Pathophysiology of Plasma Membrane PI4P Generation

 描述(申请人提供)：磷脂酰肌醇的磷酸化代谢物，称为磷脂酰肌醇，在广泛的生理过程中起主要的构成和调节作用。它们的产生和相互转化是通过肌醇环3、4和5位的磷酸化和去磷酸化，由大量的激酶和磷酸酶介导的。这种代谢的一个特别重要的酶是磷脂酰肌醇4-激酶IIIa(PI4KIIIa)，因为这种蛋白负责在质膜上产生大量磷脂酰肌醇-4-磷酸(PI4P)。这个PI4P池是质膜特性的关键决定因素，也是大多数细胞PI(4，5)P2的前体，因此也是其下游代谢产物，如PI(3，4，5)P3、DAG和IP3的前体。因此，PI4KIIIa的功能影响了许多基本过程，包括胞外内吞作用、肌动蛋白动力学、离子和其他物质在质膜上的运输、信号转导和细胞周期的调节。PI4KIIIa是从酵母到人类保守的蛋白质复合体的一部分。激酶PI4KIIIa本身(在所有物种中由单个基因表达)对细胞生命是必不可少的，而辅助因子(哺乳动物中的EFR3、TTC7和FAM126，这三个基因都由一对基因编码)的缺陷与人类疾病有关。PI4KIIIa复合体对于细胞生理学的基本重要性，以及它与病理条件的新出现的联系，有力地证明了理解其结构和功能的必要性。我们将使用一种结合细胞生物学、结构生物学和生物化学的综合方法在分子、细胞和生物水平上研究PI4KIIIa复合体的功能和调节。在目标1中，为了获得关于PI4KIIIa辅助因子如何将激酶靶向PM并调节其活性的机械性见解，我们将研究PI4KIIIa复合体的分子结构。我们将获得复杂组件或其子组件的晶体结构，然后利用生化和电子显微镜(EM)信息将整个复合体拼凑在一起。EFR3、TTC7和TTC7/FAM126亚组分的结构已经从初步工作中获得，结合生化和基于细胞的实验，确定了PI4KIIIa重新聚集到PM的分子机制他们进一步提出了一种调节复杂组装的机制，我们将研究这一机制，以及辅助因子在调节PI4KIIIa催化活性中的作用。目标2是互补的，与生物化学 并对细胞提取物和活细胞(小鼠和人类来源的细胞)中的这些蛋白质进行成像研究，以确定哺乳动物生物体中该复合体及其亚单位在其自然环境中的动力学。在这个目标中，我们将检验我们的假设，即这些亚基的耗尽会导致质膜PI4P合成的缺陷，并进一步证明这种缺陷是人类疾病发病的基础。

 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.