pFind Studio: a computational solution for mass spectrometry-based proteomics
2023
Journal of Agricultural and Food Chemistry2023. Valentina Ciaravolo et al.
CNR, Prote Metabol & Mass Spectrometry Lab, ISPAAM, I-80055 Portici, Italy
ABSTRACT:To investigate thiol-disulfide interchange reactionsin heatedmilk yielding non-native intramolecular rearranged and intermolecularcross-linked proteins, a proteomic study based on nanoLC-ESI-Q-Orbitrap-MS/MSand dedicated bioinformatics was accomplished. Raw milk samples heatedfor different times and various commercial dairy products were analyzed.Qualitative experiments on tryptic digests of resolved protein mixturesassigned the corresponding disulfide-linked peptides. Results confirmedthe limited data available on few milk proteins, generated the widestinventory of components (63 in number) involved in thiol-disulfideexchange processes, and provided novel structural information on S-S-bridgedmolecules. Quantitative experiments on unresolved protein mixturesfrom both sample typologies estimated the population of moleculesassociated with thiol-disulfide reshuffling processes. Disulfide-linkedpeptides associated with native intramolecular S-S bonds generallyshowed a progressive reduction depending on heating time/harshness,whereas those related to specific non-native intramolecular/intermolecularones showed an opposite quantitative trend. This was associated witha temperature-dependent augmented reactivity of definite native proteinthiols and S-S bridges, which determined the formation of non-nativerearranged monomers and cross-linked oligomers. Results provided novelinformation for possibly linking the nature and extent of thiol-disulfideexchange reactions in heated milk proteins to the corresponding functionaland technological characteristics, with possible implications on fooddigestibility, allergenicity, and bioactivity.
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Analytical Chemistry2023. Xinwei Li et al.
Chinese Acad Sci, Dalian Inst Chem Phys, Collaborat Innovat Ctr Chem Energy Mat iChEM, State Key Lab Catalysis, Dalian 116023, Peoples R China; Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog Res & Anal Ctr, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Peoples R China
ABSTRACT:Photosynthesis, as the core of solar energy biotransformation,is driven by photosynthetic membrane protein complexes in plants andalgae. Current methods for intracellular photosynthetic membrane proteincomplex analysis mostly require the separation of specific chloroplastsor the change of the intracellular environment, which causes the missingof real-time and on-site information. Thus, we explored a method for in vivo crosslinking and mapping of photosynthetic membraneprotein complexes in the chloroplasts of living Chlamydomonasreinhardtii (C. reinhardtii) cells under cultural conditions. Poly(lactic-co-glycolic acid) (PLGA) and poly(lactic-co-glycolicacid)-poly(ethylene glycol) (PLGA-PEG) nanoparticles were fabricatedto deliver bis(succinimidyl)propargyl with a nitro compound (BSPNO)into the chloroplasts to crosslink photosynthetic membrane proteincomplexes. After the in vivo crosslinked proteincomplexes were extracted and digested, mass spectrometry was employedto detect lysine-specific crosslinked peptides for further elucidatingthe protein conformations and interactions. With this method, theweak interactions between extrinsic proteins in the luminal side (PsbLand PsbH) and the core subunits (CP47 and CP43) in photosyntheticprotein complexes were directly captured in living cells. Additionally,the previously uncharacterized protein (Cre07.g335700) was bound to the light-harvesting proteins, which was related tothe biosynthesis of light-harvesting antennae. These results indicatedthat in vivo analysis of photosynthetic protein complexesbased on crosslinker nanocarriers was expected to not only figureout the difficulty in the study of photosynthetic protein complexesin living cells but also provide an approach to explore transientand weak interactions and the function of uncharacterized proteins.
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Available at SSRN 44458872023. DariaEzeria et al.
VIB-VUB Center for Structural Biology, Vlaams Instituut Voor Biotechnologie, Brussels B-1050, Belgium
ABSTRACT:
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Probiotics and Antimicrobial Proteins2023. Michelle Rossouw et al.
Stellenbosch Univ, Dept Microbiol, Private Bag X1, ZA-7602 Matieland, South Africa
ABSTRACT:Antimicrobial peptides or bacteriocins are excellent candidates for alternative antimicrobials, but high manufacturing costs limit their applications. Recombinant gene expression offers the potential to produce these peptides more cost-effectively at a larger scale. Saccharomyces cerevisiae is a popular host for recombinant protein production, but with limited success reported on antimicrobial peptides. Individual recombinant S. cerevisiae strains were constructed to secrete two class IIa bacteriocins, plantaricin 423 (PlaX) and mundticin ST4SA (MunX). The native and codon-optimised variants of the plaA and munST4SA genes were cloned into episomal expression vectors containing either the S. cerevisiae alpha mating factor (MF alpha 1) or the Trichoderma reesei xylanase 2 (XYNSEC) secretion signal sequences. The recombinant peptides retained their activity and stability, with the MF alpha 1 secretion signal superior to the XYNSEC secretion signal for both bacteriocins. An eight-fold increase in activity against Listeria monocytogenes was observed for MunX after codon optimisation, but not for PlaX-producing strains. After HPLC-purification, the codon-optimised genes yielded 20.9 mg/L of MunX and 18.4 mg/L of PlaX, which displayed minimum inhibitory concentrations (MICs) of 108.52 nM and 1.18 mu M, respectively, against L. monocytogenes. The yields represent a marked improvement relative to an Escherichia coli expression system previously reported for PlaX and MunX. The results demonstrated that S. cerevisiae is a promising host for recombinant bacteriocin production that requires a simple purification process, but the efficacy is sensitive to codon usage and secretion signals.
