identify an African trypanosome receptor for mammalian factor H, a negative regulator of the alternative pathway. Structural studies show how the receptor binds ligand, leaving inhibitory domains of factor H free to inactivate complement C3b deposited on the trypanosome surface.
[more...]
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Integrative structure and function of the yeast exocyst complex
Protein Science. 2020. Ganesan, SJ et al. Univ Calif San Francisco, Dept Bioengn & Therapeut Sci, Byers Hall,1700 4th St,Suite 503B, San Francisco, CA 94158 USA.
ABSTRACT: Exocyst is an evolutionarily conserved hetero-octameric tethering complex that plays a variety of roles in membrane trafficking, including exocytosis, endocytosis, autophagy, cell polarization, cytokinesis, pathogen invasion, and metastasis. Exocyst serves as a platform for interactions between the Rab, Rho, and Ral small GTPases, SNARE proteins, and Sec1/Munc18 regulators that coordinate spatial and temporal fidelity of membrane fusion. However, its mechanism is poorly described at the molecular level.
[more...]
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Molecular topology of RNA polymerase I upstream activation factor
Molecular and Cellular Biology. 2020. Knutson, BA et al. SUNY Upstate Med Univ, Dept Biochem & Mol Biol, Syracuse, NY 13210 USA.
ABSTRACT: Upstream activation factor (UAF) is a multifunctional transcription factor in Saccharomyces cerevisiae that plays dual roles in activating RNA polymerase I (Pol I) transcription and repression of Pol II. For Pol I, UAF binds to a specific upstream element in the ribosomal DNA (rDNA) promoter and interacts with two other Pol I initiation factors, the TATA-binding protein (TBP) and core factor (CF). We used an integrated combination of chemical cross-linking mass spectrometry (CXMS), molecular genetics, protein biochemistry, and structural modeling to understand the topological framework responsible for UAF complex formation.
[more...]
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Evidence of allosteric coupling between substrate binding and adx recognition in the vitamin D carbon-24 hydroxylase CYP24A1
Biochemistry. 2020. Kumar, A et al. Univ Buffalo, Jacobs Sch Med, Dept Biochem, Buffalo, NY 14203 USA.
ABSTRACT: Metabolic inactivation of 1,25(OH)2D3 requires molecular recognition between the mitochondrial enzyme cytochrome P450 24A1 (CYP24A1) and its cognate redox partner adrenodoxin (Adx). Recent evidence supports a model of CYP24A1 function in which substrate binding and Adx recognition are structurally linked. However, the details of this allosteric connection are not clear. In this study, we utilize chemical cross-linking coupled to mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and CYP24A1 functional assays to inform a working model of a CYP24A1Adx complex.
[more...]
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Fragment mass spectrum prediction facilitates site localization of phosphorylation
Journal of Proteome Research. 2020. Yang, Y et al. Fudan Univ, Dept Chem, Shanghai 200000, Peoples R China.
ABSTRACT: Liquid chromatography tandem mass spectrometry (LCMS/MS) has been the most widely used technology for phosphoproteomics studies. As an alternative to database searching and probability-based phosphorylation site localization approaches, spectral library searching has been proved to be effective in the identification of phosphopeptides. However, incompletion of experimental spectral libraries limits the identification capability. Herein, we utilize MS/MS spectrum prediction coupled with spectral matching for site localization of phosphopeptides.
[more...]
Use: pDeep; pNovo



Jasmonate-independent regulation of digestive enzyme activity in the carnivorous butterwort Pinguicula$\times$ Tina
Journal of experimental botany. 2020. Kocab, O et al. Palacky Univ, Fac Sci, Ctr Reg Hana Biotechnol & Agr Res, Dept Biophys, Slechtitelu 27, CZ-78371 Olomouc, Czech Republic
ABSTRACT: Carnivorous plants within the order Caryophyllales use jasmonates, a class of phytohormone, in the regulation of digestive enzyme activities. We used the carnivorous butterwort Pinguicula x Tina from the order Lamiales to investigate whether jasmonate signaling is a universal and ubiquitous signaling pathway that exists outside the order Caryophyllales. We measured the electrical signals, enzyme activities, and phytohormone tissue levels in response to prey capture. Mass spectrometry was used to identify proteins in the digestive secretion.
[more...]
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Identification and dereplication of endophytic Colletotrichum strains by MALDI TOF mass spectrometry and molecular networking
Scientific reports. 2020. Barthelemy, M et al. Univ Paris Saclay, Inst Chim Subst Nat, CNRS, UPR 2301, Ave Terrasse, F-91198 Gif Sur Yvette, France.
ABSTRACT: The chemical diversity of biologically active fungal strains from 42 Colletotrichum, isolated from leaves of the tropical palm species Astrocaryum sciophilum collected in pristine forests of French Guiana, was investigated. The collection was first classified based on protein fingerprints acquired by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) correlated with cytotoxicity. Liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-HRMS/MS) data from ethyl acetate extracts were acquired and processed to generate a massive molecular network (MN) using the MetGem software.
[more...]
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Development of a Sample-Preparation Workflow for Sulfopeptide Enrichment: From Target Analysis to Challenges in Shotgun Sulfoproteomics
Analytical Chemistry. 2020. Capriotti, AL et al. Sapienza Univ Roma, Dept Chem, I-00185 Rome, Italy.
ABSTRACT: Protein tyrosine O-sulfation is an important post-translational modification, as it has been correlated to inflammation, virus infection, and signal pathways. Nevertheless, methods for the characterization of protein sulfation by sulfopeptide enrichment are currently limited. In this Article, two standard compounds, representative of mono- and disulfated peptides, were used to compare the enrichment capabilities of five sorbent materials: two commercial weak anion-exchange mixed-mode sorbents (Strata X-AW and Oasis WAX) and three phosphopeptide enrichment materials based on affinity chromatography to either immobilized metals (IMAC) or metal oxides, i.e., Fe3+, TiO2, or Ti4+.
[more...]
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A new opening for the tricky untargeted investigation of natural and modified short peptides
Talanta. 2020. Cerrato, A et al. Sapienza Univ Roma, Dept Chem, Piazzale Aldo Moro 5, I-00185 Rome, Italy.
ABSTRACT: Short peptides are of extreme interest in clinical and food research fields, nevertheless they still represent a crucial analytical issue. The main aim of this paper was the development of an analytical platform for a considerable advancement in short peptides identification. For the first time, short sequences presenting both natural and post-translationally modified amino acids were comprehensively studied thanks to the generation of specific databases. Short peptide databases had a dual purpose.
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Identification Of A Novel Histone Derived Antimicrobial Peptide In Airway Surface Liquid.
FASEB JOURNAL. 2020. Biggart, MGS et al. St George’s University Of London
ABSTRACT: The airway surface liquid (ASL), a protective layer secreted by the airway epithelium, represents the first line of defence against inhaled infectious material. It contains a complex array of secreted proteins and peptides that aid the neutralisation and removal of inhaled microbes and toxicants. The ASL also contains many proteases that can cleave proteins to generate further bioactive peptides. We therefore hypothesised that the airway ASL sustained an extensive peptidome containing novel bioactive peptides.
[more...]
Use: pNovo



