pFind Studio: a computational solution for mass spectrometry-based proteomics
2021
Advanced Biology2021. 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. Integrating GECX with OpenUaa, the direct interactome of thioredoxin is identified in Escherichia coli cells, yielding 289 crosslinked peptides and corresponding to 205 direct binding protein of thioredoxin. These identified direct binders provide evidence for thioredoxin's regulation of redox state and mitochondria energy metabolism. When identifying endogenous conjugation of small ubiquitin-like modifier (SUMO), OpenUaa also markedly improves coverage of SUMOylated peptides by approximate to 92%, revealing new SUMO targets. GECX-OpenUaa will enable efficient identification of direct interactomes of various proteins in live cells.
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JOURNAL OF PHYSICAL CHEMISTRY B2021. 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. Multiscale molecular dynamics simulations in conjunction with nuclear magnetic resonance (NMR) and cross-linking mass spectrometry (XLMS) measurements are used to explore the interactions of aS with an anionic lipid bilayer. The simulations and NMR measurements together reveal a break in the helical structure of the central non-amyloid-beta component (NAC) region of aS in the vicinity of residues 65-70, which may facilitate subsequent oligomer formation. Coarse-grained simulations of aS starting from the structure of aS when bound to a detergent micelle reveal the overall pattern of protein contacts to anionic lipid bilayers, while subsequent all-atom simulations provide details of conformational changes upon membrane binding. In particular, simulations and NMR data for liposome-bound aS indicate incipient beta-strand formation in the NAC region, which is supported by intramolecular contacts seen via XLMS and simulations. Markov state models based on the all-atom simulations suggest a mechanism of conformational change of membrane-bound alpha S via a dynamic helix break in the region of residue 65 in the NAC region. The emergent dynamic model of membrane-interacting alpha S advances our understanding of the mechanism of PD, potentially aiding the design of novel therapeutic approaches.
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CCS Chemistry2021. 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. The dynamic disassembly of the PSII complex, as well as the degradation fragments from both extrinsic and intrinsic protein PSII subunits, were feasibly probed during the loss of O2-evolving activity. Some long-term controversial issues, including the activity loss of PSII complex occurs before the detachment of the Mn4CaO5cluster, were clarified. Finally, a detailed route map of dynamic PSII photodamage was outlined at the molecular level for the first time, which markedly enhanced our understanding of the molecular mechanism of photodamage.
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MBio2021. 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. Moreover, we characterize the molecular mechanism of ColB association with its OM receptor FepA by applying a combination of photoactivated cross-linking, mass spectrometry, and structural modeling. We demonstrate that complex formation is coincident with large-scale conformational changes in the colicin. Thereafter, active transport of ColB through FepA involves the colicin taking the place of the N-terminal half of the plug domain that normally occludes this iron transporter.IMPORTANCE Decades of excessive use of readily available antibiotics has generated a global problem of antibiotic resistance and, hence, an urgent need for novel antibiotic solutions. Bacteriocins are protein-based antibiotics produced by bacteria to eliminate closely related competing bacterial strains. Bacteriocin toxins have evolved to bypass the complex cell envelope in order to kill bacterial cells. Here, we uncover the cellular penetration mechanism of a well-known but poorly understood bacteriocin called colicin B that is active against Escherichia coll. Moreover, we demonstrate that the colicin B-import pathway can be exploited to deliver conjugated DNA cargo into bacterial cells. Our work leads to a better understanding of the way bacteriocins, as potential alternative antibiotics, execute their mode of action as well as highlighting how they might even be exploited in the genomic manipulation of Gram-negative bacteria.
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The Journal of biological chemistry2021. Scheer, Elisabeth et al.
