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Technology Project 3

PI: Robert Cotter       Co-PI: Jennifer Van Eyk


Tool Development for Cys and Lys Modifications

Part A. Modifications of Cys residues

 In the heart, superoxide anions (O2.-) can be generated by leakage of electrons from the mitochondrial respiratory chain, by NAD(P)H oxidases in the sarcolemma, by xanthine oxidase (mainly extracellular), or by uncoupled nitric oxide synthase with the production of hydrogen peroxide and the highly reactive hydroxyl radical. Reactive nitrogen species (RNS) are generated by the reaction of nitric oxide (NO) with oxygen or O2.- and include nitrogen trioxide, peroxynitrite, nitrogen dioxide and nitroxyl (HNO).  Cys residues are particularly targeted by these species due to their extreme sensitivity to oxidation.  Our recent proteomic analyses has revealed a panoply of PTM of mitochondrial protein targets in a pacing animal model of heart failure including sites of altered phosphorylation, S-nitrosylation, and tyrosine nitration. To date, there is no complete database for cardiac myocytes regarding Cys PTMs, and even less information about which amino acid residues are modified. This is due, at least in part, to a lack of proteomic tools. Over the past year, we concentrated on optimizing methods for cysteine modifications as protein thiols play important regulatory and structural roles for proteins and are susceptible to ROS and RNS-induced modifications.

Overall Research Goal: The goal is to expand the global enrichment strategies for the specific quantification of a number of ROS and RNS-induced PTM of myocardium and mitochondria focusing initially on S-nitrosylation and disulfide-bonds with an eye on other Cys-modifications for future work.

Part B.  Modification of Lys residues  (Cotter)

Lys is an amino acid that can undergo a number of PTMs, which alter both its hydrophobicity and charge status including acetylation, ubiquitylation, and SUMOylation all of which have been minimally analyzed in cardiac muscle. This is due to lack of global proteomic tools, which we propose to develop in this contract.  Acetylation is the most common modification in histones, and is regulated by histone acetyl transferases (HATs) and histone deacetylases (HDACs) such as the sirtuins. It has also been apparent that there are many other substrates in cardiac myocytes for these same enzymes. We propose to develop tools to quantify and characterize these regulatory proteins.

Overall research plan: Our goal is to develop proteomic tools to enrich, identify and quantify protein with a number of lysine modifications concentrating initially on acetylation and its regulation then proceeding to optimize detection of ubiquitylation and SUMOylation in cardiac muscle.