Identifying sialylation linkages at the glycopeptide level by glycosyltransferase labeling assisted mass spectrometry (GLAMS)
Analytical chemistry. 2020. Zhu, H et al. Georgia State Univ, Dept Chem, Atlanta, GA 30303 USA.
ABSTRACT: Precise assignment of sialylation linkages at the glycopeptide level is of importance in bottom-up glycoproteomics and an indispensable step to understand the function of glycoproteins in pathogen-host interactions and cancer progression. Even though some efforts have been dedicated to the discrimination of alpha 2,3/alpha 2,6-sialylated isomers, unambiguous identification of sialoglycopeptide isomers is still needed. Herein, we developed an innovative glycosyltransferase labeling assisted mass spectrometry (GLAMS) strategy.
[more...]
Use: pParse; pGlyco



A streamlined pipeline for multiplexed quantitative site-specific N-glycoproteomics
Nature Communications. 2020. Fang, P et al. Max Planck Inst Biophys Chem, Bioanalyt Mass Spectrometry Grp, D-37077 Gottingen, Germany.
ABSTRACT: Regulation of protein N-glycosylation is essential in human cells. However, large-scale, accurate, and site-specific quantification of glycosylation is still technically challenging. We here introduce SugarQuant, an integrated mass spectrometry-based pipeline comprising protein aggregation capture (PAC)-based sample preparation, multi-notch MS3 acquisition (Glyco-SPS-MS3) and a data-processing tool (GlycoBinder) that enables confident identification and quantification of intact glycopeptides in complex biological samples.
[more...]
Use: pGlyco; pParse



基于多头注意力机制和残差神经网络的肽谱匹配打分算法
Journal of Computer Applications. 2020. 闵鑫 et al. 山东理工大学计算机科学与技术学院
ABSTRACT: Peptide spectrum match scoring algorithm plays a key role in the peptide sequence identification,and the traditional scoring algorithm cannot effectively make full use of the peptide fragmentation pattern to perform scoring. In order to solve the problem,a multi-classification probability sum scoring algorithm combined with the peptide sequence information representation called deepscore- alpha was proposed. In this algorithm,the second scoring was not performed with the consideration of global information,and there was no limitation on the similarity calculation method of theoretical mass spectrum and experimental mass spectrum.
[more...]
Use: pParse; pDeep



Large-scale identification of N-linked intact glycopeptides in human serum using HILIC enrichment and spectral library search
Molecular & cellular proteomics : MCP. 2020. Shu, Qingbo et al. Laboratory of Protein and Peptide Pharmaceuticals & Proteomics Laboratory, Institute of Biophysics, Chinese Academy of Sciences
ABSTRACT: Large-scale identification of N-linked intact glycopeptides by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in human serum is challenging because of the wide dynamic range of serum protein abundances, the lack of a complete serum N-glycan database and the existence of proteoforms. In this regard, a spectral library search method was presented for the identification of N-linked intact glycopeptides from N-linked glycoproteins in human serum with target-decoy and motif-specific false discovery rate (FDR) control.
[more...]
Use: pParse; pGlyco



Virus-receptor interactions of glycosylated SARS-CoV-2 spike and human ACE2 receptor
Cell host & microbe. 2020. Zhao, P et al. Univ Georgia, Dept Biochem & Mol Biol, Complex Carbohydrate Res Ctr, Athens, GA 30602 USA.
ABSTRACT: The SARS-CoV-2 betacoronavirus uses its highly glycosylated trimeric Spike protein to bind to the cell surface receptor angiotensin converting enzyme 2 (ACE2) glycoprotein and facilitate host cell entry. We utilized glycomics-informed glycoproteomics to characterize site-specific microheterogeneity of glycosylation for a recombinant trimer Spike mimetic immunogen and for a soluble version of human ACE2. We combined this information with bioinformatics analyses of natural variants and with existing 3D structures of both glycoproteins to generate molecular dynamics simulations of each glycoprotein both alone and interacting with one another.
[more...]
Use: pGlyco



