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Protein interactions, post-translational modifications and topologies in human cells.

ABSTRACT: The unique and remarkable physicochemical properties of protein surface topologies give rise to highly specific biomolecular interactions, which form the framework through which living systems are able to carry out their vast array of functions. Technological limitations undermine efforts to probe protein structures and interactions within unperturbed living systems on a large scale. Rapid chemical stabilization of proteins and protein complexes through chemical cross-linking offers the alluring possibility to study details of the protein structure to function relationships as they exist within living cells. Here we apply the latest technological advances in chemical cross-linking combined with mass spectrometry to study protein topologies and interactions from living human cells identifying a total of 368 cross-links. These include cross-links from all major cellular compartments including membrane, cytosolic and nuclear proteins. Intraprotein and interprotein cross-links were also observed for core histone proteins, including several cross-links containing post-translational modifications which are known histone marks conferring distinct epigenetic functions. Excitingly, these results demonstrate the applicability of cross-linking to make direct topological measurements on post-translationally modified proteins. The results presented here provide new details on the structures of known multi-protein complexes as well as evidence for new protein-protein interactions.

SUBMITTER: Chavez JD

PROVIDER: S-EPMC3650351 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Protein interactions, post-translational modifications and topologies in human cells.

Chavez Juan D JD Weisbrod Chad R CR Zheng Chunxiang C Eng Jimmy K JK Bruce James E JE

Molecular & cellular proteomics : MCP 20130125 5

The unique and remarkable physicochemical properties of protein surface topologies give rise to highly specific biomolecular interactions, which form the framework through which living systems are able to carry out their vast array of functions. Technological limitations undermine efforts to probe protein structures and interactions within unperturbed living systems on a large scale. Rapid chemical stabilization of proteins and protein complexes through chemical cross-linking offers the alluring ...[more]

PMID: 23354917

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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.

Dataset Information

Protein interactions, post-translational modifications and topologies in human cells.

Publications

Protein interactions, post-translational modifications and topologies in human cells.

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