Protein Interactions, Post-translational Modifications and Topologies in Human Cells

The results presented here provide new details on the structures of known multi-protein complexes as well as evidence for new protein-protein interactions.

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. 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. Proteins are the principal operatives within cells, involved in carrying out essentially all biological functions. A complex network of intra- and intermolecular interactions, post-translational modifications and abundance levels is required to maintain the delicate balance of function essential for life. Subtle changes within this network can give rise to specific biological responses to environmental factors, onset of disease, normal aging, and other biological processes. Therefore, direct experimental observation of protein structures and interactions in relation to biological function is paramount to improved understanding of living systems. Chemical cross-linking has long been used as a method of fixation to preserve biological samples in the fields of histology and pathology (1Hopwood D. Fixatives and fixation: a review.Histochem. J. 1969; 1: 323-360Crossref PubMed Scopus (173) Google Scholar). Protein interactions and topologies have also been studied with chemical cross-linking methods for many years (2Dutton A. Adams M. Singer S.J. Bifunctional imidoesters as cross-linking reagents.Biochem. Biophys. Res. Commun. 1966; 23: 730-739Crossref PubMed Scopus (81) Google Scholar, 3Kluger R. Alagic A. Chemical cross-linking and protein-protein interactions-a review with illustrative protocols.Bioorg. Chem. PubMed Scopus Google Scholar, The of with the Chem. PubMed Google Scholar). Chemical cross-linking with mass spectrometry used mass with mass for used mass with mass for is as a to study protein structures and interactions in complex biological systems A. A. M. R. protein structures chemical mass and PubMed Scopus Google Scholar). Technological advances in and are to the of to study protein topologies and interactions on a large in complex biological systems. 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