Inositol lipid regulation of membrane fusion and fission

描述（由申请人提供）：这是通知号（NOT-OD-09-058）的GM50403的竞争性修订，通知标题：NIH宣布竞争性修订申请的恢复法案资金的可用性。密歇根州受到经济危机的打击尤其严重。此外，去年辉瑞关闭了其最大的研究园区；位于安娜堡。这导致2000多名高技能研究人员被解雇，其中许多人仍然失业。本申请寻求雇用其中三名调查员，并扩大我们对父母补助金的研究。本基金的总体目标是研究磷脂酰肌醇(3,5)-二磷酸（pi3,5p2），这是一种丰度非常低且相对未被研究的信号脂质。PI3,5P2存在于从酵母到人类的所有真核生物中。我们发现PI3,5P2的缺失导致小鼠围产期死亡和大量神经变性。此外，在2%的ALS患者中发现PI3、5P2信号通路存在轻微缺陷。父母资助的重点是研究酵母中的PI3,5P2，希望这些研究能够揭示PI3,5P2在人类中的功能。亲本资助的目的是1)确定酵母中PI3,5P2的水平是如何调节的，2)确定酵母中PI3,5P2的下游效应蛋白。作为Aim 2的一部分，我们发现了270多个PI3,5P2候选结合蛋白。我们目前正在设计最佳方法来确定哪些候选物是PI3,5P2的真正下游效应物。目前的候选基因代表了大量的内体相关事件，包括参与多个膜运输步骤的蛋白质，以及空泡atp酶的亚基，其他转运蛋白和大量未表征的蛋白质。我们的目标是确定哪些通路需要PI3,5P2才能发挥作用。在这里，我们寻求扩大这些研究，并跟进一系列新的和意想不到的候选人。我们发现一种主要的细胞调节剂TOR激酶和TORC1调节剂Kog1/Raptor特异性地与pi3,5p2结合。重要的是，Alan Saltiel博士及其同事（密歇根大学）有独立的证据表明哺乳动物猛禽结合PI3,5P2；PI3,5P2是脂肪细胞中Tor1激活所必需的。因此，我们建议扩大我们目前的研究，并实现以下三个目标。1)发现新的TORC1下游靶点，2)确定PI3、5P2是否在Tor1功能的上游和/或下游，3)确定Tor1和Kog1上每个结合PI3、5P2的位点，并测试Gtr1/Gtr2 （RagA-D）是否直接结合PI3、5P2。

DESCRIPTION (provided by applicant): This is a Competitive Revision for GM50403 of Notice Number (NOT-OD-09-058), and Notice Title: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications. The state of Michigan has been particularly hard hit by the economic crisis. Moreover, last year Pfizer closed its largest research campus; located here in Ann Arbor. This resulted in the termination of over 2000 highly skilled researchers, many of whom remain unemployed. This application seeks to employ three of these investigators, and also to expand our studies of the parent grant. The overall goal of the parent grant is to study phosphatidylinositol (3,5)-bisphosphate (PI3,5P2), a very low abundance, and relatively unstudied signaling lipid. PI3,5P2 is found in all eukaryotes, from yeast to humans. We found that depletion of PI3,5P2 leads to perinatal lethality and massive neurodegeneration in mice. Moreover, minor defects in the PI3,5P2 signaling pathway are found in 2% of ALS patients. The focus of the parent grant is to study PI3,5P2 in yeast, in the hopes that these studies will reveal insights into PI3,5P2 function in humans as well. The Aims of the parent grant are 1) determine how levels of PI3,5P2 are regulated in yeast, and 2) determine proteins that are downstream effectors of PI3,5P2 in yeast. As part of Aim 2, we uncovered over 270 PI3,5P2 candidate binding proteins. We are currently devising the best approaches to determine which candidates are bona fide downstream effectors of PI3,5P2. The current candidates represent a large number of endosomal related events including proteins involved in multiple membrane trafficking steps, and also subunits of the vacuolar ATPase, other transporters and a significant number of uncharacterized proteins. Our goal is to define which pathways require PI3,5P2 for their function(s). Here we seek to expand these studies and follow up on a set of new and unexpected candidates. We found that a major cell regulator, the TOR kinase, as well as the TORC1 regulator, Kog1/Raptor, bind specifically and avidly to PI3,5P2. Importantly, Dr. Alan Saltiel and co-workers (U. Michigan) have independent evidence that mammalian Raptor binds PI3,5P2; and that PI3,5P2 is required for the activation of Tor1 in adipocytes. Thus, we propose to expand our current studies and to perform the following three aims. 1) Identify novel downstream targets of TORC1, 2) Determine whether PI3,5P2 is upstream and/or downstream of Tor1 function, and 3) Determine each site on Tor1 and Kog1 that bind PI3,5P2, and also test whether Gtr1/Gtr2 (RagA-D) directly bind PI3,5P2. PUBLIC HEALTH RELEVANCE: The overall goals of this application, are to 1) create employment opportunities for skilled scientists, and 2) expand the goals of the parent grant. We recently made the unexpected finding that TORC1, a key cellular regulator of critical medical importance, binds specifically and avidly to the signaling lipid PI3,5P2. We will exploit these findings to pursue new avenues to determine how TORC1 is regulated, and to uncover novel downstream TORC1 targets.