Molecular Studies of the Roles and Regulations of the v-ATPase V0 Sector

描述（由申请人提供）：已显示V0扇区亚基“a”在独立于V-ATP酶质子泵送活性的多种膜融合或分泌功能中发挥重要作用。这些作用包括空泡融合、神经元的突触囊泡胞吐、外泌体和形态发生素的顶端分泌中的胞吐、胰岛的胰岛素分泌的胞吐以及小胶质细胞介导的神经元降解中的吞噬体融合。所有这些分泌/膜融合事件的分子机制和调节在很大程度上是未知的。神经元亚基a1（果蝇中的V100）是SNARE介导的突触囊泡胞吐作用所必需的。我们通过生化和结构分析发现，V100是Ca ~（2+）·钙调素（Ca ~（2+）·CaM）作用的靶点。因此，我们在果蝇中进行了体内研究，证明了这种相互作用的生理意义。钙调素结合缺陷V100不能招募钙调素突触，是无法挽救神经元功能在幼虫阶段，但不影响V100蛋白的稳定性，突触定位或神经元发育。为了阐明V100在SNARE介导的膜融合中的钙调素依赖性功能的分子机制，我们表明V100可以取代三元v-/t-SNARE“核心”复合物中的v-SNARE小突触蛋白，并形成非常相似的V100/t-SNARE复合物。CaM和t-SNARE与V100的N-末端胞质区域上的不同位点紧密结合。这些发现对导致膜融合的分子事件序列及其Ca 2+依赖性调节具有重要影响。此外，果蝇神经元中VO亚基“d”（V39）的功能丧失表现出与v-SNARE小突触蛋白或V100丧失难以区分的突触传递缺陷。综上所述，这些研究结果作为总体目标的框架，以研究在分子和生理水平上的膜融合的V-ATPase V0亚基a和d的作用，并激发以下三个具体目标：目的1：确定的结构-功能关系的神经元V0亚基a1和d。目的2：研究钙调素与神经元α 1亚基及非神经元亚基同源物的结合特异性。假设Ca ~（2+）·CaM在神经元中具有特殊的调节功能。目的3：在体内研究V0亚基a1和d在果蝇突触中的CaM和SNARE依赖性功能。当这些研究完成后，我们将阐明这两个亚基在V0功能的囊泡胞吐作用的作用。特别是，我们将建立亚基a1与钙调蛋白和SNARE蛋白相互作用的分子基础。通过证明非神经元亚基a同源物也是CaM的靶点，我们将揭示CaM对其他V0亚基a融合/分泌功能的额外调节作用。总之，本提案中包含的结构、生物化学和体内研究提供了对逾期整合分子模型的严格测试，该模型将v-ATP酶V0和Ca 2 + Ca功能与SNARE介导的膜融合相协调。公共卫生相关性：突触囊泡胞吐作用是神经细胞的一个基本的重要细胞过程;它的破坏或改变可以直接影响大脑的生理活动，引起人类疾病，如神经退行性阿尔茨海默病和帕金森病，以及癫痫、自闭症、精神分裂症等。本申请中提出的研究将在生理和分子水平上研究，胞吐作用中v-ATP酶V0区的蛋白组分，特别是亚基“a”和“d”的作用和调节。与这些组分缺陷相关的其他疾病包括由于亚基a突变引起的1型糖尿病和由于亚基d突变引起的骨质疏松症。

DESCRIPTION (provided by applicant): The V0 sector subunit "a" has been shown to play important roles in diverse membrane fusion or secretion functions independent of the V-ATPase proton-pumping activity. These roles include vacuolar fusion, synaptic vesicle exocytosis of neurons, exocytosis in apical secretion of exosomes and morphogens, exocytosis of insulin secretion from pancreatic islets and phagosomal fusion in microglial-mediated neuronal degradation. The molecular mechanism and regulation of all these secretion/membrane fusion events are largely unknown. The neuronal subunit a1 (V100 in fly) is required for SNARE-mediated synaptic vesicle exocytosis. We discovered, through biochemical and structural analysis, that V100 is a target of Ca2+.calmodulin (Ca2+.CaM). Consequently, we performed in vivo studies in Drosophila that demonstrate the physiological significance of this interaction. CaM-binding deficient V100 fails to recruit CaM to synapses and is unable to rescue neuronal function during larval stages, but does not affect V100 protein stability, synaptic localization or neuronal development. To unravel the molecular mechanism of the CaM-dependent function of V100 in SNARE- mediated membrane fusion, we show that V100 can replace the v-SNARE synaptobrevin in the ternary v-/t- SNARE 'core' complex and form a remarkably similar V100/t-SNARE complex. Ca2+.CaM and t-SNAREs bind tightly to distinct sites on the N-terminal cytosolic region of V100. These findings have important ramifications for the sequence of molecular events leading to membrane fusion as well as its Ca2+-dependent regulation. In addition, loss of function of the V0 subunit "d" (V39) in fly neurons exhibits synaptic transmission defects indistinguishable from the loss of v-SNARE synaptobrevin or V100. Taken together, these findings serve as the framework of the overall goal to investigate the roles of the V-ATPase V0 subunits a and d in membrane fusion at the molecular and physiological levels and motivate the following three specific aims: Aim 1: To determine the structure-function relationships of the neuronal V0 subunits a1 and d. Aim 2: To investigate the binding specificity of CaM to neuronal subunit a1 and non-neuronal subunit homologues. The hypothesis is that Ca2+.CaM has a specialized regulatory function in neurons. Aim 3: To characterize in vivo the CaM- and SNARE-dependent functions of V0 subunit a1 and d at Drosophila synapses. When these studies are completed, we will have elucidated the roles of both subunits in V0 function in vesicle exocytosis. In particular, we will have established the molecular basis of the interactions of subunit a1 with calmodulin and SNARE proteins. By demonstrating that non-neuronal subunit a homologues are also targets of CaM, we will have uncovered additional regulatory role of CaM of other V0 subunit a fusion/secretion functions. Together, the structural, biochemical and in vivo studies encompassed in this proposal provide a rigorous test of an overdue integrative molecular model that reconciles v-ATPase V0 and Ca2+ Ca function with SNARE-mediated membrane fusion. PUBLIC HEALTH RELEVANCE: Synaptic vesicle exocytosis is a fundamentally important cellular process for nerve cells; its disruption or alteration can directly affect physiological activity of the brain, causing human disorders, such as neurodegenerative Alzheimer's and Parkinson diseases, as well as epilepsy, autism, schizophrenia, etc. The research proposed in this application will investigate, at the physiological and molecular level, the roles and regulations of the protein components, specifically subunits "a" and "d", of the V0 sector of the v-ATPase in exocytosis. Other disorders associated with defects in these components include type 1 diabetes mellitus due to mutations of subunit a and osteoporosis owing to mutations of subunit d.