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

描述（由申请方提供）：钙池操作的钙内流（SOCE）构成免疫系统细胞中的主要钙内流途径。Ca 2+内流失调与原发性免疫缺陷、心血管重塑和肿瘤转移密切相关。当STIM 1（内质网（ER）中的钙传感器）感知到ER钙储存的耗尽时，发生SOCE;作为响应，激活的STIM 1向ER-质膜（PM）连接处迁移，在那里它募集并门控PM钙通道奥赖（ORAI 1、ORAI 2和ORAI 3）。动态STIM-ORAI偶联代表了通道激活的全新范例，并且目前被靶向用于治疗免疫炎性疾病（例如，斑块状银屑病）。在我们理解这一重要生理过程的过程中，关键的障碍包括：（i）储存耗尽信号是如何从细胞中传递的。 内质网腔至胞浆;（ii）STIM 1如何差异性地偶联到ORAI 1和ORAI 3，这两种主要的奥赖蛋白对药理学刺激有不同的反应并引起不同的信号传导表型;和（iii）迄今为止未表征的调节剂如何产生专用于钙内流的ER-PM连接。本提案的总体目标是应对这些尚未应对的挑战。在目标1中，我们将使用生物化学，蛋白质工程和化学生物学方法来建立STIM 1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，经常被忽视的单跨膜结构域可能作为关键的决定因素，在中继信号通过ER膜，并有助于在细胞质侧的STIM 1的构象转换。在目标2中，我们将首次以原子分辨率对STIM 1-ORAI 1和STIM 1-ORAI 3耦合进行结构比较。将设计一种用于在膜间界面定量解剖SOCE的模型系统和一种用于无创控制斑点形成和钙通量的新工程“光遗传学”工具，并用于辅助结构-功能研究和获得STIM 1-奥赖偶联的化学计量和调节信息。ER-PM结的新兴意义最近受到高度关注。然而，由于缺乏适当的工具和方法，这种专门的细胞区室的机械解剖受到很大阻碍。在目标3中，我们将通过双管齐下的方法克服这一障碍：（i）完整的ER-PM连接的蛋白质组学图谱，这是通过空间限制的原位蛋白质标记，以及（ii）基于双分子荧光互补的筛选。我们使用这种策略的试点研究已经揭示了以前未被识别的STIM 1结合伴侣蛋白在斑点。我们将进一步扩展这一点，以确定其他新的调节剂，并通过基因组编辑产生相应的细胞系，这将用于定义这些调节剂在调节SOCE，斑点形成，ER形态和T细胞活化中的作用。总之，我们期望通过我们的研究获得的新的机制和结构见解将导致有效治疗自身免疫性疾病和预防移植排斥反应的进展。其他涉及钙信号传导和膜间通讯的研究领域也将从中获益。

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.