Institut de Gntique et de Biologie Molculaire et Cellulaire (IGBMC), Centre National de la Recherche Scientifique, UMR7104, Institut National de la Sant et de la Recherche Mdicale, U964, Universit 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. Structural studies showed that TAF8 forms a histone fold pair with TAF10 in lobe B and participates in connecting lobe B to lobe C. To better understand the role of TAF8 in TFIID, we have investigated the requirement of the different regions of TAF8 for the invitro assembly of lobe B and C and the importance of certain TAF8 regions for mouse embryonic stem cell (ESC) viability. We have identified a region of TAF8 distinct from the histone fold domain important for assembling with the 5TAF core complex in lobe B. We also delineated four more regions of TAF8 each individually required for interacting with TAF2 in lobe C. Moreover, CRISPR/Cas9-mediated gene editing indicated that the 5TAF core-interacting TAF8 domain and the proline-rich domain of TAF8 that interacts with TAF2 are both required for mouse embryonic stem cell survival. Thus, our study defines distinct TAF8 regions involved in connecting TFIID lobe B to lobe C that appear crucial for TFIID function and consequent ESC survival. Copyright 2021 The Authors. Published by Elsevier Inc. All rights reserved.
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PNAS2021. 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. Our atomic model further reveals a twofold symmetric brake pad-shaped structure, in which Rsph4a and Rsph9 form a compact body extended laterally with two long arms of twisted Rsph1 beta-sheets and potentially connected dorsally via Rsph3b to the RS stalk. Furthermore, our modeling suggests that the core complex contacts the periodic CP projections either rigidly through its tooth-shaped Rsph4a regions or elastically through both arms for optimized RS-CP interactions and mechanosignal transduction.
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Science Signaling2021. 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. Here, using cross-linking mass spectrometry and functional studies, we gained mechanistic insights into PTEN:P-Rex2 complex assembly and coinhibition. We found that PTEN was anchored to P-Rex2 by interactions between the PDZ-interacting motif in the PTEN C-terminal tail and the second PDZ domain of P-Rex2. This interaction bridged PTEN across the P-Rex2 surface, preventing PI(3,4,5)P-3 hydrolysis. Conversely, PTEN both allosterically promoted an autoinhibited conformation of P-Rex2 and blocked its binding to G beta gamma. In addition, we observed that the PTEN-deactivating mutations and P-Rex2 truncations combined to drive Rac1 activation to a greater extent than did either single variant alone. These insights enabled us to propose a class of gain-of-function, cancer-associated mutations within the PTEN:P-Rex2 interface that uncouple PTEN from the inhibition of Rac1 signaling.
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JACS2021. 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 CC 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 domainswell characterized on their ownare poorly understood in their arrangement, conformational dynamics, and interplay in the intricate quaternary structure of modular PKSs. Here, we characterize module 2 from the 6-deoxyerythronolide B synthase by small-angle X-ray scattering and cross-linking mass spectrometry with coarse-grained structural modeling. The results of this hybrid approach shed light on the solution structure of a cis-AT type PKS module as well as its inherent conformational dynamics. Supported by a directed evolution approach, we also find that acyl carrier protein (ACP)-mediated substrate shuttling appears to be steered by a nonspecific electrostatic interaction network.
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Nature communications2021. 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. Various sites on the target (alpha -amine or epsilon -amines) react with the anhydride, but reaction is blocked if the partner does not dock. Ligation is efficient at pH 7.0, with half-time less than 2min. We arm Transforming Growth Factor-alpha with SPM, enabling specific covalent coupling to Epidermal Growth Factor Receptor at the cell-surface. NeissLock harnesses distinctive protein chemistry for high-yield covalent targeting of endogenous proteins, advancing the possibilities for molecular engineering. Covalent conjugation of endogenous protein complexes offers many opportunities for fundamental and clinical research. Based on a bacterial protein domain that forms a reactive anhydride in the presence of Ca2+, the authors here develop a system that enables the covalent capture of endogenous binding partners.
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Analytical Chemistry2021. 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)". CADI is able to significantly reduce sample complexity by depleting 90% of the linear peptides while keeping the disulfide-bonded peptides. Furthermore, all CADI data can be directly analyzed by widely used protein database search engines, such as Mascot and MaxQuant. Our data show that CADI is able to sensitively identify disulfide bonds in peptides and proteins. However, CADI has not yet achieved a satisfied in-depth coverage on complex mammalian cell lysates due to the limited enzymatic activity of carboxypeptidase Y and low occurrences of disulfide bonds in a proteome. Altogether, CADI is a useful method that can get disulfide-linked peptides enriched and analyzed with regular search engines. CADI holds great potentials to deepen the analysis of disulfide bond and other types of cross-linked peptides on the proteome scale.
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