Intracellular Cholesterol Transport

胆固醇在决定生物膜性能方面起着至关重要的作用。控制 细胞器中的胆固醇水平取决于鲜为人知的类固醇运输机制。小说 已经发展了定量研究细胞器间甾醇运输的方法，这导致了 细胞器间运输的精确模型。初步数据显示，一种广泛表达的甾醇- 受调控的可溶性固醇转运蛋白STARD4在胆固醇的非囊泡转运中发挥着重要作用。 并对其作用和转运机制进行了研究。STARD4是复合体的重要组成部分 类固醇调节网络。最近的研究表明，STARD4负责大约25%的固醇运输 在U2OS细胞的两个主要池--质膜和内吞循环室之间。 STARD4的作用也将在巨噬细胞中进行评估，巨噬细胞是一种胆固醇代谢起作用的细胞类型。 在动脉粥样硬化的发展中起主要作用，并在肝细胞模型HepG2细胞中发挥重要作用。在预赛中 研究发现，STARD4与磷脂酰肌醇磷酸(PIP)具有独特的相互作用。 STARD4从膜中提取甾醇的速度在膜中含有 PI(4，5)P2，但PI(4，5)P2对接受StARD4甾醇的膜无影响。相反， 受体膜中的PI(3，5)P2或PI5P可使∼转移速度提高10倍，但这些PIP不起作用 供体膜。PI3P也有类似的作用，但影响较小。STARD4在固醇转运到晚期中的作用 将分析内噬小体、脂滴和自噬小体(即具有良好受体PIP的细胞器)。 STARD4与膜相互作用的分子动力学模拟正在确定其潜在的位置 与PIP的互动。这些位点突变的影响将在脂质体中检测到脂质体中的甾醇。 转运试验。PIP敏感性改变的突变STARD4分子将在细胞中表达，以确定 对甾醇运输和分布的影响。在初步研究中，通过分子动力学鉴定突变体 在脂质体试验中得到证实，并且发现它们确实影响类固醇在 细胞器。X射线结晶学已经确定了有结合和没有结合的人类STARD4的差异 甾醇。核磁共振波谱被用来分析STARD4蛋白质的动力学和确定 与含有不同PIP的脂类相互作用。

Cholesterol plays an essential role in determining the properties of biological membranes. Control of cholesterol levels in organelles depends on sterol transport mechanisms that are poorly understood. Novel methods to study sterol transport among organelles quantitatively have been developed, and this is leading to precise models for transport among organelles. Preliminary data show that a widely expressed, sterol- regulated, soluble sterol transporter, STARD4, plays an important role in nonvesicular transport of cholesterol, and its role and mechanism of transport will be studied. STARD4 is an important component of a complex sterol regulatory network. Recent work showed that STARD4 is responsible for about 25% of sterol transport between two major pools – the plasma membrane and the endocytic recycling compartment - in U2OS cells. The role of STARD4 will also be assessed in macrophages, a cell type in which cholesterol metabolism plays a major role in development of atherosclerosis, and in HepG2 cells, a model for hepatocytes. In preliminary studies it has been found that STARD4 has unique interactions with phosphatidylinositol phosphates (PIPs). STARD4 extraction of sterol from membranes is accelerated nearly 10-fold when the membranes contain PI(4,5)P2, but there is no effect of PI(4,5)P2 in membranes accepting sterol from STARD4. Conversely, PI(3,5)P2 or PI5P in acceptor membranes accelerates transfer ∼10-fold with no effect when these PIPs are in donor membranes. PI3P has a similar but smaller effect. The role of STARD4 in sterol transport to late endosomes, lipid droplets, and autophagosomes (i.e., organelles with good acceptor PIPs) will be analyzed. Molecular dynamics simulations of interactions of STARD4 with membranes is identifying potential sites of interaction with PIPs. The effects of mutations of these sites will be assayed in a liposome to liposome sterol transport assay. Mutant STARD4 molecules with altered PIP sensitivity will be expressed in cells to determine effects on sterol transport and distribution. In preliminary studies, mutants identified by molecular dynamics were confirmed by tests in the liposome assay, and it was found that they do affect transport of sterol among organelles. X-ray crystallography has identified differences between human STARD4 with and without bound sterol. NMR spectroscopy is being used to analyze STARD4 protein dynamics and to determine the sites of interaction with lipids containing various PIPs.