Mass spectrometry analysis of newly emerging coronavirus HCoV-19 spike protein and human ACE2 reveals camouflaging glycans and unique post-translational modifications
Engineering (Beijing, China). 2020. Sun, Zeyu et al. State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Disease, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310011, China
ABSTRACT: The COVID-19 pandemic has led to worldwide efforts to understand the biological traits of the newly identified HCoV-19 virus. In this mass spectrometry (MS)-based study, we reveal that out of 21 possible glycosites in the HCoV-19 S protein, 20 are completely occupied by N-glycans, predominantly of the oligomannose type. All seven glycosylation sites in human angiotensin I converting enzyme 2 (hACE2) were found to be completely occupied, mainly by complex N-glycans. However, glycosylation did not directly contribute to the binding affinity between HCoV-19 S and hACE2.
[more...]
Use: pGlyco



Relative retention time estimation improves N-glycopeptide identifications by LC--MS/MS
Journal of proteome research. 2020. Klein, J et al. Boston Univ, Program Bioinformat, Boston, MA 02215 USA.
ABSTRACT: Glycopeptides identified by tandem mass spectrometry rely on the identification of the peptide backbone sequence and the attached glycan(s) by the incomplete fragmentation of both moieties. This may lead to ambiguous identifications where multiple structures could explain the same spectrum equally well due to missing information in the mass spectrum or incorrect precursor mass determination. To date, approaches to solving these problems have been limited, and few inroads have been made to address these issues.
[more...]
Use: pGlyco



SugarPy facilitates the universal, discovery-driven analysis of intact glycopeptides
Bioinformatics. 2020. Schulze, S et al. Univ Penn, Dept Biol, Leidy Labs, Philadelphia, PA 19104 USA.
ABSTRACT: Motivation: Protein glycosylation is a complex post-translational modification with crucial cellular functions in all domains of life. Currently, large-scale glycoproteomics approaches rely on glycan database dependent algorithms and are thus unsuitable for discovery-driven analyses of glycoproteomes. Results: Therefore, we devised SugarPy, a glycan database independent Python module, and validated it on the glycoproteome of human breast milk. We further demonstrated its applicability by analyzing glycoproteomes with uncommon glycans stemming from the green alga Chlamydomonas reinhardtii and the archaeon Haloferax volcanii.
[more...]
Use: pGlyco



Characterization of N-linked intact glycopeptide signatures of plasma IgGs from patients with prostate carcinoma and benign prostatic hyperplasia for diagnosis pre-stratification
Analyst. 2020. Zhang, Y et al. Sichuan Univ, West China Hosp, MOH, Key Lab Transplant Engn & Immunol, Chengdu 610041, Peoples R China.
ABSTRACT: The discovery of novel non-invasive biomarkers for discriminating between prostate carcinoma (PCa) patients and benign prostatic hyperplasia (BPH) patients is necessary to reduce the burden of biopsies, avoid overdiagnosis and improve quality of life. Previous studies suggest that abnormal glycosylation of immunoglobulin gamma molecules (IgGs) is strongly associated with immunological diseases and prostate diseases. Hence, characterizing N-linked intact glycopeptides of IgGs that correspond to theN-glycan structure with specific site information might enable a better understanding of the molecular pathogenesis and discovery of novel signatures in preoperative discrimination of BPH from PCa.
[more...]
Use: pGlyco



N-glycan structures of target cancer biomarker characterized by two-dimensional gel electrophoresis and mass spectrometry
Analytica Chimica Acta. 2020. Liu, S et al. Shanghai Jiao Tong Univ, Sch Life Sci & Biotechnol, Joint Int Res Lab Metab & Dev Sci, State Key Lab Microbial Metab, Shanghai 200240, Peoples R China.
ABSTRACT: Glycoproteins are important biomarkers for cancers, while most glycoproteomics biomarkers suffering from low sensitivity and specificity due to their uncharacterized glycan structures. AZGP1 is a potential biomarker for salivary diagnostics of lung cancer, which is used as a model glycoprotein in this study for method development. We initially analyzed salivary N-glycoproteome by using lectin affinity chromatography and more than 300 N-glycoproteins were identified, including AZGP1. 7 gel spots of AZGP1 were resolved by two-dimensional gel electrophoresis and further confirmed by two-dimensional western blot as well as mass spectrometry.
[more...]
Use: pGlyco



Glycomics-informed glycoproteomic analysis of site-specific glycosylation for SARS-CoV-2 spike protein
STAR protocols. 2020. Rosenbalm, Katelyn E et al. Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, USA
ABSTRACT: This protocol describes an integrated approach for analyzing site-specific N- and O-linked glycosylation of SARS-CoV-2 spike protein by mass spectrometry. Glycoproteomics analyzes intact glycopeptides to examine site-specific microheterogeneity of glycoproteins. Glycomics provides structural characterization on any glycan assignments by glycoproteomics. This procedure can be modified and applied to a variety of N- and/or O-linked glycoproteins. Combined with bioinformatics, the glycomics-informed glycoproteomics may be useful in generating 3D molecular dynamics simulations of certain glycoproteins alone or interacting with one another.
[more...]
Use: pGlyco



