EXCEED THE SPACE PROVIDED. A common feature of many stress-inducing stimuli that contribute to cancer and chemical toxicity and is the generation of reactive electrophiles that modify specific protein targets. Recent work indicates that protein-modifying stresses also trigger changes in posttranslational modification by the ubiquitination and small ubiquitin-like modifier (sumoylation) pathways. These pathways have emerged as key regulators of protein turnover, protein-protein interactions and transcription factors. This project will characterize the ubiquitination and sumoylation response to alkylating agents and test the hypothesis that protein alkylation is a trigger for protein modification by ubiquitin or sumo proteins. We will approach this problem with new mass spectrometry (MS)-based analytical proteomics methods to quantify changes in cellular proteomes in response to stress. The three Specific Aims of the proposed research are 1)We will characterize changes in ubiquitination and sumoylation status of proteomes in HEK 293 cells exposed to the prototypical alkylating agent 4-hydroxy-2-nonenal (HNE). Ubiquitinated and sumoylated proteins will be identified by LC-MS analysis of the immunoprecipitated protein mixtures and Sequest-assisted analysis of the LC-MS data. 2) We will map ubiquitin and sumo modifications of targeted proteins at the evel of amino acid sequence with LC-MS and the SALSA algorithm, which will detect ubiquitin or sumo-derived amino acid sequence tags on target protein lysine residues. 3) We will examine the relationship between protein covalent modification and ubiquitination or sumoylation. Prototypical electrophiles tethered to biotin affinity tags will be used to trap adducted proteins and their possible ubiquitin or sumo conjugates. These studies will evaluate the mechanisms by which protein damage triggers changes in protein turnover and provide new insights into how cellular proteomes respond to environmental stresses. PERFORMANCE SITE ========================================Section End===========================================
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