New technologies to understand the interplay of protein modifications in human diseases

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This application is primarily centered on the development of novel mass spectrometry (MS) strategies and data mining approaches to profile the dynamic changes in protein modifications affecting important cellular processes such as transcriptional regulation, protein translocation and degradation that are often misregulated during cancer development. In mammalian cells, a large array of enzymes affects the biological activities of proteins through the covalent attachment of more than 200 different types of post-translational modifications (PTMs), many of them targeting the same amino acid (e.g. lysine residues can be modified by methylation, acetylation, ubiquitylation or sumoylation). Although MS has been a key technology in providing precise information on the nature and site of PTMs, these tasks remain challenging in view of the low abundance and the dynamic nature of these modifications. These difficulties are compounded with the lack of convenient affinity chromatography approaches to enrich modified proteins from complex cell extracts and determine the interdependence between different types of modifications. To address these challenges, we will develop specific enrichment and detection methods to identify and profile protein phosphorylation and sumoylation in human cells, and apply these technologies to determine paralog-specific SUMO substrates and how they are regulated in response to As2O3 and cytokines, which are known to affect protein sumoylation and subsequent proteasome degradation. The tools developed will be use to identify novel substrates, determine changes in site occupancy and correlate protein modifications with specific enzyme activities (e.g. modified sumoylated substrates targeted by specific E3 SUMO ligase or SUMO proteases). We anticipate that developments made as part of this proposal will not only expand the scope and application of large-scale proteomics and drug discovery programs but will also provide further insights on the regulation and significance of these modifications in human diseases.