Atherosclerosis and heart disease are the leading cause of death in the industrial world. A major contributor to the pathogenesis of this disease is high levels of low density lipoprotein in serum, a condition referred to as hypercholesteremia. A major focus of pharmacological treatments of hypercholesteremia is HMG-CoA reductase, the enzyme that catalyzes the conversion of HMG-CoA to mevalonate. This early step in the pathway of cholesterol synthesis is regulated by a complex interaction of multiple regulatory influences including regulation of transcription, translation, and enzyme turnover. Although inhibitors of HMG-CoA reductase are huge financial successes, the effectiveness of these drugs is marginal because inhibiting the activity of this enzyme induces the synthesis of additional enzyme through complex and poorly understood regulatory mechanisms. The goal of this research is to establish the molecular mechanisms regulating the sterol biosynthetic pathway in the genetically and biochemically tractable Saccharomyces cerevisiae. This information will give valuable insights into the roles played by the many intermediates in this pathway. In addition these insights may allow more effective therapies to be developed. In the last grant period, all three levels of regulation of HMG-CoA reductase were discovered. Insights into the genetic mechanisms governing some aspects of this regulation were made, and some novel and surprising connections were discovered between intermediates of the sterol biosynthetic pathway and the transcription of genes encoding prenylated proteins. In addition, the discovery of protein prenylation occurred in the last grant period which provides a link between the cholesterol biosynthetic pathway and the biology of Ras-mediated tumors. The research proposed in this grant application is a logical extension of the discoveries of the last period with an emphasis on mechanisms underlying the regulation. The foci of the proposed research are 1) to determine if there is a regulatory response of HMG-CoA reductase synthesis to sterols in yeast? 2) How is transcription of HMG1 and HMG2 , the structural genes for HMG-CoA reductase regulated by heme? (3) Development of yeast strains and conditions that allow efficient mevalonate uptake from the medium in order to manipulate the physiology of the pathway and to allow metabolic labeling with mevalonate. (4) Determine the mechanism regulating the half- life of HMG2. (5) Establishment of an in vitro assay for regulated degradation. (6) Determine how the flux through the sterol pathway controls the level of Ras and a-factor mRNA levels and map the signal tying mRNA levels to the sterol pathway. and (7) Determine whether specific prenyl lipids confer unique properties to prenylated proteins and how the functional differences between farnesylated and geranylgeranyl Ras2 is mediated.
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