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

描述（由申请方提供）：细胞内细胞器之间的脂质分布存在显著差异。胆固醇占质膜中脂质分子的约30%，并且也在内吞再循环室（ERC）中富集。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转运机制在膜之间移动。然而，只有一小部分从质膜内化的膜组分到达ER，表明ER中的胆固醇传感将是非常缓慢和低效的，如果它依赖于囊泡运输。有大量的证据表明，细胞中的非囊泡固醇转运率很高。由于胆固醇在水中的溶解度非常低，非囊泡转运需要与载体蛋白结合。类固醇生成急性调节相关脂质转移（START）结构域蛋白参与了甾醇的非囊泡运输的几个途径。在可溶性START蛋白中，STARD 4已显示以酰基-CoA：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，并且在转录水平上受胆固醇控制。然而，介导STARD 4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StARD 4活性和分布的机制，以维持胆固醇稳态。目的1将评估STARD 4膜相互作用的机制并鉴定介导相互作用的区域。先前对与胆固醇复合的星星结构域的分子动力学模拟表明，胆固醇吸收和释放需要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甾醇-蛋白质复合物形成的这些过程，以及确定甾醇-STARD 4复合物的结构。目的2分析STARD 4甾醇转运的脂质特异性。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，调节STARD 4的定位和活性。此外，我们将使用荧光共振能量转移固醇转移测定和停流动力学分析来鉴定STARD 4的固醇转移中的限速步骤。为了确定STARD 4维持胆固醇稳态所需的细胞因子，我们将开发一个固醇转运动力学模型来评估STARD 4固醇在质膜和ERC之间转运的作用。此外，我们将分析特定的磷脂酰肌醇磷酸在靶向STARD 4的特定膜，以促进固醇转移使用荧光显微镜技术的目的3的作用。

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.