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Human cells adapt to translational errors by modulating protein synthesis rate and protein turnover.

ABSTRACT: Deregulation of tRNAs, aminoacyl-tRNA synthetases (aaRS) or tRNA modifying enzymes, increase the level of protein synthesis errors (PSE) and are associated with several diseases, but the cause-effect mechanisms of these pathologies remain elusive. To clarify the role of PSE in human biology, we have engineered a HEK293 cell line to overexpress a wild type (Wt) tRNASer and two tRNASer mutants that misincorporate serine at non-cognate codon sites. Then, we followed long-term adaptation to PSE of such recombinant cells by analysing cell viability, protein synthesis rate and activation of protein quality control mechanisms (PQC). Engineered cells showed higher level of misfolded and aggregated proteins; activated the ubiquitin-proteasome system (UPS) and the unfolded protein response (UPR), indicative of proteotoxic stress. Adaptation to PSE involved increased protein turnover, UPR up-regulation and altered protein synthesis rate. Gene expression analysis showed that engineered cells presented recurrent alterations in the endoplasmic reticulum, cell adhesion and calcium homeostasis. Herein, we unveil new phenotypic consequences of protein synthesis errors in human cells and identify the protein quality control processes that are necessary for long-term adaptation to PSE and proteotoxic stress. Our data provide important insight on how chronic proteotoxic stress may cause disease and highlight potential biological pathways that support the association of PSE with disease.

SUBMITTER: Varanda AS

PROVIDER: S-EPMC6948982 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Post-translational modifications (PTMs) on the N-terminal tails of histones and histone variants regulate distinct transcriptional states and nuclear events. Whereas the functional effects of specific PTMs are the current subject of intense investigation, most studies characterize histone PTMs/variants in a non-temporal fashion and very few studies have reported kinetic information about these histone forms. Previous studies have used radiolabeling, fluorescence microscopy and chromatin immunoprecipitation to determine rates of histone turnover, and have found interesting correlations between increased turnover and increased gene expression. Therefore, histone turnover is an understudied yet potentially important parameter that may contribute to epigenetic regulation. Understanding turnover in the context of histone modifications and sequence variants could provide valuable additional insight into the function of histone replacement.In this study, we measured the metabolic rate of labeled isotope incorporation into the histone proteins of HeLa cells by combining stable isotope labeling of amino acids in cell culture (SILAC) pulse experiments with quantitative mass spectrometry-based proteomics. In general, we found that most core histones have similar turnover rates, with the exception of the H2A variants, which exhibit a wider range of rates, potentially consistent with their epigenetic function. In addition, acetylated histones have a significantly faster turnover compared with general histone protein and methylated histones, although these rates vary considerably, depending on the site and overall degree of methylation. Histones containing transcriptionally active marks have been consistently found to have faster turnover rates than histones containing silent marks. Interestingly, the presence of both active and silent marks on the same peptide resulted in a slower turnover rate than either mark alone on that same peptide. Lastly, we observed little difference in the turnover between nearly all modified forms of the H3.1, H3.2 and H3.3 variants, with the notable exception that H3.2K36me2 has a faster turnover than this mark on the other H3 variants.Quantitative proteomics provides complementary insight to previous work aimed at quantitatively measuring histone turnover, and our results suggest that turnover rates are dependent upon site-specific post-translational modifications and sequence variants.

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Human cells adapt to translational errors by modulating protein synthesis rate and protein turnover.

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