Mechanisms controlling phosphoinositide synthesis at the plasma membrane

描述（由申请人提供）：磷酸肌醇PI 4 P、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，包括胞外/胞吞作用、肌动蛋白组装和几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、许多癌症和几种神经系统疾病。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 <$（PI 4 KIII <$）催化的。尽管这种酶在磷酸肌醇代谢网络中起着重要作用，但令人惊讶的是，对其调控知之甚少。为了了解自然界如何控制PI 4 KIII <$的亚细胞定位和活性，从而调节其控制下的磷酸肌醇池，我在我的初步工作中表征了两种PI 4 KIII <$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EFR 3/Rolling blackout与可溶性三肽衔接子TTC 7合作，将PI 4 KIII <$募集到质膜，即其作用部位。最近，我发现了第三种潜在的PI 4 KIII调节剂，hyccin，一种功能未知的蛋白质，与脑白色物质疾病（称为髓鞘形成不足和先天性白内障（HCC））有关。指导这项工作的首要假设是EFR 3，TTC 7和hycin形成了PI 4KIII蛋白-蛋白相互作用网络的核心，该网络控制质膜上的PI 4P合成。目的1描述了阐明EFR 3的基本细胞功能的研究，并测试EFR 3是神经元突触处磷酸肌醇代谢的主调节剂的假设。目的2概述了生物化学和化学生物学实验，以定义PI 4KIII？/TTC 7/EFR 3复合物的组装及其在质膜上的时空调节的原理。目的3描述了探索hyccin与PI 4KIII、TTC 7和EFR 3之间的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PI 4P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理解PI 4P合成调控如何与更广泛的代谢网络（例如，通过偶联到产生PI（4，5）P2、PI（3，4，5）P3、IP 3和二酰基甘油的下游酶）。同样，我提出的将hyccin功能与PI 4P代谢联系起来的研究代表了朝着理解HCC发病机制的长期目标迈出的第一步，这可能为这种和其他脑白质营养不良的潜在治疗提供线索。

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.