THE MOLECULAR BASIS OF ADRENARCHE: DISSECTION OF P450C17

本提案是对K08DK02387项目《肾上腺素的分子基础：P450c17活性的解剖》的补充。在母体奖01年和02年的工作已经产生了一个完整的人类P450c17 （17α -羟化酶/ 17,20 -裂解酶）的计算机模型，该模型已经确定了P450c17与氧化还原伴侣蛋白相互作用的重要残基。对该模型和序列比对的分析揭示了我们认为是活性位点上的残基负责将17 α -羟化酶或21-羟化酶活性传递到P450结构模板上。我们将通过构建P450c17-P450c21杂交蛋白来验证这一预测。我们还表明，细胞色素b5 （b5）通过作为P450c17 P450-OR复合物的变弹性促进剂而不是作为替代电子供体，选择性地增强了人类P450c17的17,20-裂解酶活性。基于我们对牛和大鼠细胞色素b5结构的分析，我们建议研究细胞色素b5的截断和突变形式，以确定人类细胞色素b5刺激P450c17的17,20裂解酶活性的最小功能成分。这些研究将确定P450c17和b5的功能区域，这些功能区域不同地参与17 α -羟化酶和17,20-裂解酶活性的调节，这将为这些活性的药理学操作提供新的方法。在认识到P450c17在性类固醇生物合成中的核心作用的同时，确定肾上腺素的DHEA-S产生的增加也不可能发生，除非P450scc作用于肾上腺线粒体中的胆固醇所产生的孕烯醇酮能够逃避位于整个肾上腺细胞的3 -羟基类固醇脱氢酶/delta5-4-异构酶II型（3betaHSDII）的作用，并结合微粒体P450c17进行两次连续的转换。我们假设3betaHSDII的翻译后修饰可以调节3betaHSDII的亚细胞定位，并且亚细胞定位而不是酶的数量单独控制类固醇前体沿delta5和delta4途径的通量。因此，我们建议确定3betaHSDII是磷酸化还是酰化。我们将使用酵母表达系统来研究3betaHSDII在模型肾上腺细胞系统中的定位动力学和调控。在完成这项工作后，PI将测试一种新的调节机制的假设，以确定沿着特定类固醇生成途径的通量，并开始一个调查领域，以补充他在该提案下的先前工作，并确定他自己的独立调查路线。

This proposal is to supplement grant K08DK02387, The molecular basis of adrenarche: dissection of P450c17 activities. The work during years 01 and 02 of the parent award have yielded a completed computer model of human P450c17 (17alpha- hydroxylase/17, 20-lyase) that has identified important residues for the interaction of P450c17 with redox partner proteins. Analyses of this model and of sequence alignments reveal what we believe to be the residues in the active site responsible for conveying either 17alpha-hydroxylase or 21-hydroxylase activity onto the P450 structural template. We will test this prediction by constructing P450c17-P450c21 hybrid proteins. We have also shown that cytochrome b5 (b5) selectively augments the 17,20- lyase activity of human P450c17 by acting as an allosteric facilitator of the P450c17 P450-OR complex rather that serving as an alternate electron donor. Based on our analysis of the structure of bovine and rat cytochromes b5, we propose to study truncated and mutant forms of cytochrome b5 to identify the minimal functional component of human b5 that stimulates the 17,20-lyase activity of P450c17. These studies will define functional regions of P450c17 and of b5 that differentially participate in the regulation of the 17alpha-hydroxylase and 17,20-lyase activities, which will enable novel approaches to pharmacological manipulation of these activities. While recognizing the central role of P450c17 in the biosynthesis of sex steriods, it is also true that the rise in DHEA-S production that defines adrenarche cannot occur unless pregnenolone, generated by the action of P450scc on cholesterol in the adrenal mitochondria, can escape the action of 3beta-hydroxysteroid dehydrogenase/delta5-4-isomerase type II (3betaHSDII), located throughout the adrenal cell, and bind to microsomal P450c17 for two successive turnovers. We hypothesize that a post- translational modification of 3betaHSDII can regulate the subcellular localization of 3betaHSDII and that subcellular localization rather than quantity of enzyme alone controls the flux of steroid precursors down the delta5 and delta4 pathways. We therefore propose to determine if 3betaHSDII is phosphorylated or acylated. We will use our yeast expression system to study the dynamics and regulation of 3betaHSDII localization in a model adrenal cell system. In completing this work, the PI will test a hypothesis of a novel regulatory mechanism for determining flux along specific steroidogenic pathways and commence an area of investigation that complements his previous work under this proposal and defines his own line of independent investigation.