Post-translational regulation of CYP17 activity

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DESCRIPTION (provided by applicant): Polycystic ovary syndrome (PCOS) is the most common reproductive endocrine disease in women of reproductive age. Approximately three-quarters of women with anovulatory infertility have PCOS, thus accounting for approximately one-third of women with secondary amenorrhea and approximately 90% of women with oligomenorrhea. Other consequences of PCOS are hirsutism, markedly increased incidence of recurrent early pregnancy loss, an estimated 11-fold increased risk of myocardial infarction between the ages of 50-61 years, and an increased risk of endometrial cancer at a young age. A consistent finding in women with PCOS is that the ovaries produce abnormally high amounts of androgens. There is good evidence to conclude that elevated androgens interfere with selection of dominant follicles and cause PCOS. The key enzyme required for androgen biosynthesis is known as CYP17. The enzyme has two activities, one of which, the C17-20 lyase activity, is increased by phosphorylation. The phosphorylation sites have not been identified and there is little known about how phosphorylation increases C17-20 lyase activity. In preliminary studies, we have confirmed that phosphorylation increases C17-20 lyase activity and that there is evidence to support the hypothesis that CYP17 phosphorylation is increased in polycystic ovaries. The purpose of this project is to identify the phosphorylation sites on CYP17, to identify kinases and phosphatases that regulate CYP17 phosphorylation, to begin to identify intracellular signaling mechanisms that regulate CYP17 phosphorylation and the hormones that stimulate them, and to determine the role of these mechanisms in ovarian hyperandrogenism. To accomplish these goals we will use molecular modeling techniques to predict and prioritize phosphorylation sites and kinase recognition sites. We will then confirm the identity of these sites using mass spectrometry, site-directed mutagenesis and cellular expression techniques. From the results of these experiments we will refine the molecular model by determining the effects of phosphorylation on substrate and product docking in the active site. The results of these experiments will enable us to identify targets at the active site and other locations on CYP17 and in the intracellular signaling systems regulating CYP17 phosphorylation for therapeutic intervention. We expect to utilize the molecular model to design new Pharmaceuticals to treat not only ovarian hyperandrogenism, but also other diseases of androgen excess such as prostate cancer and cardiovascular diseases.