Post-translational regulation of CYP17 activity

描述(申请人提供)：多囊卵巢综合征(PCOS)是育龄妇女最常见的生殖内分泌疾病。大约四分之三的无排卵性不孕症妇女患有多囊卵巢综合征，因此约占继发性闭经妇女的三分之一，约90%的月经稀少妇女。多囊卵巢综合征的其他后果包括多毛症、反复早孕丢失的发生率显著增加、50-61岁之间心肌梗死的风险估计增加11倍，以及年轻时子宫内膜癌的风险增加。在患有多囊卵巢综合征的女性中，一个一致的发现是卵巢产生了异常高水平的雄激素。有很好的证据表明，雄激素升高干扰了优势卵泡的选择，并导致PCOS。雄激素生物合成所需的关键酶是细胞色素P17。该酶有两个活性，其中一个是C17-20裂解酶活性，通过磷酸化提高。磷酸化位点尚未确定，关于磷酸化如何增加C17-20裂解酶活性也知之甚少。在初步研究中，我们已经证实，磷酸化增加了C17-20裂解酶的活性，并且有证据支持这样的假设，即多囊卵巢中的CYP17磷酸化增加。本项目的目的是确定细胞色素P17的磷酸化位点，确定调节细胞色素P17磷酸化的激酶和磷酸酶，开始确定细胞内调节细胞色素P17磷酸化的信号机制和刺激它们的激素，并确定这些机制在卵巢高雄激素血症中的作用。为了实现这些目标，我们将使用分子建模技术来预测和优先处理磷酸化位点和激酶识别位点。然后，我们将使用质谱仪、定点突变和细胞表达技术来确认这些位点的身份。根据这些实验的结果，我们将通过确定磷酸化对底物和活性部位的产物对接的影响来完善分子模型。这些实验的结果将使我们能够识别CYP17上活性部位和其他位置的靶点，以及调节CYP17磷酸化的细胞内信号系统中的靶点，以便进行治疗干预。我们希望利用分子模型来设计新的药物，不仅可以治疗卵巢高雄激素症，还可以治疗其他雄激素过剩的疾病，如前列腺癌和心血管疾病。

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.