STRUCTURE AND ACTION OF STEROIDOGENIC REGULATORY PROTEIN

描述（取自应用程序） 类固醇激素是普遍存在的生理过程的调节剂， 第二，人的一生是怎样的？ 物种第一步，利率限制和严格管制的步骤， 所有类固醇激素的生物合成是胆固醇转化为 通过线粒体细胞色素P450 SCC系统对双烯醇酮进行修饰。P450 SCC活性 是由组织特异性和神经调节的 P450 SCC基因的转录。然而，急性，快速，调节 类固醇生成，如在应激反应中发生在10-15分钟内， 调节胆固醇进入线粒体的水平。这种胆固醇 流量由类固醇生成急性调节蛋白（星星）调节。星星是 在所有表现出急性反应的类固醇生成组织中发现， 突变导致潜在致命的先天性类脂质肾上腺增生。 然而，一些制造类固醇的组织（胎盘，大脑）缺乏星星， 星星的作用机制尚不清楚。这种不依赖于StAR的类固醇合成 可能与MLN 64有关，MLN 64是一种普遍表达的蛋白质， 在结构上与追回星星有关。本项目将扩大我们的知识， 追回星星和MLN 64的结构和职能 星星和MLN-64的胆固醇转运活性的体外测定，2） 调整我们建立的细菌表达和纯化程序 星星的N-62同源物与MLN-64的StAR样结构域N234 MLN-64 3） 通过圆二色性（CD）表征N-234 MLN-64的折叠， 傅里叶变换红外光谱（FTIR）和荧光光谱， 4)比较N-62星星和N-234 MLN-64在氘下的动力学和折叠 交换质谱法，和5）通过以下测定N234 MLN-64的结构： 多维高分辨率核磁共振（NMR）光谱。 实现这些目标将提供发展所需的结构信息， 详细了解星星和MLN 64如何调节胆固醇运动 从线粒体外膜到线粒体内膜。

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.