Pathophysiology of Plasma Membrane PI4P Generation

 性状（由申请方提供）：磷脂酰肌醇的磷酸化代谢产物，称为磷酸肌醇，在多种生理过程中发挥主要的组成和调节作用。它们的产生和相互转化，通过肌醇环的3、4和5位的磷酸化和去磷酸化，由大量激酶和磷酸酶介导。对于这种代谢特别重要的酶是磷脂酰肌醇4-激酶IIIa型（PI 4KIIIa），因为这种蛋白质负责在质膜上产生大量磷脂酰肌醇-4-磷酸（PI 4P）。该PI 4P库是质膜特性的关键决定因素，并且是大多数细胞PI（4，5）P2的前体，因此也是其下游代谢物，例如PI（3，4，5）P3、DAG和IP 3。因此，PI 4KIIIa的功能影响许多基本过程，包括外吞作用、肌动蛋白动力学、离子和其它物质跨质膜的转运、信号转导和细胞周期的调节。PI 4KIIIa是从酵母到人类保守的蛋白质复合物的一部分。激酶PI 4 KIIIa本身（在所有物种中由单个基因表达）对于细胞生命至关重要，而辅助因子（EFR 3、TTC 7和FAM 126，这三种因子均由哺乳动物中的一对基因编码）的缺陷与人类疾病有关。PI 4KIIIa复合物对细胞生理学的根本重要性及其与病理条件的新兴联系有力地证明了了解其结构和功能的必要性。我们将使用一种综合的方法，结合细胞生物学，结构生物学和生物化学来研究PI 4KIIIa复合物在分子，细胞和生物体水平上的功能和调节。在目的1中，为了获得关于PI 4KIIIa辅助因子如何将激酶靶向PM并在那里调节其活性的机制见解，我们将研究PI 4KIIIa复合物的分子结构。我们将获得复杂成分或其晶体结构，然后利用生物化学和电子显微镜（EM）信息将整个复合物拼凑在一起。已经从初步工作中获得的EFR 3、TTC 7和TTC 7/FAM 126亚组装体的结构，以及生物化学和基于细胞的实验，定义了PI 4KIIIa募集到PM的分子机制。他们进一步提出了一个机制，我们将调查，沿着的作用，辅助因子在调节PI 4 KIIIa的催化活性的调节复杂的装配。目标2与生物化学是互补的 以及对这些蛋白质在细胞提取物和活细胞（小鼠和人衍生细胞）中的成像研究，以确定复合物及其亚基在哺乳动物生物体中的天然环境中的动力学。在这一目标中，我们将测试我们的假设，这些亚基的耗尽导致质膜PI 4P合成的缺陷，并且，进一步，这种缺陷是人类疾病发病机制的基础。

 DESCRIPTION (provided by applicant): Phosphorylated metabolites of phosphatidylinositol, called phosphoinositides, play major constitutive and regulatory roles in a wide variety of physiological processes. Their generation and interconversions, via phosphorylation and dephosphorylation of the 3, 4 and 5 position of the inositol ring, are mediated by a large number of kinases and phosphatases. An especially important enzyme for this metabolism is phosphatidylinositol 4-kinase type IIIa (PI4KIIIa), as this protein is responsible for the generatin of the bulk of phosphatidylinositol-4-phosphate (PI4P) at the plasma membrane. This PI4P pool is a key determinant of plasma membrane identity and the precursor of the majority of cellular PI (4, 5) P2 and thus also its downstream metabolites, such as PI (3, 4, 5) P3, DAG and IP3. Thus, the function of PI4KIIIa impacts numerous fundamental processes, including exo-endocytosis, actin dynamics, transport of ions and other substances across the plasma membrane, signal transduction and regulation of the cell cycle. PI4KIIIa is part of a protein complex conserved from yeast to humans. The kinase PI4KIIIa itself (expressed by a single gene in all species) is essential for cell life, whereas defects in accessory factors (EFR3, TTC7 and FAM126, all three encoded by a pair of genes in mammals) have been implicated in human diseases. The fundamental importance of the PI4KIIIa complex for cellular physiology together with its emerging connections to pathological conditions argues forcefully for the need to understand its structure and function. We will use a comprehensive approach that combines cell biology, structural biology and biochemistry to investigate the function and regulation of the PI4KIIIa complex at molecular, cellular, and organismal levels. In Aim 1, to obtain mechanistic insights as to how the PI4KIIIa accessory factors target the kinase to the PM and regulate its activity there, we will investigate the molecular architecture of the PI4KIIIa complex. We will obtain crystal structures for complex components or their subassemblies and then piece together the entire complex from these, making use of biochemical and electron microscopy (EM) information. Structures for EFR3, TTC7 and a TTC7/FAM126 subassembly already available from preliminary work, together with biochemical and cell-based experiments, define the molecular mechanism for PI4KIIIa recruitment to the PM. They further suggest a mechanism for regulating complex assembly that we will investigate, along with the roles of the accessory factors in modulating the catalytic activity of PI4KIIIa. Aim 2 is complementary, with biochemistry and imaging studies of these proteins in cell extracts and living cells (mouse and human derived cells) to determine the dynamics of the complex and of its subunits in their natural context in mammalian organisms. In this aim, we will test our hypotheses that depletion of these subunits causes a defect in plasma membrane PI4P synthesis, and, further, that this defect underlies disease pathogenesis in humans.