pFind Studio: a computational solution for mass spectrometry-based proteomics
2022
Life Science Alliance2022. Spittler, Didier et al.
Department of Genetics and Development, Columbia University Irving Medical Center, New York, NY, USA
ABSTRACT:HIV-1 Rev mediates the nuclear export of intron-containing viral RNA transcripts and is essential for viral replication. Rev is imported into the nucleus by the host protein importin beta (Impbeta), but how Rev associates with Impbeta is poorly understood. Here, we report biochemical, mutational, and biophysical studies of the Impbeta/Rev complex. We show that Impbeta binds two Rev monomers through independent binding sites, in contrast to the 1:1 binding stoichiometry observed for most Impbeta cargos. Peptide scanning data and charge-reversal mutations identify the N-terminal tip of Rev helix alpha2 within Rev's arginine-rich motif (ARM) as a primary Impbeta-binding epitope. Cross-linking mass spectrometry and compensatory mutagenesis data combined with molecular docking simulations suggest a structural model in which one Rev monomer binds to the C-terminal half of Impbeta with Rev helix alpha2 roughly parallel to the HEAT-repeat superhelical axis, whereas the other monomer binds to the N-terminal half. These findings shed light on the molecular basis of Rev recognition by Impbeta and highlight an atypical binding behavior that distinguishes Rev from canonical cellular Impbeta cargos.
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Life Science Alliance2022. Spittler, Didier et al.
Department of Genetics and Development, Columbia University Irving Medical Center, New York, NY, USA
ABSTRACT:HIV-1 Rev mediates the nuclear export of intron-containing viral RNA transcripts and is essential for viral replication. Rev is imported into the nucleus by the host protein importin beta (Impbeta), but how Rev associates with Impbeta is poorly understood. Here, we report biochemical, mutational, and biophysical studies of the Impbeta/Rev complex. We show that Impbeta binds two Rev monomers through independent binding sites, in contrast to the 1:1 binding stoichiometry observed for most Impbeta cargos. Peptide scanning data and charge-reversal mutations identify the N-terminal tip of Rev helix alpha2 within Rev's arginine-rich motif (ARM) as a primary Impbeta-binding epitope. Cross-linking mass spectrometry and compensatory mutagenesis data combined with molecular docking simulations suggest a structural model in which one Rev monomer binds to the C-terminal half of Impbeta with Rev helix alpha2 roughly parallel to the HEAT-repeat superhelical axis, whereas the other monomer binds to the N-terminal half. These findings shed light on the molecular basis of Rev recognition by Impbeta and highlight an atypical binding behavior that distinguishes Rev from canonical cellular Impbeta cargos.
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Proceedings of the National Academy of Sciences of the United States of America2022. Hart, Jonathan R et al.
Department of Molecular Medicine, Scripps Research Institute, La Jolla, CA 92037
ABSTRACT:Nanobodies and chemical cross-linking were used to gain information on the identity and positions of flexible domains of PI3Kalpha. The application of chemical cross-linking mass spectrometry (CXMS) facilitated the identification of the p85 domains BH, cSH2, and SH3 as well as their docking positions on the PI3Kalpha catalytic core. Binding of individual nanobodies to PI3Kalpha induced activation or inhibition of enzyme activity and caused conformational changes that could be correlated with enzyme function. Binding of nanobody Nb3-126 to the BH domain of p85alpha substantially improved resolution for parts of the PI3Kalpha complex, and binding of nanobody Nb3-159 induced a conformation of PI3Kalpha that is distinct from known PI3Kalpha structures. The analysis of CXMS data also provided mechanistic insights into the molecular underpinning of the flexibility of PI3Kalpha.
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Proceedings of the National Academy of Sciences of the United States of America2022. Rahman, Sanim et al.
Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD 21205
ABSTRACT:
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Science Advances2022. Han, Wenyu et al.
State Key Laboratory of Molecular Biology, National Center for Protein Science Shanghai, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai 200031,China
ABSTRACT:The cytoskeletal proteins tubulin and actin are the obligate substrates of TCP-1 ring complex/Chaperonin containing TCP-1 (TRiC/CCT), and their folding involves co-chaperone. Through cryo-electron microscopy analysis, we present a more complete picture of TRiC-assisted tubulin/actin folding along TRiC adenosine triphosphatase cycle, under the coordination of co-chaperone plp2. In the open S1/S2 states, plp2 and tubulin/actin engaged within opposite TRiC chambers. Notably, we captured an unprecedented TRiC-plp2-tubulin complex in the closed S3 state, engaged with a folded full-length beta-tubulin and loaded with a guanosine triphosphate, and a plp2 occupying opposite rings. Another closed S4 state revealed an actin in the intermediate folding state and a plp2. Accompanying TRiC ring closure, plp2 translocation could coordinate substrate translocation on the CCT6 hemisphere, facilitating substrate stabilization and folding. Our findings reveal the folding mechanism of the major cytoskeletal proteins tubulin/actin under the coordination of the biogenesis machinery TRiC and plp2 and extend our understanding of the links between cytoskeletal proteostasis and related human diseases.
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Proceedings of the National Academy of Sciences of the United States of America2022. Zhao, Mohan et al.
