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: 9257436
• 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/9257436
• 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; Complement; Complex; Couples; Coupling; Cytoplasm; Cytoplasmic Tail; Cytosol; Development; Disease; Dissection; Endoplasmic Reticulum; Engineering; Exhibits; Fluorescence; Genetic; 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; Mammalian Cell; Mediating; Membrane; Membrane Proteins; Methods; Mission; Modeling; Molecular; Molecular Conformation; Morphology; Neoplasm Metastasis; Pathogenesis; Pathway interactions; Patients; Pharmacology; 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; Stimulus; Structure; T-Cell Activation; Tail; Techniques; Testing; Therapeutic; Tissues; Transmembrane Domain; Tumor Cell Migration; United States National Institutes of Health; base; cell type; congenital immunodeficiency; disability; disorder prevention; effective therapy; genome editing; human disease; immune activation; in vivo; innovation; insight; loss of function mutation; mouse model; nanodisk; neglect; new therapeutic target; novel; novel therapeutics; optogenetics; protein complex; public health relevance; reconstitution; release of sequestered calcium ion into cytoplasm; response; screening; sensor; targeted treatment; 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）：商店经营的钙进入（SOCE）构成免疫系统细胞中的主要钙涌入途径。 CA2+流入失调的非原发性免疫缺陷，心血管重塑和肿瘤转移。 SOCE发生时，STIM1是内质网中的钙传感器（ER），感觉耗尽了ER钙储存；作为响应，活化的stim1向ER型质膜膜（PM）迁移，在该连接处，它在其中募集和大门pm钙通道ORAI（Orai1，Orai2和Orai3）。动态刺激耦合代表了通道激活的全新范式，目前正以治疗免疫炎症性疾病（例如斑块牛皮癣）。我们了解这一重要生理过程的进展的关键障碍包括：（i）商店耗尽信号是如何从 ER管腔进入细胞质； （ii）STIM1如何与Orai1和Orai3差异化，这是对药理学刺激并引起不同信号表型的两个主要ORAI蛋白； （iii）如何由迄今未表征的调节剂产生专用于钙流入的ER-PM连接。该提案的总体目标是应对这些未满足的挑战。在AIM 1中，我们将使用生化，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的不可替代作用。我们的初步研究表明，经常被忽略的单个跨膜结构域可以作为跨ER膜中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AIM 2中，我们将在原子分辨率下提供STIM1-ORAI1和Stim1-Orai3耦合之间的第一个结构比较。将设计一个模型系统，用于在膜间界面进行定量解剖和一种新的工程“光遗传学”工具，用于非侵入性控制点的形成和钙通量，并用于帮助结构功能研究，并获得有关结构功能研究的研究，并获得有关stoichiomement和contoichiometric和调节性信息im1-orai耦合。 ER-PM连接的新兴意义最近受到了很高的关注。然而，缺乏适当的工具和方法会极大地阻碍这种专业的细胞室的机械解剖。在AIM 3中，我们将通过采用两管齐下的方法来克服这一障碍：（i）完整的ER-PM连接的蛋白质组学映射，这是通过空间限制的原位蛋白质标记而实现的，并且（ii）基于双分子荧光进行筛选互补。我们使用此策略的试点研究已经在Puncta公布了以前未被认可的STIM1结合伴侣蛋白。我们将进一步扩展这一点，以确定其他新型调节剂，并通过基因组编辑产生相应的细胞系，该基因组编辑将用于定义这些调节剂在调节SOCE，PUNCTA形成，ER形态和T细胞激活中的作用。综上所述，我们期望通过我们的研究获得的新机械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。进一步的好处将带来其他涉及钙信号传导和膜间通信的研究领域。