Glycoproteomic analysis of human urinary exosomes
Analytical Chemistry. 2020. Brown, CJ et al. Indiana Univ, Dept Chem, Bloomington, IN 47401 USA.
ABSTRACT: Exosomes represent a class of secreted biological vesicles, which have recently gained attention due to their function as intertissue and interorganism transporters of genetic materials, small molecules, lipids, and proteins. Although the protein constituents of these exosomes are often glycosylated, a large-scale characterization of the glycoproteome has not yet been completed. This study identified 3144 unique glycosylation events belonging to 378 glycoproteins and 604 unique protein sites of glycosylation.
[more...]
Use: pGlyco



An ultrafast and highly efficient enrichment method for both N-Glycopeptides and N-Glycans by bacterial cellulose
Analytica Chimica Acta. 2020. Wu, MX et al. Fudan Univ, Dept Chem, Shanghai 200032, Peoples R China.
ABSTRACT: A powerful and fast glycopeptide/glycan enrichment method is critical for the efficiency and throughput of mass spectrometry (MS)-based glycoproteomic and glycomic analyses, especially for large-scale sample analysis. Here, we report an ultrafast and effective method for both intact N-glycopeptide and N-glycan enrichment and apply it to human serum samples. In this method, a natural hydrophilic material, bacterial cellulose (BC), was adopted and fully optimized for enrichment. This method offers the following advantages: (i) The enrichment material has natural hydrophilicity and is low-cost, biocompatible, biodegradable and easily accessible; (ii) the whole enrichment procedure is remarkably simple and fast.
[more...]
Use: pGlyco



One-step synthesis of hydrophilic microspheres for highly selective enrichment of N-linked glycopeptides
Analytica Chimica Acta. 2020. Zhang, N et al. Chinese Acad Sci, Dalian Inst Chem Phys, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Peoples R China.
ABSTRACT: A polyacrylamide-based hydrophilic microsphere with a lot of hydroxyl groups on surface (PAM-OH HMS) was prepared in one step. The synthetic process was simple reverse suspension polymerization without any chemical derivation or grafting steps. The properties of obtained HMS were characterized by scanning electron microscopy (SEM), static water contact angle measurement, and FT-IR. The abundant hydroxyl groups on the surface make the material highly good hydrophilic and thus it was utilized for N-glycopeptides enrichment.
[more...]
Use: pGlyco



Computational classification of core and outer fucosylation of N-glycoproteins in human plasma using collision-induced dissociation in mass spectrometry
Rapid Communications in Mass Spectrometry. 2020. Jeong, HK et al. Korea Basic Sci Inst, Res Ctr Bioconvergence Anal, 162 Yeongudanji Ro, Cheongju 28119, South Korea.
ABSTRACT: Rationale Glycoprotein fucosylation, one of the major posttranslational modifications, is known to be highly involved in proteins related to various cancers. Fucosylation occurs in the core and/or outer sites of N-glycopeptides. Elucidation of the fucosylation type of N-glycoproteins is therefore important. However, it has remained a challenge to classify the fucosylation types of N-glycopeptides using collision-induced dissociation (CID) tandem mass (MS/MS) spectra. Methods The relative intensities of the Y1F, Y2F, Y3F, and Y4F product ions in the CID-MS/MS spectra of the IgG N-glycopeptides were measured for core fucosylation.
[more...]
Use: pGlyco



Peptide release after simulated infant in vitro digestion of dry heated cow’s milk protein and transport of potentially immunoreactive peptides across the Caco-2 cell monolayer
Nutrients. 2020. Zenker, HE et al. Wageningen Univ & Res Ctr, Food Qual & Design Grp, NL-6708 WG Wageningen, Netherlands.
ABSTRACT: Dry heating of cow's milk protein, as applied in the production of "baked milk", facilitates the resolution of cow's milk allergy symptoms upon digestion. The heating and glycation-induced changes of the protein structure can affect both digestibility and immunoreactivity. The immunological consequences may be due to changes in the peptide profile of the digested dry heated milk protein. Therefore, cow's milk protein powder was heated at low temperature (60 degrees C) and high temperature (130 degrees C) and applied to simulated infant in vitro digestion.
[more...]
Use: pGlyco



Quantitative analysis of $\alpha$-1-antitrypsin glycosylation isoforms in HCC patients using LC-HCD-PRM-MS
Analytical Chemistry. 2020. Yin, HD et al. Hong Kong Polytech Univ, Shenzhen Res Inst, Shenzhen, Peoples R China.
ABSTRACT: The change in glycosylation of serum proteins is often associated with the development of various diseases and thus can be used for diagnosis. In this study, a liquid chromatography-tandem mass spectrometry-based method is used for accurate structural analysis and quantification of site-specific glycoforms of serum alpha-1-antitrypsin (A1AT) in early-stage HCC and cirrhosis patients. Serum protein A1AT was purified from patient sera by immunoprecipitation with anti-A1AT antibody conjugated agarose beads, and the isolated A1AT protein was digested and analyzed by LC-MS/MS.
[more...]
Use: pGlyco



Quantitative analysis of global protein stability rates in tissues
Scientific reports. 2020. McClatchy, DB et al. Scripps Res Inst, Dept Mol Med, La Jolla, CA 92037 USA.
ABSTRACT: Protein degradation is an essential mechanism for maintaining proteostasis in response to internal and external perturbations. Disruption of this process is implicated in many human diseases. We present a new technique, QUAD (Quantification of Azidohomoalanine Degradation), to analyze the global degradation rates in tissues using a non-canonical amino acid and mass spectrometry. QUAD analysis reveals that protein stability varied within tissues, but discernible trends in the data suggest that cellular environment is a major factor dictating stability.
[more...]
Use: pQuant



