Mechanisms controlling phosphoinositide synthesis at the plasma membrane

质膜上磷酸肌醇合成的控制机制

• 批准号: 8678102
• 负责人: JEREMY BASKIN
• 金额: $ 9万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8678102
• 关键词: 1,2-diacylglycerol; 1-Phosphatidylinositol 4-Kinase; Actins; Anabolism; Binding; Biochemical; Biochemical Pathway; Biology; Brain; Brain Diseases; Cell membrane; Cell physiology; Cells; Cellular biology; Chemicals; Collaborations; Commit; Complex; Coupling; Diabetes Mellitus; Diglycerides; Disease; Drosophila genus; Electron Transport Complex III; Endocytosis; Enzymes; Goals; Growth Factor; Impairment; Learning; Light; Lipids; Location; Malignant Neoplasms; Mediating; Membrane Proteins; Metabolism; Molecular; Nature; Nervous system structure; Neurons; Pathogenesis; Peripheral; Phase; Phosphatidylinositols; Physiological; Physiological Processes; Play; Process; Property; Proteins; Recruitment Activity; Regulation; Role; Series; Signal Pathway; Signal Transduction; Site; Sum; Synapses; Techniques; Testing; White Matter Disease; Work; adapter protein; congenital cataract; genetic regulatory protein; insight; leukodystrophy; lipid metabolism; multidisciplinary; mutant; nervous system disorder; novel; palmitoylation; phosphoinositide-3,4,5-triphosphate; protein protein interaction; public health relevance; research study; spatiotemporal; stoichiometry; synaptic function; white matter

DESCRIPTION (provided by applicant): The phosphoinositides PI4P, PI(4,5)P2, and PI(3,4,5)P3 orchestrate numerous physiological processes occurring at the plasma membrane, including exo/endocytosis, actin assembly, and several growth factor signaling pathways, and dysregulation of the metabolism of these lipids is causative of many diseases, including diabetes, numerous cancers, and several neurological disorders. The first committed step in the biosynthesis of these phosphoinositides is catalyzed by the PI 4-kinase Type III¿ (PI4KIII¿). Despite the fundamental role that this enzyme plays in the phosphoinositide metabolic network, surprisingly little is known about its regulation. To understand how nature controls the subcellular localization and activity of PI4KIII¿ and hence regulates the phosphoinositide pools under its control, I have in my preliminary work characterized two PI4KIII¿ regulatory proteins and begun to study their molecular properties and physiological functions. I showed that the palmitoylated, peripheral membrane protein EFR3/Rolling blackout cooperates with a soluble tetratricopeptide adaptor, TTC7, to recruit PI4KIII¿ to the plasma membrane, its site of action. Recently, I identified a third potential PI4KIII¿ regulator, hyccin, a protein of unknown function implicated in a brain white matter disease termed hypomyelination and congenital cataracts (HCC). The overarching hypothesis guiding this work is that EFR3, TTC7, and hyccin form the core of a PI4KIII¿ protein-protein interaction network that controls PI4P synthesis at the plasma membrane. Aim 1 describes studies to elucidate fundamental cellular functions of EFR3 and test the hypothesis that EFR3 is a master regulator of phosphoinositide metabolism at the neuronal synapse. Aim 2 outlines biochemical and chemical biology experiments to define the principles governing the assembly of the PI4KIII¿/TTC7/EFR3 complex and its spatiotemporal regulation at the plasma membrane. Aim 3 describes studies to probe the physical and functional connection between hyccin and PI4KIII¿, TTC7, and EFR3. In sum, these multidisciplinary studies will elucidate fundamental principles that regulate the PI4P synthetic machinery at the plasma membrane. More broadly, the approaches I develop and the principles that I elucidate will be applicable to the long-term goal of understanding how regulation of PI4P synthesis connects to the broader metabolic network (e.g., via coupling to downstream enzymes that generate PI(4,5)P2, PI(3,4,5)P3, IP3, and diacylglycerol). As well, my proposed studies to connect hyccin function to PI4P metabolism represent a first step toward the long-term goal of understanding the mechanisms of pathogenesis of HCC, which may shed light on potential therapies for this and other leukodystrophies.

描述（由申请人提供）：磷酸肌醇 PI4P、PI(4,5)P2 和 PI(3,4,5)P3 协调发生在质膜上的许多生理过程，包括胞吐/内吞、肌动蛋白组装和多种生长因子信号传导途径，这些脂质代谢的失调是许多疾病的原因，包括 糖尿病、多种癌症和多种神经系统疾病。这些磷酸肌醇生物合成的第一个关键步骤是由 PI 4-激酶 III 型 (PI4KIII¿) 催化。尽管这种酶在磷酸肌醇代谢网络中发挥着重要作用，但令人惊讶的是，人们对其调节知之甚少。为了了解大自然如何控制 PI4KIII¿ 的亚细胞定位和活性，从而调节其控制下的磷酸肌醇库，我在前期工作中表征了两种 PI4KIII¡ 调节蛋白，并开始研究它们的分子特性和生理功能。我发现棕榈酰化的外周膜蛋白 EFR3/Rolling blackout 与可溶性四肽接头 TTC7 合作，将 PI4KIII 招募到质膜（其作用位点）。最近，我发现了第三种潜在的 PI4KIII 调节剂，hyccin，一种功能未知的蛋白质，与称为髓鞘形成不足和先天性白内障 (HCC) 的大脑白质疾病有关。指导这项工作的总体假设是 EFR3、TTC7 和 hyccin 形成 PI4KIII 蛋白质-蛋白质相互作用网络的核心，控制质膜上的 PI4P 合成。目标 1 描述了阐明 EFR3 基本细胞功能并测试 EFR3 是神经元突触磷酸肌醇代谢主要调节因子这一假设的研究。目标 2 概述了生化和化学生物学实验，以确定 PI4KIII¿/TTC7/EFR3 复合物的组装及其在质膜上的时空调节的原理。目标 3 描述了探索 hyccin 与 PI4KIII¿、TTC7 和 EFR3 之间物理和功能联系的研究。总之，这些多学科研究将阐明调节质膜 PI4P 合成机制的基本原理。更广泛地说，我开发的方法和我阐明的原则将适用于了解 PI4P 合成的调节如何与更广泛的代谢网络连接的长期目标（例如，通过与生成 PI(4,5)P2、PI(3,4,5)P3、IP3 和二酰基甘油的下游酶偶联）。此外，我提出的将 hyccin 功能与 PI4P 代谢联系起来的研究代表了朝着了解 HCC 发病机制的长期目标迈出的第一步，这可能会为这种疾病和其他脑白质营养不良的潜在疗法带来启示。