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Analytica Chimica Acta2023. Zhong, Bowen et al.
Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog R&A Ctr, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Liaoning, Peoples R China
ABSTRACT:Comprehensive interactome analysis of targeted proteins is important to understand how proteins work together in regulating functions. Commonly, affinity purification followed by mass spectrometry (AP-MS) has been recognized as the most often used technique for studying protein-protein interactions (PPIs). However, some proteins with weak interactions, which are responsible for key roles in regulation, are easily broken during cell lysis and purification through an AP approach. Herein, we have developed an approach termed in vivo cross-linking-based affinity purification and mass spectrometry (ICAP-MS). By this method, in vivo cross-linking was introduced to covalently fix intracellular PPIs in their functional states to assure all PPIs could be integrally maintained during cell disruption. In addition, the chemically cleavable crosslinkers which were employed enabled unbinding of PPIs for in-depth identification of components within the interactome and biological analysis, while allowing binding of PPIs for cross-linking-mass spectrometry (CXMS)-based direct interaction determination. Multi-level information on targeted PPIs network can be obtained by ICAP-MS, including composition of interacting proteins, as well as direct interacting partners and binding sites. As a proof of concept, the interactome of MAPK3 from 293A cells was profiled with 6.15-fold improvement in identification than by conventional AP-MS. Meanwhile, 184 cross-link site pairs of these PPIs were experimentally identified by CXMS. Furthermore, ICAP-MS was applied in the temporal profiling of MAPK3 interactions under activation by cAMP-mediated pathway. The regulatory manner of MAPK pathways was presented through the quantitative changes of MAPK3 and its interacting proteins at different time points after activation. Therefore, all reported results demonstrated that the ICAP-MS approach may provide comprehensive information on interactome of targeted protein for functional exploration.
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ACS Chemical Neuroscience2023. Gayani Wijegunawardena et al.
Univ Calif San Francisco, Weill Inst Neurosci, Dept Neurol, San Francisco, CA 94158 USA; Univ Calif San Francisco, Inst Neurodegenerat Dis, San Francisco, CA 94158 USA; Wichita State Univ, Dept Chem & Biochem, Wichita, KS 67260 USA
ABSTRACT:Mutations in a microglia-associated gene TREM2increase the risk of Alzheimer's disease. Currently, structuraland functional studies of TREM2 mainly rely on recombinant TREM2 proteinsexpressed from mammalian cells. However, using this method, it isdifficult to achieve site-specific labeling. Here, we present thetotal chemical synthesis of the 116 amino acid TREM2 ectodomain. Rigorousstructural analysis ensured correct structural fold after refolding.Treating microglial cells with refolded synthetic TREM2 enhanced microglialphagocytosis, proliferation, and survival. We also prepared TREM2constructs with defined glycosylation patterns and found that glycosylationat N79 is critical to the thermal stability of TREM2. This methodwill provide access to TREM2 constructs with site-specific labeling,such as fluorescent labeling, reactive chemical handles, and enrichmenthandles, to further advance our understanding of TREM2 in Alzheimer'sdisease.
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Nature Communications2023. Zhennan Zhao et al.
Chinese Acad Sci, Inst Biophys, Natl Lab Biomacromol, Beijing 100101, Peoples R China; Southern Univ Sci & Technol, Cryo EM Ctr, Dept Biol, Shenzhen 518055, Peoples R China; Chinese Acad Med Sci & Peking Union Med Coll, Peking Union Med Coll Hosp, Clin Res Inst, State Key Lab Complex Severe & Rare Dis, Beijing 100730, Peoples R China; South China Agr Univ, Coll Marine Sci, Guangdong Lab Lingnan Modern Agr, Guangzhou 510642, Peoples R China; Univ Chinese Acad Sci, Beijing 100049, Peoples R China; Chinese Acad Sci, Inst Microbiol, CAS Key Lab Pathogen Microbiol & Immunol, Beijing 100101, Peoples R China
ABSTRACT:High morbidity and mortality in aquatic have been caused by iridovirids worldwide. Here the authors present a near-atomic SGIV capsid structure. Functional assays further reveal the relationships between identified capsid proteins and viral assembly.Singapore grouper iridovirus (SGIV), one of the nucleocytoviricota viruses (NCVs), is a highly pathogenic iridovirid. SGIV infection results in massive economic losses to the aquaculture industry and significantly threatens global biodiversity. In recent years, high morbidity and mortality in aquatic animals have been caused by iridovirid infections worldwide. Effective control and prevention strategies are urgently needed. Here, we present a near-atomic architecture of the SGIV capsid and identify eight types of capsid proteins. The viral inner membrane-integrated anchor protein colocalizes with the endoplasmic reticulum (ER), supporting the hypothesis that the biogenesis of the inner membrane is associated with the ER. Additionally, immunofluorescence assays indicate minor capsid proteins (mCPs) could form various building blocks with major capsid proteins (MCPs) before the formation of a viral factory (VF). These results expand our understanding of the capsid assembly of NCVs and provide more targets for vaccine and drug design to fight iridovirid infections.
