pFind Studio: a computational solution for mass spectrometry-based proteomics
2020
Cell host & microbe2020. 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. syringae strains that cannot detoxify SFN. Chemoproteomics analyses showed that SFN covalently modified the cysteine at position 209 of HrpS, a key transcription factor controlling TTSS gene expression. Site-directed mutagenesis and functional analyses further confirmed that Cys209 was responsible for bacterial sensitivity to SFN in vitro and sensitivity to plant defenses conferred by the aliphatic glucosinolate pathway. Collectively, these results illustrate a previously unknown mechanism by which plants disarma pathogenic bacterium.
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NATURE PROTOCOLS2020. 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. We therefore adapted this approach and developed a new chemoproteomic platform, termed 'QTRP' (quantitative thiol reactivity profiling), that relies on the ability of a commercially available thiol-reactive probe IPM (2-iodo-N-(prop-2-yn-1-yl)acetamide) to covalently label, enrich and quantify the reactive cysteinome in cells and tissues. Here, we provide a detailed and updated workflow of QTRP that includes procedures for (i) labeling of the reactive cysteinome from cell or tissue samples (e.g., control versus treatment) with IPM, (ii) processing the protein samples into tryptic peptides and tagging the probe-modified peptides with isotopically labeled azido-biotin reagents containing a photo-cleavable linker via click chemistry reaction, (iii) capturing biotin-conjugated peptides with streptavidin beads, (iv) identifying and quantifying the photo-released peptides by mass spectrometry (MS)-based shotgun proteomics and (v) interpreting MS data by a streamlined informatic pipeline using a proteomics software, pFind 3, and an automatic post-processing algorithm. We also exemplified here how to use QTRP for mining H2O2-sensitive cysteines and for determining the intrinsic reactivity of cysteines in a complex proteome. We anticipate that this protocol should find broad applications in redox biology, chemical biology and the pharmaceutical industry. The protocol for sample preparation takes 3 d, whereas MS measurements and data analyses require 75 min and <30 min, respectively, per sample. Proteomic cysteines can undergo redox reactions and electrophile-derived modifications. In QTRP, a thiol-reactive probe is used to covalently label, enrich and quantify the reactive cysteinome in cultured cells and tissue samples.
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Nature communications2020. 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. Comparison of these structures shows a progressive maturation for the functional regions, such as peptidyl transferase centre and peptide exit tunnel, and illustrate a sequence of factor-assisted rRNA maturation events. These data facilitate our understanding of the global conservation of ribosome assembly in eukaryotes and species-specific features of human assembly factors. Ribosome biogenesis in eukaryotes is a complex process that involves more than 200 protein factors. Here the authors present a structural analysis of a collection of human pre-60S structures sampled through a nuclear export adaptor NMD3, representing structural snapshots of pre-60S particles immediately before and after passing through nuclear pore complex.
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Nature communications2020. 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. We further identify multiple trans-acting factors that bind these IRES-like elements to initiate translation. Using mass-spectrometry data, hundreds of circRNA-coded peptides are identified, most of which have low abundance due to rapid degradation. As judged by mass-spectrometry, 50% of translatable endogenous circRNAs undergo rolling circle translation, several of which are experimentally validated. Consistently, mutations of the IRES-like element in one circRNA reduce its translation. Collectively, our findings suggest a pervasive translation of circRNAs, providing profound implications in translation control.Unbiased screen of random sequences identified many short IRES-like elements to drive circular RNA translation and hundreds of rolling circle translation events, suggesting a pervasive cap-independent translation in human transcriptome.
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Elife2020. 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. This region harbors an Atg1-binding domain and a homodimerization domain. Dimerizing Atg1 is the main role of Atg11, as it can be bypassed by artificially dimerizing Atg1. In an Atg1 dimer, only one Atg1 molecule needs to be catalytically active, suggesting that Atg1 activation can be achieved through cis-autophosphorylation. We propose that mediating Atg1 oligomerization and activation may be a conserved function of Atg11/FIP200 family proteins and cis-autophosphorylation may be a general mechanism of Atg1 activation.
