Covalent binding of reactive intermediates to target proteins is an important mechanisms of chemical toxicity. Although this mechanism of cytotoxicity has been recognized for more than 20 years, there is still very little known about the actual protein targets for reactive species. Our objective is to understand how reactive intermediates kill cells by combining covalently with specific target proteins. The long term goal is to understand the nature of the cellular response to this type of damage with regard to cell death and repair of protein damage. These are fundamental issues in Toxicology which cannot be answered until model biological system in which the protein targets have been identified are available. The experiments proposed in this application are a first step toward that goal. We believe that combining sound chemical and biochemical knowledge of toxic mechanisms will lead to development of better therapeutic strategies to treat kidney damage. Activation of nephrotoxic cysteine conjugates (NCC) by the beta-lyase pathway is an important mechanism of nephrotoxicity for environmental chemicals. These toxins form reactive intermediates which covalently modify proteins in the mitochondria of the proximal tubule epithelium resulting in cell death. The broad goal of this project is to test the hypothesis that """"""""Reactive species derived from metabolism of NCC damage specific proteins targets which leads to a loss of vital cellular functions and cell death."""""""" The specific objectives are to determine, i) the chemical structure of NCC-derived protein adducts and raise antibodies which recognize them, 2) to purify and identify protein targets, and 3) to investigate the mechanisms through which modification of target proteins kills cells in vivo and in vitro. To accomplish these goals we will, 1) use NMR and mass spectrometry to obtain adduct structures, 2) purify adducted proteins and obtaining amino acid sequence data, and 3) use antibodies against NCC-adducts to investigate mechanisms of cytotoxicity in vivo and in vitro.
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