STRUCTURE AND ACTION OF STEROIDOGENIC REGULATORY PROTEIN

描述(取自应用程序) 类固醇激素是生理过程中普遍存在的调节器，它是 对于个体的生存和对 物种。第一步，速度限制和激素调节 所有类固醇激素的生物合成是将胆固醇转化为 通过线粒体细胞色素P450scc系统产生孕烯醇酮。P450scc活性 是由组织特异性和荷尔蒙调节的 P450scc基因的转录。然而，监管的急性，快速， 类固醇的生成，与应激反应一样，在10-15分钟内发生，是 在胆固醇进入线粒体的水平上进行调节。这种胆固醇 流量受类固醇合成急性调节蛋白(STAR)调节。明星是 在所有表现出急性反应的类固醇生成组织中发现，并 突变会导致潜在的致命性先天性类脂肾上腺增生。 然而，一些制造类固醇的组织(胎盘、大脑)缺乏星形，而 STAR的作用机制尚不清楚。这种不依赖于恒星的类固醇生成 可能与MLN64有关，MLN64是一种普遍表达的蛋白质， 在结构上与恒星有关。本项目将扩大我们对 STAR和MLN64结构和功能通过五个目标1)建立 StAR和MLN-胆固醇转运活性的体外测定(2) 适应我们已建立的细菌表达和纯化程序 与Mln-，N234Mln-的类星结构域的N-62同源 用圆二色谱(CD)表征N-234MlN-的折叠 傅里叶变换红外光谱(FTIR)和荧光光谱， 4)比较了N-62STAR和N-234MlN-的动力学和重离子折叠 交换质谱学，5)确定了N234Mln-的结构 多维高分辨率核磁共振波谱。 实现这些目标将提供发展所需的结构性信息 详细了解STAR和MLN64如何调节胆固醇运动 从外到内的线粒体膜。

DESCRIPTION (taken from the application) Steroid hormones are ubiquitous regulators of physiologic process that are mandatory for the survival of the individual and the propagation of the species. The first, rate limiting and hormonally regulated step in the biosynthesis of all steroid hormones is the conversion of cholesterol to pregnenolone by the mitochondrial cytochrome P450scc system. P450scc activity is regulated chronically by the tissue-specific and hormonally regulated transcription of the P450scc gene. However, the acute, rapid, regulation of steroidogenesis, which occurs in 10-15 minutes as in the stress responses, is regulated at the level of cholesterol entry into mitochondria. This cholesterol flow is regulated by the Steroidogenic Acute Regulatory protein (StAR). StAR is found in all steroidogenic tissues that exhibit an acute response, and StAR mutations cause potentially lethal congenital lipoid adrenal hyperplasia. However, some tissues that make steroids (placenta, brain) lack StAR, and the mechanism of StAR's action is unknown. This StAR-independent steroidogenesis may be associated with MLN64, a ubiquitously expressed protein that is structurally related to StAR. The present project will expand our knowledge of the structure and function of StAR and MLN64 through five aims 1) Establish an in vitro assay for the cholesterol-transport activity of StAR and MLN-64, 2) Adapt our established procedure for the bacterial expression and purification of N-62 homologue of StAR to the StAR-like domain of MLN-64, N234 MLN-64 3) Characterize the folding of N-234 MLN-64 by circular dichroism (CD), Fourier-transform infrared spectroscopy (FTIR), and fluorescence spectroscopy, 4) Compare the dynamics and folding of N-62 StAR and N-234 MLN-64 by deuterium exchange mass spectrometry, and 5) Determine the structure of N234 MLN-64 by multidimensional high resolution nuclear magnetic resonance (NMR) spectroscopy. Fulfilling these aims will provide the structural information needed to develop a detailed understanding of how StAR and MLN64 regulate cholesterol movement from the outer to inner mitochondrial membrane.