In the last few years, it has been demonstrated by this group and others that rat liver peroxisomes participate in cholesterol synthesis. Peroxisomes contain the following enzymes and proteins involved in cholesterol synthesis: 1) 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme of cholesterol synthesis; 2) the thiolase activity necessary for the initial step in cholesterol synthesis; and 3) cellular sterol carrier protein 2 (SCP-2), a protein required for several steps in cholesterol utilization and biosynthesis. In addition, peroxisomes can convert mevalonic acid to cholesterol in the presence of cytosolic proteins. These unexpected findings raise interesting questions about the characteristics of the proteins involved in peroxisomal cholesterol synthesis, the fate of peroxisome-synthesized cholesterol, and the regulation and function of the peroxisomal system. This proposal will focus on the following: 1. Purification of peroxisomal HMG-CoA reductase and production of polyclonal and monoclonal antibodies that are specific for the peroxisomal reductase; 2. Cloning of the cDNA for the rat and/or mouse peroxisomal HMG-CoA reductase and determination of its nucleotide sequence; 3. Cloning of the human peroxisomal HMG-CoA reductase cDNA. We have shown that the peroxisomal HMG-CoA reductase constitutes the majority of the immunoreactive material in the human hepatoma cell line HepG2; 4. Analysis of the regulation of the peroxisomal HMG-CoA reductase. Since peroxisomes are essential for life, and have also been shown to be involved in cholesterol metabolism, it is critical to obtain answers to the following questions. What is the structure of the peroxisomal HMG-CoA reductase and how is it regulated? What is the significance of the cholesterol synthesis system in peroxisomes? What is the pathway of peroxisomal cholesterol synthesis? What is the fate of the cholesterol synthesized by peroxisomes: is it used for bile acid synthesis, steroid hormone synthesis, vitamin D, or lipoproteins? Does peroxisomal cholesterol synthesis occur in tissues other than the liver? Only a systematic study of the proteins involved in cholesterol synthesis in peroxisomes will begin to answer the above questions. It is essential to understand the function of the peroxisomal reductase and the fate of the cholesterol synthesized by peroxisomes before we can begin to assess the significance and the role of peroxisomal cholesterol synthesis in regulation of total cholesterol homeostasis.
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