Inositol lipid regulation of membrane fusion and fission

描述（由申请人提供）：这是GM 50403的竞争性修订，通知编号（NOT-OD-09-058），通知标题：NIH宣布恢复法案资金可用于竞争性修订申请。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;位于这里的安阿伯。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。本申请旨在雇用其中三名调查员，并扩大我们对父母补助金的研究。总的目标是研究磷脂酰肌醇（3，5）-二磷酸（PI 3，5 P2），一个非常低的丰度，相对未研究的信号脂质。PI 3，5 P2存在于从酵母到人类的所有真核生物中。我们发现PI 3，5 P2的缺失导致小鼠围产期死亡和大量神经退行性变。此外，在2%的ALS患者中发现PI 3，5 P2信号通路的轻微缺陷。该基金的重点是研究酵母中的PI 3，5 P2，希望这些研究也能揭示PI 3，5 P2在人类中的功能。该研究的目的是：1）确定酵母中PI 3，5 P2的水平是如何调节的，2）确定酵母中PI 3，5 P2的下游效应蛋白。作为目标2的一部分，我们发现了超过270种PI 3，5 P2候选结合蛋白。我们目前正在设计最好的方法来确定哪些候选人是真正的PI 3，5 P2下游效应子。目前的候选者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，以及液泡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途径需要PI 3，5 P2的功能。在这里，我们试图扩大这些研究，并跟进一组新的和意想不到的候选人。我们发现，一个主要的细胞调节因子TOR激酶，以及TORC 1调节因子Kog 1/Raptor，特异性地和贪婪地结合到PI 3，5 P2。重要的是，艾伦·萨尔蒂尔博士和他的同事（美国）。Michigan）有独立的证据表明哺乳动物Raptor与PI 3，5 P2结合;并且PI 3，5 P2是脂肪细胞中Tor 1激活所必需的。因此，我们建议扩大我们目前的研究，并执行以下三个目标。1)鉴定TORC 1的新下游靶标，2）确定PI 3，5 P2是否是Tor 1功能的上游和/或下游，和3）确定Tor 1和Kog 1上结合PI 3，5 P2的每个位点，并且还测试Gtr 1/Gtr 2（RagA-D）是否直接结合PI 3，5 P2。 公共卫生相关性：这项申请的总体目标是：1）为熟练的科学家创造就业机会，2）扩大父母补助金的目标。我们最近取得了意想不到的发现，TORC 1是一种具有重要医学意义的关键细胞调节因子，它特异性地与信号脂质PI 3，5 P2结合。我们将利用这些发现来寻求新的途径来确定TORC 1是如何调节的，并发现新的下游TORC 1靶点。

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.