Temporal Quantitative Profiling of Newly Synthesized Proteins during A$\beta$ Accumulation
Journal of Proteome Research. 2020. Ma, YH et al. Scripps Res Inst, Dept Chem Physiol & Mol & Cellular Neurobiol, La Jolla, CA 92037 USA.
ABSTRACT: Accumulation of aggregated amyloid beta (A beta) in the brain is believed to impair multiple cellular pathways and play a central role in Alzheimer's disease pathology. However, how this process is regulated remains unclear. In theory, measuring protein synthesis is the most direct way to evaluate a cell's response to stimuli, but to date, there have been few reliable methods to do this. To identify the protein regulatory network during the development of A beta deposition in AD, we applied a new proteomic technique to quantitate newly synthesized protein (NSP) changes in the cerebral cortex and hippocampus of 2-, 5-, and 9-month-old APP/PS1 AD transgenic mice.
[more...]
Use: pQuant



In silico spectral libraries by deep learning facilitate data-independent acquisition proteomics
Nature communications. 2020. Yang, Y et al. Fudan Univ, Dept Chem, Shanghai Stomatol Hosp, Shanghai 200000, Peoples R China.
ABSTRACT: Data-independent acquisition (DIA) is an emerging technology for quantitative proteomic analysis of large cohorts of samples. However, sample-specific spectral libraries built by data-dependent acquisition (DDA) experiments are required prior to DIA analysis, which is time-consuming and limits the identification/quantification by DIA to the peptides identified by DDA. Herein, we propose DeepDIA, a deep learning-based approach to generate in silico spectral libraries for DIA analysis. We demonstrate that the quality of in silico libraries predicted by instrument-specific models using DeepDIA is comparable to that of experimental libraries, and outperforms libraries generated by global models.
[more...]
Use: pDeep



Full-spectrum prediction of peptides tandem mass spectra using deep neural network
Analytical chemistry. 2020. Liu, KY et al. Indiana Univ, Sch Informat Comp & Engn, Bloomington, IN 47405 USA.
ABSTRACT: The ability to predict tandem mass (MS/MS) spectra from peptide sequences can significantly enhance our understanding of the peptide fragmentation process and could improve peptide identification in proteomics. However, current approaches for predicting high-energy collisional dissociation (HCD) spectra are limited to predict the intensities of expected ion types, that is, the a/b/c/x/y/z ions and their neutral loss derivatives (referred to as backbone ions). In practice, backbone ions only account for <70% of total ion intensities in HCD spectra, indicating many intense ions are ignored by current predictors.
[more...]
Use: pDeep



DeepRescore: leveraging deep learning to improve peptide identification in immunopeptidomics
Proteomics. 2020. Li, K et al. Baylor Coll Med, Lester & Sue Smith Breast Ctr, Houston, TX 77030 USA.
ABSTRACT: The identification of major histocompatibility complex (MHC)-binding peptides in mass spectrometry (MS)-based immunopeptideomics relies largely on database search engines developed for proteomics data analysis. However, because immunopeptidomics experiments do not involve enzymatic digestion at specific residues, an inflated search space leads to a high false positive rate and low sensitivity in peptide identification. In order to improve the sensitivity and reliability of peptide identification, a post-processing tool named DeepRescore is developed.
[more...]
Use: pDeep



Hybrid spectral library combining DIA-MS data and a targeted virtual library substantially deepens the proteome coverage
IScience. 2020. Lou, RH et al. ShanghaiTech Univ, iHuman Inst, Shanghai 201210, Peoples R China.
ABSTRACT: Data-independent acquisition mass spectrometry (DIA-MS) is a powerful technique that enables relatively deep proteomic profiling with superior quantification reproducibility. DIA data mining predominantly relies on a spectral library of sufficient proteome coverage that, in most cases, is built on data-dependent acquisition-based analysis of the same sample. To expand the proteome coverage for a pre-determined protein family, we report herein on the construction of a hybrid spectral library that supplements a DIA experiment-derived library with a protein family-targeted virtual library predicted by deep learning.
[more...]
Use: pDeep




2019




Quick and clean: Cracking sentences encoded in E. coli by LC–MS/MS, de novo sequencing, and dictionary search
EuPA Open Proteomics. 2019. Niu, Lili et al. Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark
ABSTRACT: In this study, we faced the challenge of deciphering a protein that has been designed and expressed by E. coli in such a way that the amino acid sequence encodes two concatenated English sentences. The letters 'O' and 'U' in the sentence are both replaced by 'K' in the protein. The sequence cannot be found online and carried to-be-discovered modifications. With limited information in hand, to solve the challenge, we developed a workflow consisting of bottom-up proteomics, de novo sequencing and a bioinformatics pipeline for data processing and searching for frequently appearing words.
[more...]
Use: pNovo; pFind



