Reduced nitric oxide (NO) bioactivity is a hallmark of hypercholesterolemia (HC), and is believed to accelerate the progression of atherosclerosis. Our long-term goal is to determine the role of oxidant stress induced by HC and the mechanisms by which it impairs NO bioactivity, in the last funding period, we have made major advances in 1) identifying and understanding the key role of smooth muscle sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) in mediating the decrease in intracellular Ca2+ caused by NO, and 2) demonstrating that SERCA function is impaired, and that it is a direct target of oxidants in atherosclerotic rabbit and human aorta. Recent experiments indicate that high concentrations of reactive oxygen and nitrogen species (RNS) tyrosine-nitrate, oxidize thiols, and inactivate SERCA. However, in normal arteries, low physiological concentrations of NO form RNS that are responsible for reversibly forming glutathione-cysteine adducts (S-glutathiolation) on SERCA, resulting in activation of the enzyme. The hypothesis for the next funding period is that abnormally high levels and prolonged exposure to oxidants that arise during HC are responsible for irreversibly oxidizing cysteine thiols or other amino acid residues on SERCA, and as a result interfere with the normal NO-induced activation of this regulatory enzyme that lowers intracellular Ca2+. There are three aims: #1) To determine precise oxidative modifications in SERCA purified from atherosclerotic rabbit, pig, and human aorta. Chemical analyses and mass spectrometry will allow us to better characterize tyrosine nitration, cysteine thiol oxidation and other oxidant modifications in SERCA in atherosclerotic rabbit, pig and human aorta. #2) To determine how oxidative modifications of SERCA in atherosclerosis interfere with its NO-induced S-glutathioiation and its physiological function to decrease intracellular calcium. #3) To determine the identity of other oxidant-modified proteins in atherosclerotic arteries. Building on our work on SERCA as a paradigm, we will use proteomic methods to identify these proteins in rabbit, pig and human aorta, determine if these proteins participate in the response to NO via S-glutathiolation, and assess if their response to NO is impaired in atherosclerotic aorta.
These aims will be met with a variety of new chemical techniques that we have developed and will benefit from the newly established Boston University Cardiovascular Proteomics Center. ? ?
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