Molecular Biology of Steroidogenic P450 Enzymes

描述（由申请人提供）：类固醇生物合成始于胆固醇，并通过充分描述的途径进行。类固醇生成的快速诱导需要类固醇生成急性调节蛋白（星星），其促进胆固醇从线粒体外膜（OMM）向内膜（IMM）的移动。我们提出了熔融球模型的星星的行动，这表明，星星经历了酸诱导的构象变化的OMM，它应该发挥其活性。我们已经确定，星星只作用于线粒体外膜，只有羧基末端的α-螺旋的星星与膜相互作用，这一C-末端螺旋的运动是一个重要组成部分的星星的熔融球过渡，这种运动是必不可少的星星的活动。我们现在提出进一步的实验来剖析星星与胆固醇和OMM的其他组分的复杂相互作用。目标1是“确定星星如何在OMM上”暂停“。星星比其他蛋白质更慢地进入线粒体，其活性水平与其在OMM上停留的时间成正比。我们将研究这种“暂停”的机制，通过识别星星在此进入过程中被切割的位点，并通过使用缺失扫描诱变来识别发挥暂停活性的前导结构域，缓慢进入线粒体。目的2是“鉴定OMM上与星星相互作用的蛋白质。大量数据表明，外周苯二氮卓受体（PER），一种18 kDa的OMM蛋白，参与胆固醇输入过程。我们将研究PBR的功能作用及其与星星的潜在物理相互作用，并进行实验以确定其他潜在的StAR相互作用蛋白，无论其身份如何。目的3：研究外周苯二氮卓受体（PBR）在星星作用中的作用。PBR是胆固醇进入类固醇生成线粒体所必需的，星星触发了这种进入，但这两种必需蛋白质之间的潜在相互作用仍不清楚。我们将确定PBR的膜拓扑结构，通过将PBR纳入脂质体和确定可用于蛋白水解的结构域，我们将确定结合到PBR羧基末端的胆固醇是否构成动力学上不同的“不稳定”类固醇生成胆固醇池。实现这些目标将大大推进我们对所有肾上腺和性腺类固醇激素产生中这一不可或缺的初始步骤的理解。

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.