结核分枝杆菌 H37Rv 新基因 Rv2742 克隆表达及纯化
Sheng wu gong cheng xue bao = Chinese journal of biotechnology. 2019. 赵加玲 et al. Key Laboratory of Combinational Biosynthesis and Drug Discovery(Wuhan University),Ministry of Education,School of Pharmaceutical Science,Wuhan University,Wuhan 430072,China
ABSTRACT: Rv2742 是本课题组前期基于蛋白质基因组学策略从结核分枝杆菌Mycobacterium tuberculosis H37Rv 中发现、鉴定的遗漏注释基因。文中旨在建立结核分枝杆菌H37Rv 漏注释蛋白Rv2742 的可溶性诱导表达、纯化体系,为进一步探索Rv2742 基因参与的生物学功能奠定基础。前期实验发现构建的pGEX-4T-2-Rv2742、pET-28a-Rv2742、pET-32a-Rv2742 及pMAL-c2X-Rv2742 原核表达载体均无法实现目的蛋白的诱导表达。但经密码子优化后,仅有pMAL-c2X-Rv2742 载体能够实现目的蛋白的可溶性诱导表达。此外,通过比较不同宿主菌、温度及IPTG 浓度对目的蛋白表达量的影响,发现目的蛋白在Rosetta (DE3)中,16 ℃及 0.5 mmol/L IPTG 诱导条件下表达量最高。直链淀粉树脂 (Amylose resin) 亲和层析柱纯化获得较纯的产物,经LC-MS/MS 验证确认是Rv2742 融合蛋白肽段序列。成功获得结核分枝杆菌H37Rv 新基因Rv2742的重组蛋白,可进一步开展其潜在相互作用及免疫原性研究工作。.
Use: pFind



Open-pFind enhances the identification of missing proteins from human testis tissue
Journal of proteome research. 2019. Sun, JS et al. Sun Yat Sen Univ, Sch Life Sci, State Key Lab Biocontrol, Guangzhou 510275, Guangdong, Peoples R China.
ABSTRACT: In recent years, high-throughput technologies have contributed to the development of a more precise picture of the human proteome. However, 2129 proteins remain listed as missing proteins (MPs) in the newest neXtProt release (2019-02). The main reasons for MPs are a low abundance, a low molecular weight, unexpected modifications, membrane characteristics, and so on. Moreover, >50% of the MS/MS data have not been successfully identified in shotgun proteomics. Open-pFind, an efficient open search engine, recently released by the pFind group in China, might provide an opportunity to identify these buried MPs in complex samples.
[more...]
Use: pFind; pDeep



A thiazolidine formation-based approach for ultrafast and highly efficient solid-phase extraction of N-Glycoproteome
Analytica Chimica Acta. 2019. Cai, Y et al. Fudan Univ, Shanghai Canc Ctr, Shanghai 200032, Peoples R China.
ABSTRACT: For mass spectrometry (MS)-based N-glycoproteomics, selective enrichment of N-glycopeptides prior to MS analysis is a crucial step to reduce sample complexity. Enrichment based on covalent coupling is as an increasingly attractive strategy due to the unbiased and highly specific features. However, most of current covalent coupling reactions for N-glycopeptides enrichment are still limited by long coupling time and harsh coupling conditions. Herein, we developed a thiazolidine formation-based approach for ultrafast and highly efficient solid-phase extraction of N-Glycoproteome.
[more...]
Use: pFind



Open search unveils modification patterns in formalin-fixed, paraffin-embedded thermo HCD and SCIEX TripleTOF shotgun proteomes
International Journal of Mass Spectrometry. 2019. Tabb, DL et al. Stellenbosch Univ, Dept Biomed Sci, Div Mol Biol & Human Genet, Bioinformat Unit,South African TB Bioinformat Ini, Cape Town, South Africa.
ABSTRACT: The application of database search algorithms with very wide precursor mass tolerances for the "Open Search" paradigm has brought new efforts at post-translational modification discovery in shotgun proteomes. This approach has motivated the acceleration of database search tools by incorporating fragment indexing features. In this report, we compare open searches and sequence tag searches of high-resolution tandem mass spectra to seek a common "palette" of modifications when analyzing multiple formalin-fixed, paraffin-embedded (FFPE) tissues from Thermo Q-Exactive and SCIEX TripleTOF instruments.
[more...]
Use: pBuild; pFind; pParse



O-GlcNAcylation of Thr12/Ser56 in short-form O-GlcNAc transferase (sOGT) regulates its substrate selectivity
Journal of Biological chemistry. 2019. Liu, L et al. Nankai Univ, Coll Pharm, Tianjin 300353, Peoples R China.
ABSTRACT: O-GlcNAcylation is a ubiquitous protein glycosylation playing different roles on variant proteins. O-GlcNAc transferase (OGT) is the unique enzyme responsible for the sugar addition to nucleocytoplasmic proteins. Recently, multiple O-GlcNAc sites have been observed on short-form OGT (sOGT) and nucleocytoplasmic OGT (ncOGT), both of which locate in the nucleus and cytoplasm in cell. Moreover, O-GlcNAcylation of Ser(389) in ncOGT (1036 amino acids) affects its nuclear translocation in HeLa cells. To date, the major O-GlcNAcylation sites and their roles in sOGT remain unknown.
[more...]
Use: pFind



Direct proteomic mapping of cysteine persulfidation
Antioxidants & redox signaling. 2019. Fu, L et al. Beijing Inst Life, Natl Ctr Prot Sci Beijing, State Key Lab Prote, Beijing Proteome Res Ctr, Beijing 102206, Peoples R China.
ABSTRACT: Aims: Cysteine persulfidation (also called sulfhydration or sulfuration) has emerged as a potential redox mechanism to regulate protein functions and diverse biological processes in hydrogen sulfide (H2S) signaling. Due to its intrinsically unstable nature, working with this modification has proven to be challenging. Although methodological progress has expanded the inventory of persulfidated proteins, there is a continued need to develop methods that can directly and unequivocally identify persulfidated cysteine residues in complex proteomes.
[more...]
Use: pFind; pQuant



