Unbiased translation proteomics upon cell stress.
Ontology highlight
ABSTRACT:
SUBMITTER: Klann K
PROVIDER: S-EPMC7469668 | biostudies-literature | 2020
REPOSITORIES: biostudies-literature
Publications
Unbiased translation proteomics upon cell stress.
Klann Kevin K Münch Christian C
Molecular & cellular oncology 20200518 4
The mammalian target of rapamycin and the integrated stress response are central cellular hubs regulating translation upon stress. The precise proteins and pathway specificity of translation targets of these pathways remained largely unclear. We recently described a new method for quantitative translation proteomics and found that both pathways control translation of the same sets of proteins. ...[more]
Similar Datasets
Project description:Mass spectrometry (MS)-based quantitative interaction proteomics has successfully elucidated specific protein-protein, DNA-protein, and small molecule-protein interactions. Here, we developed a gel-free, sensitive, and scalable technology that addresses the important area of RNA-protein interactions. Using aptamer-tagged RNA as bait, we captured RNA-interacting proteins from stable isotope labeling by amino acids in cell culture (SILAC)-labeled mammalian cell extracts and analyzed them by high-resolution, quantitative MS. Binders specific to the RNA sequence were distinguished from background by their isotope ratios between bait and control. We demonstrated the approach by retrieving known and novel interaction partners for the HuR interaction motif, H4 stem loop, "zipcode" sequence, tRNA, and a bioinformatically-predicted RNA fold in DGCR-8/Pasha mRNA. In all experiments we unambiguously identified known interaction partners by a single affinity purification step. The 5' region of the mRNA of DGCR-8/Pasha, a component of the microprocessor complex, specifically interacts with components of the translational machinery, suggesting that it contains an internal ribosome entry site.
| S-EPMC2697111 | biostudies-literature
Project description:Cells release diverse types of extracellular vesicles (EVs), which transfer complex signals to surrounding cells. Specific markers to distinguish different EVs (e.g. exosomes, ectosomes, enveloped viruses like HIV) are still lacking. We have developed a proteomic profiling approach for characterizing EV subtype composition and applied it to human Jurkat T cells. We generated an interactive database to define groups of proteins with similar profiles, suggesting release in similar EVs. Biochemical validation confirmed the presence of preferred partners of commonly used exosome markers in EVs: CD81/ADAM10/ITGB1, and CD63/syntenin. We then compared EVs from control and HIV-1-infected cells. HIV infection altered EV profiles of several cellular proteins, including MOV10 and SPN, which became incorporated into HIV virions, and SERINC3, which was re-routed to non-viral EVs in a Nef-dependent manner. Furthermore, we found that SERINC3 controls the surface composition of EVs. Our workflow provides an unbiased approach for identifying candidate markers and potential regulators of EV subtypes. It can be widely applied to in vitro experimental systems for investigating physiological or pathological modifications of EV release.
| S-EPMC8047442 | biostudies-literature
Project description:RATIONALE:Human pluripotent stem cell (hPSC)-derived cardiomyocytes exhibit the properties of fetal cardiomyocytes, which limits their applications. Various methods have been used to promote maturation of hPSC-cardiomyocytes; however, there is a lack of an unbiased and comprehensive method for accurate assessment of the maturity of hPSC-cardiomyocytes. OBJECTIVE:We aim to develop an unbiased proteomics strategy integrating high-throughput top-down targeted proteomics and bottom-up global proteomics for the accurate and comprehensive assessment of hPSC-cardiomyocyte maturation. METHODS AND RESULTS:Utilizing hPSC-cardiomyocytes from early- and late-stage 2-dimensional monolayer culture and 3-dimensional engineered cardiac tissue, we demonstrated the high reproducibility and reliability of a top-down proteomics method, which enabled simultaneous quantification of contractile protein isoform expression and associated post-translational modifications. This method allowed for the detection of known maturation-associated contractile protein alterations and, for the first time, identified contractile protein post-translational modifications as promising new markers of hPSC-cardiomyocytes maturation. Most notably, decreased phosphorylation of ?-tropomyosin was found to be associated with hPSC-cardiomyocyte maturation. By employing a bottom-up global proteomics strategy, we identified candidate maturation-associated markers important for sarcomere organization, cardiac excitability, and Ca2+ homeostasis. In particular, upregulation of myomesin 1 and transmembrane 65 was associated with hPSC-cardiomyocyte maturation and validated in cardiac development, making these promising markers for assessing maturity of hPSC-cardiomyocytes. We have further validated ?-actinin isoforms, phospholamban, dystrophin, ?B-crystallin, and calsequestrin 2 as novel maturation-associated markers, in the developing mouse cardiac ventricles. CONCLUSIONS:We established an unbiased proteomics method that can provide accurate and specific assessment of the maturity of hPSC-cardiomyocytes and identified new markers of maturation. Furthermore, this integrated proteomics strategy laid a strong foundation for uncovering the molecular pathways involved in cardiac development and disease using hPSC-cardiomyocytes.
