Protein Radical, Protein Nitration, Mitochondria, and AD

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DESCRIPTION (Taken from the Investigator's Abstract) The causative factors of Alzheimer's disease (AD) involve aging, genetics, and environmental exposures. At the neuronal level, a defect of energy metabolism associated with mitochondrial cytochrome C oxidase (CcO) deficiency was marked in AD brain. The mitochondrial abnormality caused by oxidative damage of CcO has been suggested as a part of causative events in AD. The long-term objective of this proposed research is to identify the molecular mechanism related to oxidative damage of AD-associated mitochondrial CcO. The proteincentered radical(s) in CcO, as detected with ESR spin-trapping technique, is formed by reacting with peroxides, such as hydrogen peroxide, organic peroxides, and lipid peroxides. Available evidence indicates that a specific tyrosine in mitochondrial cytochrome C may be nitrated to form 3- nitrotyrosine. The candidate hypothesizes that a specific amino acid residue(s) in CcO is involved in the radical formation and that 3- nitrotyrosine can be detected when the purified CcO is exposed to a reactive nitrogen species (RNS) such as nitric oxide (NO) or peroxynitrite. The radioisotope-labeled spin trap, [14C]-labeled 2-methyl-2-nitrosopropane (MNP), will be synthesized and used to trap the protein-centered radical(s) in CcO. The subunit (location) of CcO associated with the spin trap will be visualized by SDS-PAGE and radioisotope distribution. NO and peroxynitrite will be used to test the nitration of tyrosine in purified CcO. The nitration of CcO will be characterized by immunoblotting using the antibody against 3-nitrotyrosine. Peptide chemistry/mapping and mass spectrometry will be used to identify specific amino residue(s) associated with the above events of oxidative damage. The functional domains involved in the above radical formation and nitration will be identified. The essential role of the identified domains will be studied by immunochemistry. The results will provide fundamental information concerning the pathological relevance of neurodegenerative disease, such as AD, associated with mitochondrial dysfunction caused by oxidative damage.