Zebrafish prmt5 arginine methyltransferase is essential for germ cell development
DEVELOPMENT. 2019. Zhu, JJ et al. Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan 430072, Hubei, Peoples R China.
ABSTRACT: Protein arginine methyltransferase 5 (Prmt5), a type II arginine methyltransferase, symmetrically dimethylates arginine in nuclear and cytoplasmic proteins. Prmt5 is involved in a variety of cellular processes, including ribosome biogenesis, cellular differentiation, germ cell development and tumorigenesis. However, the mechanisms by which prmt5 influences cellular processes have remained unclear. Here, prmt5 loss in zebrafish led to the expression of an infertile male phenotype due to a reduction in germ cell number, an increase in germ cell apoptosis and the failure of gonads to differentiate into normal testes or ovaries.
[more...]
Use: pFind



Mass spectrometry--driven exploration reveals nuances of neoepitope-driven tumor rejection
JCI Insight. 2019. Ebrahimi-Nik, H et al. Univ Connecticut, Sch Med, Dept Immunol, Farmington, CT 06032 USA.
ABSTRACT: Neoepitopes are the only truly tumor-specific antigens. Although potential neoepitopes can be readily identified using genomics, the neoepitopes that mediate tumor rejection constitute a small minority, and there is little consensus on how to identify them. Here, for the first time to our knowledge, we use a combination of genomics, unbiased discovery mass spectrometry (MS) immunopeptidomics, and targeted MS to directly identify neoepitopes that elicit actual tumor rejection in mice. We report that MS-identified neoepitopes are an astonishingly rich source of tumor rejection-mediating neoepitopes (TRMNs).
[more...]
Use: pFind



Quick and clean: Cracking sentences encoded in E. coli by LC--MS/MS, de novo sequencing, and dictionary search
EuPA Open Proteomics. 2019. Niu, Lili et al. Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark
ABSTRACT: In this study, we faced the challenge of deciphering a protein that has been designed and expressed by E. coli in such a way that the amino acid sequence encodes two concatenated English sentences. The letters 'O' and 'U' in the sentence are both replaced by 'K' in the protein. The sequence cannot be found online and carried to-be-discovered modifications. With limited information in hand, to solve the challenge, we developed a workflow consisting of bottom-up proteomics, de novo sequencing and a bioinformatics pipeline for data processing and searching for frequently appearing words.
[more...]
Use: pNovo; pFind



Skeletal muscle proteome analysis provides insights on high altitude adaptation of yaks
Molecular Biology Reports. 2019. Wen, WT et al. Sichuan Univ, Coll Life Sci, Minist Educ, Key Lab Bioresources & Ecoenvironm, Chengdu 610064, Sichuan, Peoples R China.
ABSTRACT: The differences in proteome profile of longissimus thoracis (LT) muscles of yak (Bos grunniens) and cattle (Bos taurus) were investigated employing isobaric tag for relative and absolute quantification (iTRAQ) approach to identify differentially expressed proteins and to understand the cellular level adaptations of yaks to high altitudes. Fifty-two proteins were differentially expressed in the two species, among which 20 were up-regulated and 32 were down-regulated in yaks. Gene ontology (GO) annotation revealed that most of the differentially expressed proteins were involved in the molecular function of protein binding, catalytic activity, and structural activity.
[more...]
Use: pFind



A chemical strategy for protease substrate profiling
Cell Chemical Biology. 2019. Griswold, AR et al. Mem Sloan Kettering Canc Ctr, Weill Cornell Grad Sch Med Sci, Pharmacol Program, New York, NY 10065 USA.
ABSTRACT: The dipeptidyl peptidases (DPPs) regulate hormones, cytokines, and neuropeptides by cleaving dipeptides after proline from their amino termini. Due to technical challenges, many DPP substrates remain unknown. Here, we introduce a simple method, termed CHOPS (chemical enrichment of protease substrates), for the discovery of protease substrates. CHOPS exploits a 2-pyridinecarboxaldehyde (2PCA)-biotin probe, which selectively biotinylates protein N-termini except those with proline in the second position.
[more...]
Use: pFind



Rapid covalent-probe discovery by electrophile-fragment screening
Journal of the American Chemical Society. 2019. Resnick, E et al. Weizmann Inst Sci, Nancy & Stephen Grand Israel Natl Ctr Personalize, Dept Organ Chem, IL-7610001 Rehovot, Israel.
ABSTRACT: Covalent probes can display unmatched potency, selectivity, and duration of action; however, their discovery is challenging. In principle, fragments that can irreversibly bind their target can overcome the low affinity that limits reversible fragment screening, but such electrophilic fragments were considered nonselective and were rarely screened. We hypothesized that mild electrophiles might overcome the selectivity challenge and constructed a library of 993 mildly electrophilic fragments. We characterized this library by a new high-throughput thiol-reactivity assay and screened them against 10 cysteine-containing proteins.
[more...]
Use: pFind



Assessing the relationship between mass window width and retention time scheduling on protein coverage for data-independent acquisition
Journal of the American Society for Mass Spectrometry. 2019. Li, WX et al. Yale Univ, Yale Canc Biol Inst, West Haven, CT 06516 USA.
ABSTRACT: Due to the technical advances of mass spectrometers, particularly increased scanning speed and higher MS/MS resolution, the use of data-independent acquisition mass spectrometry (DIA-MS) became more popular, which enables high reproducibility in both proteomic identification and quantification. The current DIA-MS methods normally cover a wide mass range, with the aim to target and identify as many peptides and proteins as possible and therefore frequently generate MS/MS spectra of high complexity.
[more...]
Use: pFind



