OXYSTEROL BINDING PROTEIN AND CHOLESTEROL METABOLISM

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The metabolism of cholesterol is a central problem in health and disease. Oxysterols are the most potent known regulators of cholesterol synthesis. They are produced naturally at various steps in steroid biosynthetic and degradative pathways, and they are readily formed through autooxidation in the body and outside it. A protein fraction containing a specific oxysterol binding site has been identified recently in many cells and tissues. We propose that this protein fraction contains a specific oxysterol binding protein (OBP). Occupancy of this cytoplasmic binding site by potent oxygenated sterols correlates, both with respect toconcentration and specificity, with those compounds capable of down-regulating 3-hydroxy-3-methyglutary1 coenzyme A reductase (HMG CoA reductase), the rate limiting enzyme in cholesterol biosynthesis. Occupancy of OBP also may correlate with inhibition of cell growth. OBP may be, therefore, an important regulatory protein in both sterol synthesis and cell growth. We propose to purify OBP from a human (CEM) cell line and from cultured mouse L cells, to prepare both mono- and poly-clonal antibodies against it, and to use these to further study the protein and to isolate and sequence its gene. We also will determine the specific and non-specific DNA binding properties of OBP. Total genomic DNA from several sources, and DNA from specific genes, including the gene from HMG CoA reductase, will be examined for binding.