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

描述（由申请人提供）：V0扇区亚基“a”已被证明在多种膜融合或独立于v - atp酶质子泵活性的分泌功能中发挥重要作用。这些作用包括空泡融合、神经元突触囊泡胞吐、外泌体和形态原顶端分泌的胞吐、胰岛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控在很大程度上是未知的。神经元亚单位a1（果蝇中的V100）是sns介导的突触囊泡胞吐所必需的。通过生化和结构分析，我们发现V100是Ca2+.calmodulin （Ca2+. cam）的靶标。因此，我们在果蝇身上进行了体内研究，证明了这种相互作用的生理意义。CaM结合缺陷的V100不能将CaM招募到突触，在幼虫期不能挽救神经元功能，但不影响V100蛋白的稳定性、突触定位或神经元发育。为了揭示V100在SNARE介导的膜融合中cam依赖功能的分子机制，我们发现V100可以取代三元v-/t- SNARE“核心”复合体中的v-SNARE突触brevin，并形成非常相似的V100/t-SNARE复合体。Ca2 +。CaM和t-SNAREs紧密结合在V100细胞质n端不同的位点上。这些发现对导致膜融合及其Ca2+依赖性调节的分子事件序列具有重要的影响。此外，果蝇神经元中V0亚基“d”（V39）的功能缺失与v-SNARE突触短链蛋白或V100的缺失难以区分。综上所述，这些发现构成了研究v - atp酶V0亚基a和d在分子和生理水平上在膜融合中的作用的总体目标框架，并激发了以下三个具体目标：目的1：确定神经元V0亚基a1和d的结构-功能关系。目的2：研究CaM与神经元亚基a1和非神经元亚基同源物的结合特异性。假设是Ca2+。CaM在神经元中具有特殊的调节功能。目的3：在体内表征果蝇突触中V0亚基a1和d的CaM和snare依赖功能。当这些研究完成后，我们将阐明这两个亚基在V0功能中在囊泡胞吐中的作用。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的靶标，我们将发现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、生化和体内研究为一个迟来的整合分子模型提供了严格的测试，该模型将v- atp酶V0和Ca2+ Ca功能与snare介导的膜融合协调起来。公共卫生相关性：突触囊泡胞吐是神经细胞的一个基本重要的细胞过程；它的破坏或改变可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。本申请拟开展的研究将在生理和分子水平上研究v- atp酶V0区蛋白组分，特别是“a”和“d”亚基在胞吐作用中的作用和调控。其他与这些成分缺陷相关的疾病包括由亚基a突变引起的1型糖尿病和由亚基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.