Nitration-induced ubiquitination and degradation control quality of ERK1
Biochemical Journal. 2019. Zhang, YY et al. Chinese Acad Sci, Inst Genet & Dev Biol, State Key Lab Mol Dev Biol, 1 West Beichen Rd, Beijing 100101, Peoples R China.
ABSTRACT: The mitogen-activated protein kinase ERK1/2 (ERKs, extracellular-regulated protein kinases) plays important roles in a wide spectrum of cellular processes and have been implicated in many disease states. The spatiotemporal regulation of ERK activity has been extensively studied. However, scarce information has been available regarding the quality control of the kinases to scavenge malfunctioning ERKs. Using site-specific mutagenesis and mass spectrometry, we found that the disruption of the conserved H-bond between Y210 and E237 of ERK1 through point mutation at or naturally occurring nitration on Y210 initiates a quality control program dependent on chaperon systems and CHIP (C-terminal of Hsp70-interacting protein)-mediated ubiquitination and degradation.
[more...]
Use: pFind



Mutual regulation of receptor-like kinase SIT1 and B'$\kappa$-PP2A shapes the early response of rice to salt stress
The Plant Cell. 2019. Zhao, Ji-Long et al. 1; College of Life Science, Hebei Normal University, Hebei Key Laboratory of 7; Molecular and Cellular Biology, Key Laboratory of Molecular and Cellular Biology 8; of Ministry of Education, Hebei Collaboration Innovation Center for Cell Signaling, 9; Shijiazhuang, Hebei 050024, China
ABSTRACT: The receptor-like kinase SIT1 acts as a sensor in rice (Oryza sativa) roots, relaying salt stress signals via elevated kinase activity to enhance salt sensitivity. Here, we demonstrate that Protein Phosphatase 2A (PP2A) regulatory subunit B'kappa constrains SIT1 activity under salt stress. B'kappa-PP2A deactivates SIT1 directly by dephosphorylating the kinase at Thr515/516, a salt-induced phosphorylation site in the activation loop that is essential for SIT1 activity. B'kappa overexpression suppresses the salt sensitivity of rice plants expressing high levels of SIT1, thereby contributing to salt tolerance.
[more...]
Use: pFind



An integrated proteomic and glycoproteomic study for differences on glycosylation occupancy in rheumatoid arthritis
Analytical and Bioanalytical Chemistry. 2019. Li, X et al. Georgia State Univ, Ctr Diagnost & Therapeut, 50 Decatur St SE, Atlanta, GA 30303 USA.
ABSTRACT: Rheumatoid arthritis (RA) is an autoimmune disease in which certain immune cells are dysfunctional and attack their own healthy tissues. There has been great difficulty in finding an accurate and efficient method for the diagnosis of early-stage RA. The present shortage of diagnostic methods leads to the rough treatments of the patients in the late stages, such as joint removing. Nowadays, there is an increasing focus on glyco-biomarkers discovery for malicious disease via MS-based strategy. In this study, we present an integrated proteomics and glycoproteomics approach to uncover the pathological changes of some RA-related glyco-biomarkers and glyco-checkpoints involved in the RA onset.
[more...]
Use: pBuild; pFind



Precision De Novo Peptide Sequencing Using Mirror Proteases of Ac-LysargiNase and Trypsin for Large-scale Proteomics*[S]
Technological Innovation and Resources. 2019. Hao Yang; Yan-Chang Li; Ming-Zhi Zhao et al. State Key Laboratory of Proteomics; Beijing Proteome Research Center; National Center for Protein Sciences Beijing; Beijing Institute of Lifeomics, Beijing 102206, China;Key Laboratory of Combinatorial Biosynthesis and Drug Discovery of Ministry of Education (Wuhan University), Wuhan University School of Pharmaceutical Sciences, Wuhan 430071, China;College of Life Sciences, Hebei University, Baoding 071002, China(X.P.); Key Lab of Intelligent Information Processing of Chinese Academy of Sciences (CAS), Institute of Computing Technology, CAS; University of Chinese Academy of Sciences; Institute of Computing Technology, CAS, Beijing 100190, China
ABSTRACT: De novo peptide sequencing for large-scale proteomics remains challenging because of the lack of full coverage of ion series in tandem mass spectra. We developed a mirror protease of trypsin, acetylated LysargiNase (Ac-LysargiNase), with superior activity and stability. The mirror spectrum pairs derived from the Ac-LysargiNase and trypsin treated samples can generate full b and y ion series, which provide mutual complementarity of each other, and allow us to develop a novel algorithm, pNovoM, for de novo sequencing.
[more...]
Use: pFind; pNovo



A multi-parallel N-glycopeptide enrichment strategy for high-throughput and in-depth mapping of the N-glycoproteome in metastatic human hepatocellular carcinoma cell lines
Talanta. 2019. Jiang, BY et al. Fudan Univ, Peoples Hosp Shanghai 5, Shanghai 200433, Peoples R China.
ABSTRACT: N-glycosylation is deeply involved in many biological processes, and approximately 50% of mammalian proteins are predicted to be glycosylated. Many large-scale studies have been carried out to reveal the glycosylation status involved in different physiological pathologies across species. However, the lack of a highly specific and high throughput N-glycosylated enrichment method not only results in extended time requirements but also limits the depth of mapping when handling a large number of samples.