The topology of protein-protein interactions in situ in eukaryotic cells

Division Of Basic Sciences - Nci

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Abstract

My previous research has focused on developing and using proteomics techniques in combination with structural biology technologies to discover the topology of protein complexes. This project will further develop crosslinking mass spectrometry (MS) to identify protein interactions in situ, to reveal their topology and, eventually, to compare how these interactions are altered during cellular stress. The complexity and dynamic range of eukaryotic proteomes present a particular challenge for generating whole cell interactomes by crosslinking MS and other technologies. The complexity of large-scale genetic tagging and difficult-to-purify protein-protein interactions (PPIs), such as those within membranes or phase-separated regions, mean that studying interactions in situ is only possible with crosslinking MS. To push the boundaries of this technology to investigating entire eukaryotes, I propose divide-and-conquer approaches to address their complexity. The data produced will highlight interactions that are difficult to study ex vivo and will guide further structural studies. Of particular initial interest is the the nucleus, as it is home to an array of regulatory complexes in central information processing that may have been too labile to be detected by previous pulldown analyzes. This should aid our understanding of the functioning of human cells and allow further work on understanding how they change during the onset of cancer.