| S-EPMC6852699 | biostudies-literature
Project description:Protein redox regulation plays important roles in many biological processes. Protein cysteine thiols are sensitive to redox changes and may function as redox switches, which turn signaling and metabolic pathways on or off to ensure speedy responses to environmental stimuli or stresses. Here we report a novel integrative proteomics method called cysTMTRAQ that combines two types of isobaric tags, cysteine tandem mass tags and isobaric tag for relative and absolute quantification, in one experiment. The method not only enables simultaneous analysis of cysteine redox changes and total protein level changes, but also allows the determination of bona fide redox modified cysteines in proteins through the correction of protein turnover. Overlooking the factor of protein-level changes in the course of protein posttranslational modification experiments could lead to misleading results. The capability to analyze protein posttranslational modification dynamics and protein-level changes in one experiment will advance proteomic studies in many areas of biology and medicine.
| S-EPMC4288258 | biostudies-literature
Project description:Post-transcriptional events regulate herpesvirus gene expression, yet few herpesvirus RNA-binding proteins have been identified. We used an unbiased approach coupling oligo(dT) affinity capture with proteomics to identify viral RNA-associated proteins during infection. Using this approach, we identified and confirmed changes in the abundance or activity of two host RNA-associated proteins, DHX9 and DDX3, in cells infected with human cytomegalovirus (HCMV). We also identified and confirmed previously unreported activities for the HCMV US22 and pp71 proteins as RNA-associated viral proteins and confirmed that a known viral RNA-binding protein, pTRS1, associates with RNA in infected cells. Further, we found that HCMV pp71 co-sedimented with polysomes, associated with host and viral RNAs, and stimulated the overall rate of protein synthesis. These results demonstrate that oligo(dT) affinity capture coupled with proteomics provides a rapid and straightforward means to identify RNA-associated viral proteins during infection that may participate in the post-transcriptional control of gene expression.
| S-EPMC4437878 | biostudies-literature
Project description:Translation of RNA to protein is a core process for any living organism. While for some steps of this process the effect on protein production is understood, a holistic understanding of translation still remains elusive. In silico modelling is a promising approach for elucidating the process of protein synthesis. Although a number of computational models of the process have been proposed, their application is limited by the assumptions they make. Ribosome profiling (RP), a relatively new sequencing-based technique capable of recording snapshots of the locations of actively translating ribosomes, is a promising source of information for deriving unbiased data-driven translation models. However, quantitative analysis of RP data is challenging due to high measurement variance and the inability to discriminate between the number of ribosomes measured on a gene and their speed of translation. We propose a solution in the form of a novel multi-scale interpretation of RP data that allows for deriving models with translation dynamics extracted from the snapshots. We demonstrate the usefulness of this approach by simultaneously determining for the first time per-codon translation elongation and per-gene translation initiation rates of Saccharomyces cerevisiae from RP data for two versions of the Totally Asymmetric Exclusion Process (TASEP) model of translation. We do this in an unbiased fashion, by fitting the models using only RP data with a novel optimization scheme based on Monte Carlo simulation to keep the problem tractable. The fitted models match the data significantly better than existing models and their predictions show better agreement with several independent protein abundance datasets than existing models. Results additionally indicate that the tRNA pool adaptation hypothesis is incomplete, with evidence suggesting that tRNA post-transcriptional modifications and codon context may play a role in determining codon elongation rates.
