Structure-function studies of the sterol transport protein, STARD4.

甾醇转运蛋白 STARD4 的结构功能研究。

• 批准号: 9058424
• 负责人: David Burton Iaea
• 金额: $ 2.59万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-05-01 至 2016-08-26
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9058424
• 关键词: Accounting; Acute; Acyl Coenzyme A; Address; Binding; Biochemical; Biological Assay; Carrier Proteins; Cell membrane; Cells; Cholesterol; Cholesterol Esters; Cholesterol Homeostasis; Complex; Endoplasmic Reticulum; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Homeostasis; In Vitro; Kinetics; Lipids; Maintenance; Mammalian Cell; Mediating; Membrane; Microscopy; Modeling; Molecular; Movement; Nuclear Magnetic Resonance; Organelles; Pathway interactions; Phosphatidylinositol Phosphates; Process; Proteins; Recycling; Role; Site; Specificity; Sterols; Structure; Surface; Techniques; Transferase; Vesicle Transport Pathway; Water; absorption; carrier mediated transport; cholesterol absorption; cholesterol biosynthesis; cholesterol trafficking; insight; interdisciplinary approach; interest; lipid transfer protein; molecular dynamics; novel; protein complex; public health relevance; trafficking

DESCRIPTION (provided by applicant): Significant differences in lipid distribution are maintained between intracellular organelles. Cholesterol comprises ~30% of the lipid molecules in the plasma membrane and is also enriched in the endocytic recycling compartment (ERC). In the endoplasmic reticulum (ER) cholesterol accounts for 5% of the lipid molecules. Cholesterol can move between membranes by vesicular and non-vesicular transport mechanisms. However, only a small fraction of membrane components internalized from the plasma membrane reach the ER, indicating that cholesterol sensing in the ER would be very slow and inefficient if it depended on vesicle transport. There is substantial evidence for high rates of no-vesicular sterol transport in cells. Since cholesterol is very poorly soluble in water, non-vesiculr transport requires binding to carrier proteins. The steroidogenic acute regulator-related lipid-transfer (START) domain containing proteins are involved in several pathways of non-vesicular trafficking of sterols. Among the soluble START proteins, STARD4 has been shown to increase cholesteryl ester accumulation in lipid droplets, in an acyl-CoA:cholesterol acyl-transferase dependent manner, and is controlled at the transcriptional level by cholesterol. However, the precise molecular mechanisms that mediate STARD4 membrane targeting, interaction and sterol extraction are unknown. This proposal will address the mechanisms that facilitate StARD4 activity and distribution required to maintain cholesterol homeostasis. Aim 1 will evaluate the mechanism of STARD4 membrane interaction and identify regions mediating interaction. Previous molecular dynamic simulations of STAR domains in complex with cholesterol have suggested that movement of the Omega-1 loop is required for sterol absorption and release. We will utilize nuclear magnetic resonance and x-ray crystallographic techniques to investigate these processes of sterol-protein complex formation as well as determine the structure of the sterol-STARD4 complex. Aim 2 we will analyze the lipid specificity of STARD4 sterol transfer in vitro. In preliminary studies, we have identified two organelle-specific anionic lipids, PI(4,5)P2 and PI(3,5)2, which modulate STARD4 localization and activity. Additionally, we will identify the rate limiting step in sterol transfer of STARD4 using fluorescence resonance energy transfer sterol transfer assays and stop-flow kinetic analysis. To determine the cellular factors required for the maintenance of cholesterol homeostasis by STARD4, we will develop a sterol transport kinetic model to evaluate the role of STARD4 sterol transport between the plasma membrane and the ERC. Additionally, we will analyze the role of specific phosphatidylinositols phosphates in targeting STARD4 to specific membranes to facilitate sterol transfer using fluorescent microscopy techniques in Aim 3.

描述（由申请人提供）：细胞内细胞器之间的脂质分布保持显着差异。胆固醇占质膜中脂质分子的约 30%，在内吞再循环室 (ERC) 中也富集。在内质网 (ER) 中，胆固醇占脂质分子的 5%。胆固醇可以通过囊泡和非囊泡运输机制在膜之间移动。然而，只有一小部分从质膜内化的膜成分到达内质网，这表明如果依赖于囊泡运输，内质网中的胆固醇感应将非常缓慢且低效。有大量证据表明细胞中非囊泡甾醇转运率很高。由于胆固醇在水中的溶解度非常差，因此非囊泡运输需要与载体蛋白结合。含有类固醇生成急性调节相关脂质转移 (START) 结构域的蛋白质参与甾醇非囊泡运输的多种途径。在可溶性 START 蛋白中，STARD4 已被证明可以以酰基辅酶 A：胆固醇酰基转移酶依赖性方式增加脂滴中胆固醇酯的积累，并在转录水平上受胆固醇控制。然而，介导 STARD4 膜靶向、相互作用和甾醇提取的精确分子机制尚不清楚。该提案将解决促进 StARD4 活性和维持胆固醇稳态所需分布的机制。目标 1 将评估 STARD4 膜相互作用的机制并确定介导相互作用的区域。先前对与胆固醇复合的 STAR 结构域的分子动力学模拟表明，Omega-1 环的运动是甾醇吸收和释放所必需的。我们将利用核磁共振和 X 射线晶体学技术来研究甾醇-蛋白质复合物形成的这些过程，并确定甾醇-STARD4 复合物的结构。目标 2 我们将分析 STARD4 甾醇体外转移的脂质特异性。在初步研究中，我们鉴定了两种细胞器特异性阴离子脂质，PI(4,5)P2 和 PI(3,5)2，它们调节 STARD4 定位和活性。此外，我们将使用荧光共振能量转移甾醇转移测定和停流动力学分析来确定 STARD4 甾醇转移中的限速步骤。为了确定 STARD4 维持胆固醇稳态所需的细胞因子，我们将开发甾醇转运动力学模型来评估 STARD4 甾醇在质膜和 ERC 之间转运的作用。此外，我们将在目标 3 中使用荧光显微镜技术分析特定磷脂酰肌醇磷酸盐在将 STARD4 靶向特定膜以促进甾醇转移中的作用。