3-hydroxy-3-methylglutaryl (HMG)-CoA reductase is a key regulatory enzyme in cholesterol, dolichol, ubiquinone, and isopentenyl adenosine biosynthesis. The molecular mechanisms through which sterols and non-sterol products derived from mevalonate regulate the level of HMG-CoA reductase, and of reductase mRNA, will be determined. HMG-CoA reductase cDNA and genomic clones will be isolated from wild type rat liver cells and used to determine HMG-CoA reductase is encoded by a single gene or a small gene family. We have established a simple model system for regulation of HMG-CoA reductase based on the water soluble cholesterol derivative methoxy polyoxyethyl (MPOE) cholesterol. The effect of MPOE cholesterol on reductase mRNA levels and on reductase gene transcription, nuclear RNA processing and degradation, and cytoplasmic mRNA stability will be determined. Biochemical experiments and available mutant cell lines will be used to determine whether oxygenated sterols (7-keto cholesterol) exhibit the same mode of actions as MPOE cholesterol. We will use similar techniques to determine whether non-sterol products derived from mevalonate regulate HMG-CoA reductase through the same mechanism as sterols. We shall investigate the possibility that HMG-CoA reductase is subject to multivalent regulation in which different end products of a biosynthetic pathway regulate expression of different members of a gene family. These studies should deepen our understanding of the molecular mechanism through which the level of HMG-CoA reductase, and ultimately the rate of cholesterol synthesis, are controlled. They will also provide a useful model for control of the level of cellular regulatory enzymes in mammalian systems.
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