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

描述（由申请人提供）：在细胞内细胞器之间保持着显著的脂质分布差异。胆固醇占质膜脂质分子的约30%，并在内吞循环室（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运输机制在细胞膜之间移动。然而，只有一小部分从质膜内化的膜成分到达内质网，这表明内质网中的胆固醇感知如果依赖于囊泡运输，将是非常缓慢和低效的。有大量证据表明细胞中无囊性固醇运输率很高。由于胆固醇很难溶于水，非囊泡运输需要与载体蛋白结合。类固醇急性调节相关脂质转移（START）结构域蛋白参与了几种非囊性固醇转运途径。在可溶性START蛋白中，STARD4已被证明以酰基辅酶a：胆固醇酰基转移酶依赖的方式增加脂滴中的胆固醇酯积累，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确切分子机制尚不清楚。该提案将解决促进维持胆固醇稳态所需的StARD4活性和分布的机制。目的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STAR结构域与胆固醇复合物的分子动力学模拟表明，Omega-1环的运动是固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这些甾醇-蛋白复合物的形成过程，并确定甾醇- stard4复合物的结构。目的2我们将分析体外STARD4甾醇转移的脂质特异性。在初步研究中，我们已经确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的定位和活性。此外，我们将利用荧光共振能量转移甾醇转移测定和停流动力学分析确定STARD4的甾醇转移速率限制步骤。为了确定STARD4维持胆固醇稳态所需的细胞因子，我们将建立一个固醇运输动力学模型来评估STARD4在质膜和ERC之间的固醇运输的作用。此外，我们将在Aim 3中使用荧光显微镜技术分析特定磷脂酰肌醇磷酸酯在将STARD4靶向到特定膜以促进甾醇转移中的作用。

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.