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: 8838431
• 负责人: David Burton Iaea
• 金额: $ 4.31万
• 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 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8838431
• 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存在显着差异。胆固醇占质膜中脂质分子的约30％，并且也富含内吞回收室（ERC）。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腔传输机制在膜之间移动。然而，只有一小部分从质膜内部内部内部到达ER的膜成分，表明ER中的胆固醇感测依赖囊泡的运输，将非常缓慢且效率低下。有大量证据表明，细胞中无抗固醇的速率高。由于胆固醇在水中的溶解度非常低，因此非氧化剂运输需要与载体蛋白结合。类固醇急性调节剂相关的脂质转移（起始）结构域含有蛋白质，参与了多种非固醇的非毒性运输途径。在可溶性起始蛋白中，已证明Stard4在脂质液滴中，以酰基-COA：胆固醇酰基转移酶依赖性方式增加胆固醇酯的积累，并通过胆固醇在转录水平控制。但是，介导Stard4膜靶向，相互作用和固醇提取的精确分子机制尚不清楚。该提案将解决促进Stard4活动和分布维持胆固醇稳态所需的机制。 AIM 1将评估Stard4膜相互作用的机理，并确定介导相互作用的区域。先前与胆固醇复合物中星形结构域的分子动力学模拟表明，晶状体吸收和释放需要Omega-1环的运动。我们将利用核磁共振和X射线晶体学技术来研究固醇 - 蛋白质复合物形成的这些过程，并确定Sterol-Stard4复合物的结构。 AIM 2我们将在体外分析Stard4固醇转移的脂质特异性。在初步研究中，我们已经确定了两个细胞器特异性阴离子脂质Pi（4,5）P2和Pi（3,5）2，它们调节了Stard4定位和活性。此外，我们将使用荧光共振转移固醇转移测定法和停止流动动力学分析来确定Stard4固醇转移的速率限制步骤。为了确定Stard4维持胆固醇稳态所需的细胞因子，我们将开发一种固醇转运动力学模型，以评估质膜和ERC之间Stard4固醇转运的作用。此外，我们将分析特定的磷脂酰肌醇在靶向stard4到特定膜中的作用，以使用AIM 3中的荧光显微镜技术促进固醇转移。