State Key Laboratory of Respiratory Disease, Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
ABSTRACT:Orphan nuclear receptor Nurr1 plays important roles in the progression of various diseases, including Parkinson's disease, neuroinflammation, Alzheimer's disease, and multiple sclerosis. It can recognize DNA as a monomer or heterodimer with retinoid X receptor alpha (RXRalpha). But the molecular mechanism of its transcriptional activity regulation is still largely unknown. Here we obtained a crystal structure of monomer Nurr1 (DNA- and ligand-binding domains, DBD and LBD) bound to NGFI-B response element. The structure exhibited two different forms with distinct DBD orientations, unveiling the conformational flexibility of nuclear receptor monomer. We then generated an integrative model of Nurr1-RXRalpha heterodimer. In the context of heterodimer, the structural flexibility of Nurr1 would contribute to its transcriptional activity modulation. We demonstrated that the DNA sequence may specifically modulate the transcriptional activity of Nurr1 in the absence of RXRalpha agonist, but the modulation can be superseded when the agonist binds to RXRalpha. Together, we propose a set of signaling pathways for the constitutive transcriptional activation of Nurr1 and provide molecular mechanisms for therapeutic discovery targeting Nurr1 and Nurr1-RXRalpha heterodimer.
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Immunity2022. Wu, Rui-Qi et al.
Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Oncology in South China
ABSTRACT:The reinvigoration of anti-tumor Tcells in response to immune checkpoint blockade (ICB) therapy is well established. Whether and how ICB therapy manipulates antibody-mediated immune response in cancer environments, however, remains elusive. Using tandem mass spectrometric analysis of modification of immunoglobulin G (IgG) from hepatoma tissues, we identified a role of ICB therapy in catalyzing IgG sialylation in the Fc region. Effector Tcells triggered sialylation of IgG via an interferon (IFN)-gamma-ST6Gal-I-dependent pathway. DC-SIGN+ macrophages represented the main target cells of sialylated IgG. Upon interacting with sialylated IgG, DC-SIGN stimulated Raf-1-elicited elevation of ATF3, which inactivated cGAS-STING pathway and eliminated subsequent type-I-IFN-triggered antitumorigenic immunity. Although enhanced IgG sialylation in tumors predicted improved therapeutic outcomes for patients receiving ICB therapy, impeding IgG sialylation augmented antitumorigenic Tcell immunity after ICB therapy. Thus, targeting antibody-based negative feedback action of ICB therapy has potential for improving efficacy of cancer immunotherapies.
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BIOCHEMISTRY2022. 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. In ammonia channeling enzymes reported thus far, a tight complex of the two subunits is observed, while the interaction of the two subunits of Methanocaldococcus jannaschii GMP synthetase (MjGMPS) is transient with the underlying mechanism of allostery and substrate channeling largely unclear. Here, we present a mechanistic model encompassing the various steps in the catalytic cycle of MjGMPS based on biochemical experiments, crystal structure, and cross-linking mass spectrometry guided integrative modeling. pH dependence of enzyme kinetics establishes that ammonia is tunneled across the subunits with the lifetime of the complex being & LE;0.5 s. The crystal structure of the XMP-bound ATPPase subunit reported herein highlights the role of conformationally dynamic loops in enabling catalysis. The structure of MjGMPS derived using restraints obtained from cross-linking mass spectrometry has enabled the visualization of subunit interactions that enable allostery under catalytic conditions. We integrate the results and propose a functional mechanism for MjGMPS detailing the various steps involved in catalysis.
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Frontiers in Molecular Biosciences2022. Wu, Chun et al.
Jinan Univ, Inst Life & Hlth Engn, MOE Key Lab Tumor Mol Biol, Guangzhou, Peoples R China; Jinan Univ, Key Lab Funct Prot Res Guangdong Higher Educ Inst, Inst Life & Hlth Engn, Guangzhou, Peoples R China
ABSTRACT:Alternative splicing (AS) isoforms create numerous proteoforms, expanding the complexity of the genome. Highly similar sequences, incomplete reference databases and the insufficient sequence coverage of mass spectrometry limit the identification of AS proteoforms. Here, we demonstrated full-length translating mRNAs (ribosome nascent-chain complex-bound mRNAs, RNC-mRNAs) sequencing (RNC-seq) strategy to sequence the entire translating mRNA using next-generation sequencing, including short-read and long-read technologies, to construct a protein database containing all translating AS isoforms. Taking the advantage of read length, short-read RNC-seq identified up to 15,289 genes and 15,906 AS isoforms in a single human cell line, much more than the Ribo-seq. The single-molecule long-read RNC-seq supplemented 4,429 annotated AS isoforms that were not identified by short-read datasets, and 4,525 novel AS isoforms that were not included in the public databases. Using such RNC-seq-guided database, we identified 6,766 annotated protein isoforms and 50 novel protein isoforms in mass spectrometry datasets. These results demonstrated the potential of full-length RNC-seq in investigating the proteome of AS isoforms.
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Journal of Proteome Research2022. Cheng, Kai et al.
Univ Ottawa, Fac Med, Sch Pharmaceut Sci, Ottawa, ON K1H 8M5, Canada
ABSTRACT:The studies of microbial communities have drawn increased attention in various research fields such as agriculture, environment, and human health. Recently, metaproteomics has become a powerful tool to interpret the roles of the community members by investigating the expressed proteins of the microbes. However, analyzing the metaproteomic data sets at genome resolution is still challenging because of the lack of efficient bioinformatics tools. Here we develop MetaLab-MAG, a specially designed tool for the characterization of microbiomes from metagenome-assembled genomes databases. MetaLab-MAG was evaluated by analyzing various human gut microbiota data sets and performed comparably or better than searching the gene catalog protein database directly. MetaLab-MAG can quantify the genome-level microbiota compositions and supports both label-free and isobaric labeling-based quantification strategies. MetaLab-MAG removes the obstacles of metaproteomic data analysis and provides the researchers with in-depth and comprehensive information from the microbiomes.
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