Therapeutic strategies for reducing serum cholesterol have been aimed at decreasing the intake or absorption of dietary cholesterol and at inhibiting the rate-limiting enzyme of de novo cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl Coenzyme A reductase (HMGR). HMGR is extremely sensitive to oxidative inactivation and intermolecular disulfide crosslinking in glutathione redox buffers (mixtures of glutathione and glutathione disulfide) that are comparable to the highly reducing intracellular environment. This leads to the hypothesis that the reversible formation of disulfide bonds is an important component of the regulation of this enzyme. The major form of HMGR found in vivo using methods to freeze the thiol/disulfide redox state of the enzyme is a disulfide-linked dimer between a full-length 97 kDa species and a partially proteolyzed fragment. Mevinolin, an HMGR inhibitor used clinically, alters the redox behavior of HMGR significantly. The long-range goal of this research program is to understand the molecular details and regulatory consequences of the thiol/disulfide redox state changes that are observed for HMGR in vivo. The coupling between the thiol/disulfide redox state of HMGR and the intracellular glutathione redox state will be investigated by manipulating the intracellular glutathione redox buffer of hepatocytes by 2-oxothiazolidine-4-carboxylate, glutathione monoethyl ester, and buthionine sulfoximine and observing the effects on cholesterol biosynthesis and the redox state and turnover rate of HMGR. The structural basis for the oxidative inactivation and disulfide crosslinking of HMGR in the presence and absence of mevinolin will be studied by specific labeling, peptide mapping, and sitedirected mutagenesis of a 58 kDa soluble, active fragment of HMGR expressed in E coli. Experiments will probe several potential mechanisms that could account for the formation of the disulfide dimers of HMGR that are observed in vivo including the effects of phosphorylation and proteolysis on the redox behavior of HMGR. The structural basis for the effect of mevinolin on the redox behavior of the enzyme will be investigated with mevinolin analogs, and experiments will search for interactions between thiol/disulfide redox state changes and other mechanisms of HMGR regulation including phosphorylation and proteolysis.
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