Molecular Biology of Steroidogenic P450 Enzymes

描述（由申请人提供）：类固醇生物合成从胆固醇开始，并通过良好描述的途径进行。快速诱导类固醇生成需要类固醇生成急性调节蛋白（StAR），它促进胆固醇从线粒体外膜（OMM）向内膜（IMM）的运动。我们提出了StAR作用的熔融球模型，该模型表明，StAR在OMM上经历了酸诱导的构象变化，在那里它应该发挥其活性。我们已经确定，StAR只作用于线粒体外膜，只有StAR的羧基末端a-螺旋与膜相互作用，c -末端螺旋的运动是StAR熔融球转变的重要组成部分，这种运动对StAR的活性至关重要。我们现在建议进一步的实验来剖析StAR与胆固醇和OMM其他成分的复杂相互作用。目标1是“确定StAR如何在OMM上暂停”。与其他蛋白质相比，StAR进入线粒体的速度更慢，其活性水平与它在OMM上停留的时间成正比。我们将通过确定在进入过程中StAR被切割的位点，并通过删除扫描诱变来确定发挥暂停活性的先导结构域，来研究这种“暂停”、缓慢的线粒体进入的机制。目标2是“识别OMM上与StAR相互作用的蛋白质”。大量数据表明外周苯二氮卓受体（PER），一种18kDa的OMM蛋白，参与胆固醇进口过程。我们将研究PBR的功能作用及其与StAR的潜在物理相互作用，并进行实验来鉴定其他可能与StAR相互作用的蛋白质，无论它们的身份如何。目的3是“表征外周苯二氮卓受体（PBR）在StAR作用中的作用”。PBR是胆固醇进入甾体源性线粒体所必需的，而StAR触发了这一进入，但这两种必需蛋白之间的潜在相互作用尚不清楚。我们将通过将PBR结合到脂质体中并识别可用于蛋白质水解的结构域来确定PBR的膜拓扑结构，我们将确定与PBR羧基端结合的胆固醇是否构成了动力学上不同的“不稳定”甾体源性胆固醇池。实现这些目标将大大促进我们对所有肾上腺和性腺类固醇激素生产中必不可少的第一步的理解。

DESCRIPTION (provided by applicant): Steroid biosynthesis begins with cholesterol and proceeds through well-described pathways. The rapid induction of steroidogenesis requires the steroidogenic acute regulatory protein (StAR), which facilitates the movement of cholesterol from the outer mitochondrial membrane (OMM) to the inner membrane (IMM). We proposed the molten globule model of StAR's action, which states that StAR undergoes an acid-induced conformational change on the OMM, where it should exert its activity. We have established that StAR acts exclusively on the outer mitochondrial membrane, that only the carboxyl-terminal a-helix of StAR interacts with the membrane, that movement of this C-terminal helix is an essential component of StAR's molten globule transition, and that this movement is essential for StAR's activity. We now propose further experiments to dissect the complex interaction of StAR with cholesterol and other components of the OMM. Aim 1 is to 'Determine how StAR 'pauses' on the OMM.' StAR is imported into mitochondria more slowly than other proteins, and its level of activity is proportional to the time it resides on the OMM. We shall investigate the mechanism of this 'paused', slow mitochondrial entry by identifying the site(s) at which StAR is cleaved during this entry, and by using deletional scanning mutagenesis to identify leader domains that exert pause activity. Aim 2 is to 'Identify proteins on the OMM that interact with StAR.' Substantial data implicate the peripheral benzodiazepine receptor (PER), an 18kDa OMM protein, in the cholesterol-import process. We shall investigate the functional role of PBR and its potential physical interaction with StAR, as well as performing experiments to identify other potential StAR-interacting proteins, irrespective of their identity. Aim 3 is to 'Characterize the role of the peripheral benzodiazepine receptor (PBR) in StAR's action.' PBR is required for cholesterol's entry into steroidogenic mitochondria, and StAR triggers this entry, but the potential interactions between these two essential proteins remain unclear. We shall determine the membrane topology of PBR by incorporating PBR into liposomes and identifying the domains accessible for proteolysis, and we shall determine if the cholesterol bound to the carboxyl-terminus of PBR constitutes the kinetically distinct pool of 'labile' steroidogenic cholesterol. Fulfilling these aims will substantially advance our understanding of this indispensable initial step in the production of all adrenal and gonadal steroid hormones.