Directing Membrane Function with Inositol Lipids in Health and Disease

项目摘要/摘要 质膜(PM)是维持生命的运输、信号和结构功能的繁忙枢纽 在细胞水平上。未能恰当地编排这些平行功能是导致发病的原因之一 很多疾病的影响。PM功能的选择性调节依赖于胞浆小叶磷脂酰肌醇(PPIN)脂类， 磷脂酰肌醇4，5-二磷酸[PI(4，5)P2]。PI(4，5)P2是主调节器，招募和/或激活 控制质膜通透性、囊泡运输、细胞骨架组装和受体驱动的数十种蛋白质 发信号。已知从癌症到罕见的单基因疾病等人类疾病与异常有关。 PI(4，5)P2水平，这是由于PI(4，5)P2合成或分解中断所致。这就是说，绝对数量的 PI(4，5)P2调节的功能使得识别哪些功能与疾病有关具有挑战性 更不用说尝试治疗干预了。因此，该实验室研究的长期目标是 对PPIN代谢如何与个体生理耦合形成详细的、机械的理解 过程；最终，我们的目标是使用这些知识来开发调整或恢复PI(4，5)P2的策略 对疾病中异常的个体功能的调节。我们在此应用程序中的目标是定义三个 PM PI(4，5)P2或其信号产物代谢与多个 生理过程。在第一个项目中，我们利用最近发现的PM PI(4，5)P2动态平衡 实验调节细胞中脂质水平的机制：这将使我们第一次能够确定 PI(4，5)P2对关键PM函数的浓度依赖性。这将揭示哪些功能受影响最大 致病性PI(4，5)P2水平，从而可能驱动疾病表型--这是设计治疗方法的关键第一步 干预措施。在第二个项目中，我们将确定脂转移蛋白(LTPS)，它与PPIN从 膜对它们被位于其他细胞器中的PPIN磷酸酶降解的影响。我们会雇佣一名合成材料 LTPS耗尽活细胞中异位线粒体PPIN的生物学方法。识别耦合到的LTP 不同的PPIN池将揭示防止单基因疾病PPIN积聚的新靶点 磷酸酶活性丧失。在第三个项目中，我们将定义由以下驱动的独特的PI3K信号场景 PI(4，5)P2的两个下游脂类产物：PIP3和PI(3，4)P2。我们将定义唯一的时空和 活细胞中这些脂类的效应蛋白激活谱，使用我们独特的分子工具精确检测 并在细胞培养中对脂质进行化学遗传学操作。至关重要的是，这将确定时空和效应者 每种脂类独一无二的蛋白质激活特性。这一知识对于消除目标上的不利影响至关重要 经过验证的PI3K疗法的效果。总体而言，在本供资周期结束时，我们预计已确定 PM PI(4，5)P2信号与个体PM耦合产生特异性的基本原理 单元格中的函数。这将是意义重大的，因为它将为实验和 最终在PI(4，5)P2调节点上对这些孤立的过程进行治疗干预。

PROJECT SUMMARY/ABSTRACT The plasma membrane (PM) is a bustling hub of transport, signaling and structural functions that sustain life at the cellular level. Failure to appropriately choreograph these parallel functions contributes to the pathogenesis of many diseases. Selective regulation of PM function relies on the cytosolic leaflet phosphoinositide (PPIn) lipid, phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. PI(4,5)P2 is a master regulator, recruiting and/or activating scores of proteins controlling PM permeability, vesicular traffic, cytoskeletal assembly and receptor-driven signaling. Human diseases from cancer to rare monogenic disorders are known to be associated with aberrant PI(4,5)P2 levels, which result from disrupted PI(4,5)P2 synthesis or breakdown. That said, the sheer number of PI(4,5)P2-regulated functions has made it challenging to identify which functions contribute to disease phenotype, let alone attempt therapeutic intervention. Therefore, the long-term goal of the lab's research is to develop a detailed, mechanistic understanding of how PPIn metabolism couples to individual physiological processes; ultimately, we aim to use this knowledge to develop strategies that modulate or restore PI(4,5)P2 regulation of individual functions that are aberrant in disease. Our goal in this application is to define three distinct mechanisms by which metabolism of PM PI(4,5)P2 or its signaling products couple to multiple physiological processes. In the first project, we leverage our recent discovery of a PM PI(4,5)P2 homeostatic mechanism to experimentally tune the lipid level in cells: this will allow us for the first time to determine the PI(4,5)P2 concentration-dependence of key PM functions. This will reveal which functions are most affected by pathogenic PI(4,5)P2 levels and thus likely drive disease phenotypes – a crucial first step in devising therapeutic interventions. In the second project, we will identify lipid transfer proteins (LTPs) that couple PPIn removal from membranes to their degradation by PPIn phosphatases located in other organelles. We will employ a synthetic biology approach in which LTPs deplete ectopic mitochondrial PPIn in live cells. Identifying LTPs coupled to different PPIn pools will reveal new targets to prevent PPIn accumulation in monogenic disease where phosphatase activity is lost. In the third project, we will define the distinct PI3K signaling landscapes driven by the two downstream lipid products of PI(4,5)P2: PIP3 and PI(3,4)P2. We will define the unique spatiotemporal and effector protein activation profiles of these lipids in live cells, using our unique molecular tools to precisely detect and chemogenetically manipulate the lipids in cell culture. Crucially, this will identify spatiotemporal and effector protein activation profiles that are unique to each lipid. This knowledge will be vital to eliminate on-target adverse effects of proven PI3K therapeutics. Collectively, at the conclusion of this funding cycle we expect to have defined fundamental principles by which specificity emerges from the coupling of PM PI(4,5)P2 signaling to individual PM functions in cells. This will be significant, because it will provide the conceptual framework for experimental and eventually therapeutic intervention in these isolated processes at the point of PI(4,5)P2 regulation.