MOLECULAR CONTROL OF CALCIUM INFLUX AT THE ER-PLASMA MEMBRANE JUNCTIONS

项目概要/摘要。 钙库操纵性钙进入（SOCE）是哺乳动物体内钙进入的主要途径， 淋巴细胞活化、肌肉收缩、基因表达和细胞代谢。钙的释放- 由ORAI-STIM组成的激活钙（CRAC）通道代表了SOCE的典型实例， 淋巴细胞SOCE的临床相关性通过两种人类疾病来举例说明， 免疫缺陷（SCID）和管状聚集性肌病（TAM），其由功能丧失或获得引起 ORAI 1和STIM 1的突变。增强的SOCE也与心血管疾病有关 和癌症转移。因此，CRAC通道已被追求为治疗性的有吸引力的药物靶点。 干预已作出巨大努力，将ORAI-STIM作为最低限度的两个组成部分， 系统耦合ER钙库消耗与跨质膜钙流入。监管 然而，专用于ORAI-STIM信令的机器仍然没有完全定义。 在这项提案中，PI旨在通过揭示两种新颖的功能来弥合这一关键的知识差距。 SOCE调节剂，位于不同的亚细胞位置，作用于ORAI-STIM的不同步骤 信号传导：ER腔内STIM的初始激活和ORAI-STIM复合物的后期稳定 在ER-PM膜接触部位（MCS），其中两个膜的紧密贴壁被 间隙距离为10-30 nm。在目标1中，基于潜在STIM 1蛋白质组学分析的初步发现， 在ER腔内的相互作用，PI将定义如何以前未被识别的多EF-手蛋白 与STIM 1的管腔结构域（EFSAM）合作，以形成STIM 1的激活和失活动力学。 SOCE。PI将采用新的“ER至PM”运输策略，将管腔结构域暴露于 细胞外侧，从而克服了对ER-居民的阈侧研究的主要障碍 信号蛋白在目标2中，利用TMEM家族蛋白作为钙调节剂的发现， 在ER-PM MCS的流入中，PI将定义该调节剂如何响应生理刺激以重塑 ER-PM连接的组装和PIP稳态以维持SOCE。创新的光遗传学的产生 工具和转基因小鼠模型来剖析钙信号和蛋白质-PIP相互作用将进一步 加速我们对这些新型调节剂的结构-功能关系的研究。 总的来说，通过拟议的研究获得的新的机制见解将导致在 不断恢复活力的钙信号领域，并在平行，产卵膜接触的活力领域 网站.从长远来看，研究中的发现可以转化为有效的发展。 靶向异常钙和磷酸肌醇信号传导的治疗剂。

Project Summary / Abstract. Store-operated calcium entry (SOCE) constitutes a major calcium entry pathway in mammals to control lymphocyte activation, muscle contraction, gene expression and cell metabolism. The calcium release- activated calcium (CRAC) channel composed of ORAI-STIM represents a prototypical example of SOCE in lymphocytes. The clinical relevance of SOCE is exemplified by two human diseases, the severe combined immunodeficiency (SCID) and tubular aggregate myopathy (TAM), which are caused by loss- or gain-of-function mutations in ORAI1 and STIM1, respectively. Augmented SOCE is also implicated in cardiovascular disorders and cancer metastasis. Therefore, CRAC channel has been pursued as an attractive drug target for therapeutic intervention. Tremendous efforts have been directed to establish ORAI-STIM as the minimal two-component system to couple ER calcium store depletion with calcium influx across the plasma membrane. The regulatory machinery dedicated to the ORAI-STIM signaling, nonetheless, still remains incompletely defined. In this proposal, the PI aims to bridge this critical knowledge gap by unveiling the functions of two novel SOCE modulators, which reside at distinct subcellular locations to act on different steps of ORAI-STIM signaling: the initial activation of STIM within the ER lumen and the later stabilization of ORAI-STIM complexes at ER-PM membrane contact sites (MCS), where the close appositions of two membranes are separated by a gap distance of 10-30 nm. In Aim 1, based on preliminary findings from proteomic profiling of potential STIM1 interactors within the ER lumen, the PI will define how a previously-unrecognized multiple EF-hand protein cooperates with the luminal domain of STIM1 (EFSAM) to shape the activation and deactivation kinetics of SOCE. The PI will employ a new “ER-to-PM” trafficking strategy to expose the luminal domain toward the extracellular side, thereby overcoming a major impediment to studies on the liminal sides of ER-resident signaling proteins. In Aim 2, capitalizing on the discovery of a TMEM family protein as a regulator of calcium influx at ER-PM MCS, the PI will define how this modulator responds to physiological stimuli to remodel the assembly of ER-PM junctions and PIP homeostasis to sustain SOCE. The generation of innovative optogenetic tools and a transgenic mouse model to dissect calcium signaling and protein-PIP interactions will further accelerate our structure-function relationship studies on these novel regulators. Overall, the new mechanistic insights gained through the proposed study will lead to advances in the constantly-revitalized field of calcium signaling, and in parallel, spawn the vibrant field of membrane contact sites. In the long run, discoveries made in the study can be translated into the development of effective therapeutics targeting aberrant calcium and phosphoinositide signaling.