| S-EPMC4537299 | biostudies-literature
Project description:The p57(Kip2) cyclin-dependent kinase inhibitor (CDKi) has been implicated in embryogenesis, stem-cell senescence and pathologies, but little is known of its role in cell cycle control. Here, we show that p57(Kip2) is targeted by the p38 stress-activated protein kinase (SAPK). Phosphorylation of p57(Kip2) at T143 by p38 enhances its association with and inhibition of Cdk2, which results in cell-cycle delay upon stress. Genetic inactivation of the SAPK or the CDKi abolishes cell-cycle delay upon osmostress and results in decreased cell viability. Oxidative stress and ionomycin also induce p38-mediated phosphorylation of p57 and cells lacking p38 or p57 display reduced viability to these stresses. Therefore, cell survival to various stresses depends on p57 phosphorylation by p38 that inhibits CDK activity. Together, these findings provide a novel molecular mechanism by which cells can delay cell cycle progression to maximize cell survival upon stress.
| S-EPMC3395087 | biostudies-other
Project description:Membrane lipids were once thought to be homogenously distributed in the 2D surface of a membrane, but the lipid raft theory suggests that cholesterol and sphingolipids partition away from other membrane lipids. Lipid raft theory further implicates these cholesterol-rich domains in many processes such as signaling and vesicle traffic. However, direct characterization of rafts has been difficult, because they cannot be isolated in pure form. In the first functional proteomic analysis of rafts, we use quantitative high-resolution MS to specifically detect proteins depleted from rafts by cholesterol-disrupting drugs, resulting in a set of 241 authentic lipid raft components. We detect a large proportion of signaling molecules, highly enriched versus total membranes and detergent-resistant fractions, which thus far biochemically defined rafts. Our results provide the first large-scale and unbiased evidence, to our knowledge, for the connection of rafts with signaling and place limits on the fraction of plasma membrane composed by rafts.
| S-EPMC156283 | biostudies-literature
Project description:When adapting to environmental stress, cells attenuate and reprogram their translational output. In part, these altered translation profiles are established through changes in the interactions between RNA-binding proteins and mRNAs. The Argonaute 2 (Ago2)/microRNA (miRNA) machinery has been shown to participate in stress-induced translational up-regulation of a particular mRNA, CAT-1; however, a detailed, transcriptome-wide understanding of the involvement of Ago2 in the process has been lacking. Here, we profiled the overall changes in Ago2-mRNA interactions upon arsenite stress by cross-linking immunoprecipitation (CLIP) followed by high-throughput sequencing (CLIP-seq). Ago2 displayed a significant remodeling of its transcript occupancy, with the majority of 3' untranslated region (UTR) and coding sequence (CDS) sites exhibiting stronger interaction. Interestingly, target sites that were destined for release from Ago2 upon stress were depleted in miRNA complementarity signatures, suggesting an alternative mode of interaction. To compare the changes in Ago2-binding patterns across transcripts with changes in their translational states, we measured mRNA profiles on ribosome/polysome gradients by RNA sequencing (RNA-seq). Increased Ago2 occupancy correlated with stronger repression of translation for those mRNAs, as evidenced by a shift toward lighter gradient fractions upon stress, while release of Ago2 was associated with the limited number of transcripts that remained translated. Taken together, these data point to a role for Ago2 and the mammalian miRNAs in mediating the translational component of the stress response.
| S-EPMC3731550 | biostudies-literature
Project description:Tactical disruption of protein synthesis is an attractive therapeutic strategy, with the first-in-class eIF4A-targeting compound zotatifin in clinical evaluation for cancer and COVID-19. The full cellular impact and mechanisms of these potent molecules are undefined at a proteomic level. Here, we report mass spectrometry analysis of translational reprogramming by rocaglates, cap-dependent initiation disruptors that include zotatifin. We find effects to be far more complex than simple "translational inhibition" as currently defined. Translatome analysis by TMT-pSILAC (tandem mass tag-pulse stable isotope labeling with amino acids in cell culture mass spectrometry) reveals myriad upregulated proteins that drive hitherto unrecognized cytotoxic mechanisms, including GEF-H1-mediated anti-survival RHOA/JNK activation. Surprisingly, these responses are not replicated by eIF4A silencing, indicating a broader translational adaptation than currently understood. Translation machinery analysis by MATRIX (mass spectrometry analysis of active translation factors using ribosome density fractionation and isotopic labeling experiments) identifies rocaglate-specific dependence on specific translation factors including eEF1ε1 that drive translatome remodeling. Our proteome-level interrogation reveals that the complete cellular response to these historical "translation inhibitors" is mediated by comprehensive translational landscape remodeling.
| S-EPMC8558842 | biostudies-literature