The enzyme 3-Hydroxy-3-Methylglutaryl Coenzyme A (HMG-CoA) Reductase plays a key role in regulating cholesterol biosynthesis. It produces mevalonate, which is required for the synthesis of cholesterol and other isoprenoids, such as ubiquinone, dolichol, and isopentenyl tRNA. A complete understanding of the mechanisms regulating synthesis of this enzyme is crucial, especially view of the fact that serum cholesterol is one of the strongest risk factors in the development of atherosclerosis. HMG-CoA reductase is subject to negative feedback regulation, and this regulation is mediated by metabolic intermediates in the cholesterol biosynthetic pathway. A complete suppression of HMG-CoA reductase synthesis in mammalian cells requires both a sterol (or an oxysterol), as well as a non- sterol, mevalonate derived intermediate. HMG-CoA reductase is a highly regulated enzyme, and it is well established that the level of HMG-CoA reductase activity is regulated through changes in the level of HMG-CoA reductase mRNA as well as through sterol mediated degradation of membrane bound enzyme. Of equal importance are studies which have established that levels of HMG-CoA reductase enzyme activity are also regulated at the level of translation, or the rate of protein synthesis from the HMG-CoA reductase mRNA. The overall goal of this project is to determine the mechanism through which oxysterols and mevalonate regulate translation of HMG-CoA reductase from its mRNA. The hamster C100 cell line will be used for experiments in specific aims land 3. This cell line expresses high levels of both mRNA and enzyme activity for HMG-CoA reductase. There are two classes of transcripts for hamster HMG-CoA reductase known as classes I and II. They differ in the length of their 5' untranslated leader sequences, and both classes code for the same protein species. C100 cells will be treated with the drug mevinolin, a transition state inhibitor of HMG-CoA reductase, and the effects of both an oxysterol (25-hydroxycholesterol) and mevalonate will be evaluated on the synthesis of HMG-CoA reductase in these cells. Experiments in specific aim #1 will determine the correlation between changes in the relative amounts of classes I and II HMG-CoA reductase transcripts nad changes in the rate of HMG-CoA reductase synthesis. The role of 5' untranslated leader sequences in regulating translation of HMG-CoA reductase mRNA will be evaluated in specific aim 2 using a heterologous protein expression system.
In specific aim 3 the role of both classes I and II HMG-CoA reductase mRNA transcripts in the initiation of translation for HMG-CoA reductase, as well as the effects of mevinolin, 25-hydroxycholestrol, and mevalonate on this initiation process, will be evaluated. The role of either soluble cytoplasmic or ribosomally associated factors involved specifically in the regulation HMG-CoA reductase translation will be evaluated in specific aim #4.
In specific aim #5 the role of mevinolin, 25-hydroxycholesterol and mevalonate in regulating the synthesis of HMG-CoA reductase at the level of translation will be evaluated in cultured primary rat hepatocytes.
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