? The goals of this proposal are to complete and extend our study designed to assess the effects of peroxynitrite (ONOO-) on microtubule proteins. Microtubules, composed of tubulin and microtubuleassociated proteins (MAPs), are a key component of the neuronal cytoskeleton and are requisite for proper neuron function. Oxidative damage to proteins and cytoskeletal abnormalities have been detected in neurons of Alzheimer's disease (AD) patients. Tau, a neuron-specific MAP, is the major component of the paired helical filaments (PHFs) found in neurofibrillary tangles (NFTs) of AD brain and tau isolated from NFTs no longer binds to microtubules. ONOO-, formed from the reaction of nitric oxide and superoxide, is a strong oxidant that can damage several amino acids in proteins. Cysteine oxidation of tubulin, the main component of microtubules, has been identified as the primary mechanism by which ONOO- inhibits microtubule polymerization.
The specific aims of this proposal are: 1. To complete the characterization and quantitation of ONOO- induced damage to microtubule proteins including tubulin, tau and MAP2; 2. To explore the reactivity of the cysteines of tubulin, tau and MAP2 through kinetic and equilibrium studies; and 3. To investigate the interactions of native and ONOO- -treated tubulin with physiologic redox regulatory elements including the thioredoxin reductase system (TRS) and nitric oxide (NO). The studies described herein will provide valuable information about the role that sulfhydryl groups of microtubule proteins play in the regulation of microtubule assembly in vivo. The results gathered will enhance our understanding of the susceptibility of tubulin, tau and MAP2 to oxidation by reactive oxygen species. This work is important because ONOO- damage to microtubule proteins may jeopardize microtubule function and may be a critical early step in the development of neurodegenerative diseases such as AD. These studies will also enhance our understanding of the protein interactions that are required for proper microtubule formation and stability. ? ?
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