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Nucleic Acids Research2023. Ellison, Mitchell A. et al.
Univ Pittsburgh, Dept Biol Sci, Pittsburgh, PA 15260 USA
ABSTRACT:The Paf1 complex (Paf1C) is a conserved transcription elongation factor that regulates transcription elongation efficiency, facilitates co-transcriptional histone modifications, and impacts molecular processes linked to RNA synthesis, such as polyA site selection. Coupling of the activities of Paf1C to transcription elongation requires its association with RNA polymerase II (Pol II). Mutational studies in yeast identified Paf1C subunits Cdc73 and Rtf1 as important mediators of Paf1C association with Pol II on active genes. While the interaction between Rtf1 and the general elongation factor Spt5 is relatively well-understood, the interactions involving Cdc73 have not been fully elucidated. Using a site-specific protein cross-linking strategy in yeast cells, we identified direct interactions between Cdc73 and two components of the Pol II elongation complex, the elongation factor Spt6 and the largest subunit of Pol II. Both of these interactions require the tandem SH2 domain of Spt6. We also show that Cdc73 and Spt6 can interact in vitro and that rapid depletion of Spt6 dissociates Paf1 from chromatin, altering patterns of Paf1C-dependent histone modifications genome-wide. These results reveal interactions between Cdc73 and the Pol II elongation complex and identify Spt6 as a key factor contributing to the occupancy of Paf1C at active genes in Saccharomyces cerevisiae.
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Biochemical and Biophysical Research Communications2023. XuhuiZhen et al.
Chinese Acad Sci, Guangzhou Inst Hlth, Guangdong Prov Key Lab Biocomp, Guangzhou 510530, Peoples R China; Chinese Acad Sci, Guangzhou Inst Biomed, Guangdong Prov Key Lab Biocomp, Guangzhou 510530, Peoples R China
ABSTRACT:The estrogen-related receptor (ERR) family members are reported to bind DNA elements as either monomer or dimer. However, to date, only one solution NMR structure of ERRI3 in complex with a half -site DNA element has been reported. To better understand the DNA regulation mechanism, we deter-mined the crystal structure of ERRy-DBD bound to a natural DR1 element in Pla2g12b promoter to 2.2 angstrom resolution. Combined with biochemical assays, we show that ERRy acts as a dimer and the C-terminal extension region undergoes conformational rearrangement when binding to the downstream DR1 element. In addition, the T-box region on the dimerization interface exhibits unique main-chain conformation. Thus, our structure presents a novel dimer interface for NR binding on DR1 DNA and provides a molecular basis for understanding the homodimer organization of ERR on DR1 elements.(c) 2023 Elsevier Inc. All rights reserved.
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Science Advances2023. Dybkov, Olexandr et al.
Max Planck Inst Multidisciplinary Sci, Cellular Biochem, Fassberg 11, D-37077 Gottingen, Germany; Free Univ Berlin, Inst Chem & Biochem, RNA Biochem, Takustr 6, D-14195 Berlin, Germany
ABSTRACT:Alternative precursor messenger RNA splicing is instrumental in expanding the proteome of higher eukaryotes, and changes in 3 ' splice site (3'ss) usage contribute to human disease. We demonstrate by small interfering RNA-mediated knockdowns, followed by RNA sequencing, that many proteins first recruited to human C* spli-ceosomes, which catalyze step 2 of splicing, regulate alternative splicing, including the selection of alternatively spliced NAGNAG 3 ' ss. Cryo-electron microscopy and protein cross-linking reveal the molecular architecture of these proteins in C* spliceosomes, providing mechanistic and structural insights into how they influence 3'ss usage. They further elucidate the path of the 3 ' region of the intron, allowing a structure-based model for how the C* spliceosome potentially scans for the proximal 3 ' ss. By combining biochemical and structural approaches with genome-wide functional analyses, our studies reveal widespread regulation of alternative 3 ' ss usage after step 1 of splicing and the likely mechanisms whereby C* proteins influence NAGNAG 3 ' ss choices.
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