THE MOLECULAR BASIS OF ADRENARCHE--DISSECTION OF P450C17

本提案是对K 08 DK 02387基金的补充，肾上腺初显的分子基础：P450 c17活性的解剖。 在01年和02年期间的工作已经产生了一个完整的人类P450 c17（17 α-羟化酶/17，20-裂解酶）的计算机模型，该模型已经确定了P450 c17与氧化还原伴侣蛋白相互作用的重要残基。该模型和序列比对的分析揭示了我们认为是负责将17 α-羟化酶或21-羟化酶活性传递到P450结构模板上的活性位点中的残基。 我们将通过构建P450 c17-P450 c21杂合蛋白来验证这一预测。 我们还表明，细胞色素b5（b5）选择性增强17，20-裂解酶活性的人P450 c17作为一个变构促进剂的P450 c17 P450-OR复合物，而不是作为一个替代的电子供体。 基于我们的牛和大鼠细胞色素b5的结构分析，我们建议研究截断和突变形式的细胞色素b5，以确定最小的功能成分的人b5刺激17，20-裂解酶活性的P450 c17。 这些研究将确定P450 c17和b5的功能区域，这些功能区域差异参与17 α-羟化酶和17，20-裂解酶活性的调节，这将使这些活性的药理学操作成为可能。 虽然认识到P450 c17在性类固醇生物合成中的中心作用，但也确实，定义肾上腺初显的DHEA-S产生的增加不可能发生，除非由P450 SCC作用于肾上腺线粒体中的胆固醇而产生的双烯醇酮可以逃避3 β-羟基类固醇脱氢酶/δ 5 -4-异构酶II型的作用（3 β HSDII），位于整个肾上腺细胞，并结合微粒体P450 c17两个连续的周转。 我们假设3 β HSDII的翻译后修饰可以调节3 β HSDII的亚细胞定位，并且亚细胞定位而不是单独的酶的量控制类固醇前体沿着δ 5和δ 4途径的流动。因此，我们建议确定3 β HSDII是磷酸化的还是酰化的。 我们将使用我们的酵母表达系统来研究模型肾上腺细胞系统中3 β HSDII定位的动力学和调节。 在完成这项工作时，PI将测试一种新的调节机制的假设，用于确定沿着特定类固醇生成途径的通量，并开始一个研究领域，补充他在本提案下的先前工作，并定义他自己的独立研究路线。

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.