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JOURNAL OF THE AMERICAN CHEMICAL SOCIETY2020. 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. We further validate the generality of this active-site residue among NRPS Ox domains. These findings not only resolve the biosynthetic pathway mediated by indigoidine synthetase but enable mechanistic insight into NRPS Ox domains.
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Plant Physiology2020. 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. Here, we report that RIN and Tomato AGAMOUS-LIKE1 (TAGL1) directly bind to the promoter region of SlFERL and further activate its expression transcriptionally, suggesting a potential role of SlFERL in fruit ripening. Overexpression of SlFERL significantly accelerated the ripening process of tomato fruit, whereas RNA interference knockdown of SlFERL resulted in delayed fruit ripening. Moreover, a surface plasmon resonance assay coupled with tandem mass spectrometry and a protein interaction assay revealed that SlFERL interacts with the key enzyme S-adenosyl-Met synthetase 1 (SlSAMS1) in the ethylene biosynthesis pathway, leading to increased S-adenosyl-Met accumulation and elevated ethylene production. Thus, SlFERL serves as a positive regulator of ethylene production and fruit ripening. This study provides clues to the molecular regulatory networks underlying fruit ripening.
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Nature microbiology2020. 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. Mechanistic characterization through viral RNA profiling and in vitro MeV polymerase assays identified a block in the initiation phase of the viral polymerase. GHP-88309 showed nanomolar potency against HPIV3 isolates in well-differentiated human airway organoid cultures, was well tolerated (selectivity index > 7,111) and orally bioavailable, and provided complete protection against lethal infection in a Sendai virus mouse surrogate model of human HPIV3 disease when administered therapeutically 48 h after infection. Recoverees had acquired robust immunoprotection against reinfection, and viral resistance coincided with severe attenuation. This study provides proof of the feasibility of a well-behaved broad-spectrum allosteric antiviral and describes a chemotype with high therapeutic potential that addresses major obstacles of anti-paramyxovirus drug development. In this Article, the authors report the discovery of a small molecule (GHP-88309) that is a new class of allosteric viral polymerase inhibitor that works against two different genera of paramyxoviruses.
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Cell Chemical Biology2020. 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. Through a combination of protein-centric and peptide-centric chemoproteomic experiments, we have identified 165 proteins with a specificity of 94% in the yeast mitochondrial matrix. In addition, we have demonstrated that Alk-Ph is useful for proximity-dependent RNA labeling in yeast, thus expanding the scope of APEX-seq. We envision that this improved APEX-labeling strategy would set the stage for the large-scale mapping of spatial proteome and transcriptome in yeast.
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Journal of proteomics2020. 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. This workflow could now be applied to the study of 141 T. cruzi modified histone peptides, which we used to investigate the dynamics of histone PTMs along the metacyclogenesis and the life cycle of T. cruzi. Global levels of histone acetylation and methylation fluctuates along metacyclogenesis, however most critical differences were observed between parasite life forms. More than 66 histone PTM changes were detected. Strikingly, the histone PTM pattern of metacyclic trypomastigotes is more similar to epimastigotes than to cellular trypomastigotes. Finally, we highlighted changes at the H4 N-terminus and at H3K76 discussing their impact on the trypanosome biology. Altogether, we have optimized a workflow easily applicable to the analysis of histone PTMs in T. cruzi and generated a dataset that may shed lights on the role of chromatin modifications in this parasite. Significance: Trypanosomes are unicellular parasites that have divergent histone sequences, no chromosome condensation and a peculiar genome/gene regulation. Genes are transcribed from divergent polycistronic regions and post-transcriptional gene regulation play major role on the establishment of transcripts and protein levels. In this regard, the fact that their histones are decorated with multiple PTMs raises interesting questions about their role. Besides, this digenetic organism must adapt to different environments changing its metabolism accordingly. As metabolism and epigenetics are closely related, the study of histone PTMs in trypanosomes may enlighten this strikingly, and not yet fully understood, interplay. From a biomedical perspective, the comprehensive study of molecular mechanisms associated to the metacyclogenesis process is essential to create better strategies for controlling Chagas disease.
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