Lipid Trafficking for Steroidogenesis

类固醇激素在细胞新陈代谢的几乎每一个方面都扮演着重要的角色，包括 调节碳水化合物、脂肪和蛋白质代谢和免疫功能(糖皮质激素)，以及 盐和水平衡和血压调节(矿质皮质激素)。他们还关键地参与了 在维持第二性征、生殖功能以及肌肉和骨骼生长方面 (睾丸激素、孕激素和雌激素)。因此，了解类固醇激素的产生具有重要意义。 对几乎所有与正常人类健康相关的特征的影响，但也与大多数疾病有关，使其 与退伍军人管理局非常相关。类固醇激素生物合成的共同前体是胆固醇，其中 限速步骤是将胆固醇从外线粒体膜转移到内线粒体膜的过程。 尽管胆固醇通过运输来产生类固醇激素一直是密集调查的对象， 胆固醇转运到线粒体膜外膜的机制仍不完全 明白了。这项提案的总体目标是阐明贩毒的根本机制。 胆固醇对类固醇合成的作用。总体目标将通过检验两个主要假设来实现。首先，我们 提出胆固醇酯选择性地从高密度脂蛋白转移到血浆的新假说 膜通过SR-B1，在细胞质一侧表现为微小的脂滴，获得脂滴- 然后引导胆固醇酯(微脂滴)结合成成熟脂类的相关蛋白质 或与现有的细胞质脂滴融合。这将通过跟踪 高密度脂蛋白中胆固醇酯进入肾上腺和性腺细胞内脂滴的运动 哪些特定的胞浆蛋白已被击倒或被抑制。其次，我们假设 胆固醇从质膜、内质网、 而线粒体的脂滴每个都涉及不同的机制，包括囊泡和非囊泡。 水泡的意思是。这一目标将使用几种不同的方法来实现。首先，一种新的无细胞 我们建立了胆固醇转运到线粒体的体外重建试验，其中含有分离的 线粒体和重组STAR将被用来评估胆固醇从 质膜、内质网和脂滴存在的特异性重组 蛋白质，包括陷阱。第二，我们将敲除特定的质膜和内质 颗粒细胞和肾上腺细胞中的网状蛋白评估其对胆固醇转运的影响 线粒体。第三，我们将使用SNARE突变小鼠来比较胆固醇稳态和运动与 体内线粒体和类固醇的生成。这些研究的结果应该会为 类固醇激素产生的关键生物学过程。

Steroid hormones play important roles in virtually every aspect of cellular metabolism, including the regulation of carbohydrate, lipid and protein metabolism and immune function (glucocorticoids), as well as salt and water balance and blood pressure regulation (mineralocorticoids). They are also critically involved in the maintenance of secondary sex characteristics, reproductive functions and muscle and bone growth (testosterone, progestins and estrogens). Thus, understanding steroid hormone production has important implications for almost every feature related to normal human health, but also to most diseases, making it quite relevant for the VA. The common precursor of steroid hormone biosynthesis is cholesterol, with the rate-limiting step being the transfer of cholesterol from the outer to the inner mitochondrial membrane. Though cholesterol trafficking for steroid hormone production has been the subject of intense investigation, the mechanisms how cholesterol traffics to the outer mitochondrial membrane remain incompletely understood. The overall goal of this proposal is to elucidate the mechanisms underlying the trafficking of cholesterol for steroidogenesis. The overall goal will be achieved by testing 2 major hypotheses. First, we propose the novel hypothesis that cholesteryl esters selectively transferred from HDL traverse the plasma membrane via SR-B1 and at the cytoplasmic side behave as micro-lipid droplets, acquiring lipid droplet- associated proteins that then direct the cholesteryl esters (micro-lipid droplets) to coalesce into mature lipid droplets or to fuse with existing cytoplasmic lipid droplets. This will be accomplished by tracing the movement of cholesteryl esters contained within HDL into lipid droplets in adrenal and gonadal cells in which specific cytosolic proteins have been knocked down or inhibited. Second, we hypothesize that the pathways through which cholesterol is transported from the plasma membrane, the endoplasmic reticulum, and lipid droplets to mitochondria each involves different mechanisms that comprise both vesicular and non- vesicular means. This aim will be accomplished using several different approaches. First, a novel cell-free in vitro reconstitution assay we have developed for cholesterol transport to mitochondria containing isolated mitochondria and recombinant StAR will be used to assess the efficiency of cholesterol delivery from the plasma membrane, the endoplasmic reticulum and lipid droplets in the presence of specific recombinant proteins, including SNAREs. Second, we will knockdown specific plasma membrane and endoplasmic reticulum proteins in granulosa and adrenal cells to assess their impact on cholesterol transport to mitochondria. Third, we will use SNARE mutant mice to compare cholesterol homeostasis and movement to mitochondria and steroidogenesis in vivo. The results from these studies should provide insights into the critical biological process of steroid hormone production.