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

内质网-质膜连接处钙内流的分子控制

• 批准号: 8765824
• 负责人: Yubin Zhou
• 金额: $ 27.65万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8765824
• 关键词: Address; Advanced Development; Area; Attention; Autoimmune Diseases; Autoimmune Process; Binding; Biochemical; Biological Models; Biology; C-terminal; Calcium; Calcium Channel; Calcium Signaling; Cardiovascular Diseases; Cardiovascular system; Cell Line; Cell membrane; Cells; Chemicals; Chronic small plaque psoriasis; Clinical; Coiled-Coil Domain; Communication; Complex; Couples; Coupling; Cytoplasm; Cytoplasmic Tail; Cytosol; Development; Disease; Dissection; Endoplasmic Reticulum; Engineering; Exhibits; Figs - dietary; Fluorescence; Genetic; Genome; Genome engineering; Goals; Graft Rejection; Heart Hypertrophy; Human; Hypersensitivity; Immune response; Immune system; Immunologic Deficiency Syndromes; In Situ; Inflammation; Inflammatory; Integral Membrane Protein; Knowledge; Label; Lead; Mammalian Cell; Maps; Mediating; Membrane; Membrane Proteins; Methods; Mission; Modeling; Molecular; Morphology; Neoplasm Metastasis; Pathogenesis; Pathway interactions; Patients; Phenotype; Physiological Processes; Pilot Projects; Positioning Attribute; Process; Protein Engineering; Protein Isoforms; Proteins; Proteomics; Public Health; Recruitment Activity; Research; Resolution; Role; Route; STIM1 gene; Severe Combined Immunodeficiency; Side; Signal Transduction; Site; Solid; Solutions; Stimulus; Structure; T-Cell Activation; Tail; Techniques; Testing; Therapeutic; Tissues; Transmembrane Domain; base; cell type; congenital immunodeficiency; disability; disorder prevention; effective therapy; human disease; in vivo; innovation; insight; loss of function mutation; mouse model; nanodisk; neglect; novel; optogenetics; protein complex; public health relevance; reconstitution; release of sequestered calcium ion into cytoplasm; response; screening; sensor; signal processing; tool; tumor; tumor growth

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.

描述（由申请人提供）：钙库操纵的钙内流（SOCE）构成免疫系统细胞中主要的钙流入途径。 Ca2+ 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 STIM1（内质网 (ER) 中的钙传感器）感知到 ER 钙储备耗尽时，就会发生 SOCE；作为响应，激活的 STIM1 迁移到 ER 质膜 (PM) 连接处，在那里募集并门控 PM 钙通道 ORAI（ORAI1、ORAI2 和 ORAI3）。动态 STIM-ORAI 耦合代表了通道激活的全新范例，目前正用于治疗免疫炎症性疾病（例如斑块型银屑病）。我们在理解这一重要的生理过程方面取得进展的关键障碍包括：（i）存储耗尽信号如何从 ER 腔通向细胞质； (ii) STIM1 如何与 ORAI1 和 ORAI3 差异耦合，这两种主要的 ORAI 蛋白对药理刺激有不同的反应并引起不同的信号表型； (iii) 迄今为止尚未表征的调节因子如何产生专门用于钙流入的 ER-PM 连接。该提案的总体目标是解决这些未解决的挑战。在目标1中，我们将利用生物化学、蛋白质工程和化学生物学方法来确定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 细胞质侧的构象转换。在目标 2 中，我们将在原子分辨率下首次对 STIM1-ORAI1 和 STIM1-ORAI3 耦合进行结构比较。将设计用于膜间界面处 SOCE 定量解剖的模型系统和用于无创控制泪点形成和钙通量的新型工程“光遗传学”工具，并用于辅助结构功能研究并获得化学计量和监管信息STIM1-ORAI 耦合。 ER-PM 连接的新兴重要性最近受到高度关注。然而，由于缺乏适当的工具和方法，对这种特殊细胞区室的机械解剖受到极大阻碍。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 ER-PM 连接的蛋白质组图谱，这可以通过空间限制的原位蛋白质标记来实现，以及（ii）基于双分子荧光的筛选互补。我们使用这种策略的初步研究已经揭示了 puncta 中以前未被识别的 STIM1 结合伴侣蛋白。我们将进一步扩展这一点，以识别更多的新型调节因子，并通过基因组编辑生成相应的细胞系，这将用于定义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。总而言之，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自身免疫性疾病的有效治疗和移植排斥的预防方面取得进展。涉及钙信号传导和膜间通讯的其他研究领域将获得进一步的好处。