Molecular control of calcium influx at the ER-plasma membrane junctions

描述(由申请人提供)：存储操作的钙进入(SOCE)构成了免疫系统细胞中主要的钙内流途径。钙离子内流失调与原发免疫缺陷、心血管重构和肿瘤转移密切相关。当内质网(ER)中的钙感受器STIM1感觉内质网(ER)钙储备耗尽时，就会发生SOCE；作为回应，激活的STIM1迁移到ER-质膜(PM)连接，在那里它招募并门控PM钙通道ORAI(ORAI1、ORAI2和ORAI3)。动态STIM-ORAI偶联代表了一种全新的通道激活范例，目前正被用于治疗免疫性炎症性疾病(例如斑块型牛皮癣)。在我们理解这一重要的生理过程的过程中，关键障碍包括：(I)储藏耗竭信号是如何从 (Ii)STIM1如何与ORAI1和ORAI3不同地偶联，这两种主要的ORAI蛋白对药物刺激的反应不同，并导致不同的信号表型；以及(Iii)专用于钙内流的ER-PM连接是如何由迄今尚未确定的调节因子产生的。这项提案的总体目标是解决这些未得到满足的挑战。在目标1中，我们将使用生化、蛋白质工程和化学生物学的方法来确定STIM1跨膜结构域在信号转导中不可替代的作用。我们的初步研究表明，经常被忽视的单个跨膜结构域可能是跨ER膜传递信号的关键决定因素，并有助于STIM1胞质侧的构象开关。在目标2中，我们将在原子分辨率下首次提供STIM1-ORAI1和STIM1-ORAI3耦合的结构比较。将设计一种定量分析膜间界面SOCE的模型系统和一种新的工程“光遗传学”工具，用于非侵入性地控制点状结构和钙离子通量，以辅助结构功能研究，并获得STIM1-ORAI偶联的化学计量学和调控信息。内质网-质膜连接的重要性最近受到高度重视。然而，由于缺乏适当的工具和方法，对这个专门的细胞隔室的机械解剖受到了极大的阻碍。在目标3中，我们将通过双管齐下的方法克服这一障碍：(I)通过空间受限的原位蛋白质标记对完整的ER-PM连接进行蛋白质组学定位，以及(Ii)基于双分子荧光互补的筛选。我们使用这一策略的初步研究已经在SPITTA上揭示了以前未识别的STIM1结合伙伴蛋白。我们将进一步扩展这一点，以确定其他新的调节因子，并通过基因组编辑产生相应的细胞系，这将被用于确定这些调节因子在调节SOCE、斑点形成、内质网形态和T细胞激活中的作用。综上所述，我们期待通过我们的研究获得的新的机制和结构方面的见解将导致在有效治疗自身免疫性疾病和预防移植排斥反应方面的进展。其他涉及钙信号转导和膜间通讯的研究领域也将进一步受益。

DESCRIPTION (provided by applicant): Store-operated calcium entry (SOCE) constitutes the primary calcium influx pathway in cells of the immune system. Dysregulated Ca2+ influx is intimately involved in primary immunodeficiency, cardiovascular remodeling, and tumor metastasis. SOCE occurs when STIM1, the calcium sensor in the endoplasmic reticulum (ER), senses depletion of ER calcium stores; in response, activated STIM1 migrates toward ER-plasma membrane (PM) junctions, where it recruits and gates the PM calcium channels ORAI (ORAI1, ORAI2 and ORAI3). Dynamic STIM-ORAI coupling represents a totally new paradigm for channel activation, and is currently being targeted for treatment of immuno- inflammatory diseases (e.g., plaque psoriasis). Critical barriers in our progress to understanding this important physiological process include: (i) how the store depletion signal is transmitted from the ER lumen to the cytoplasm; (ii) how STIM1 differentially couples to ORAI1 and ORAI3, the two major ORAI proteins that respond differently to pharmacological stimuli and cause distinct signaling phenotypes; and (iii) how ER-PM junctions dedicated to calcium influx are generated by hitherto uncharacterized regulators. The overall goal of this proposal is to tackle these unmet challenges. In Aim 1, we will use biochemical, protein engineering, and chemical biology approaches to establish the irreplaceable role of the STIM1 transmembrane domain in signal transduction. Our preliminary studies have suggested that the often-neglected single transmembrane domain may serve as the key determinant in relaying signals across the ER membrane and contribute to conformational switch in the cytoplasmic side of STIM1. In Aim 2, we will provide the first structural comparison between STIM1-ORAI1 and STIM1-ORAI3 coupling at atomic resolution. A model system for quantitative dissection of SOCE at the inter-membrane interface and a new engineered "optogenetic" tool for noninvasive control of puncta formation and calcium flux will be devised and used to aid structure-function studies and to gain stoichiometric and regulatory information on STIM1-ORAI coupling. The emerging significance of ER-PM junctions has recently received high attention. However, mechanistic dissection of this specialized cellular compartment is greatly hampered by the lack of appropriate tools and methods. In Aim 3, we will overcome this barrier by taking a two-pronged approach: (i) proteomic mapping of intact ER-PM junctions, which is made possible through spatially restricted in situ protein labeling, and (ii) screening based on bimolecular fluorescence complementation. Our pilot study using this strategy has already unveiled previously unrecognized STIM1 binding partner proteins at puncta. We will further expand this to identify additional novel regulators and generate corresponding cell lines through genome editing, which will be used to define the roles of those regulators in modulating SOCE, puncta formation, ER morphology, and T cell activation. Taken together, we expect that the novel mechanistic and structural insights gained through our study will lead to advances in effective treatment of autoimmune diseases and prevention of transplant rejection. Further benefit will accrue to other research areas that involve calcium signaling and intermembrane communication.