Understanding Store-Operated Calcium Signal Transduction

了解存储操作的钙信号转导

• 批准号: 10372049
• 负责人: CHRISTOPHER M YENGO
• 金额: $ 42.99万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10372049
• 关键词: Address; Animals; Area; Autoimmunity; B-Lymphocytes; Binding; Biological Models; Calcium; Calcium Signaling; Cardiovascular Diseases; Cardiovascular system; Cell Nucleus; Cell physiology; Cells; Cytoplasm; Defect; Development; Disease; Dysplasia; Environment; Fluorescence Resonance Energy Transfer; Functional disorder; Gene Expression; Generations; Genes; Genetic Transcription; Growth; Image; Imaging technology; Immune; Immune System Diseases; Immunity; Knowledge; Link; Malignant Neoplasms; Measurement; Mediating; Metabolism; Modeling; Molecular; Molecular Probes; Monitor; Muscle hypotonia; Mutation; Neurodegenerative Disorders; New Agents; Phenotype; Physiological; Property; Protein Isoforms; Proteins; Pump; RNA Splicing; Resolution; Role; STIM1 gene; Severe Combined Immunodeficiency; Signal Transduction; Skin; Smooth Muscle Myocytes; System; Variant; Vascular Smooth Muscle; Work; airway remodeling; cell motility; crosslink; knockout animal; molecular subtypes; nanoscale; novel therapeutics; operation; optogenetics; prevent; sensor; therapeutic target; transcriptome

Project Summary The remarkable spatial and temporal precision of Ca2+ signals finely controls a vast array of cellular functions. Ca2+ signals reflect the function of highly coordinated Ca2+ sensors and Ca2+ channels. The interaction between Ca2+ sensor STIM proteins in the ER and Orai Ca2+ channels in the PM, is crucial in Ca2+ signal generation. The two proteins physically couple in ER-PM junctions, mediating “store-operated” Ca2+ signals, essential in controlling gene expression, secretion, motility and growth. Their role in Ca2+ signaling is critical, and defects in STIM or Orai proteins cause a spectrum of disorders including severe combined immunodeficiency, muscular hypotonia, autoimmunity, skin dysplasia, and exocrine defects. Dysregulation of STIM/Orai expression is closely linked to cardiovascular and airway remodeling, neurodegenerative disorders, altered immunity, and cancer. We have defined much on the molecular properties and cellular organization of STIM and Orai proteins. We now turn toward the nanoscale ER-PM junctional environment in which they operate. Junctional Ca2+ is critical in generating both “local” Ca2+ signals and in sustaining “global” Ca2+ oscillations that extend across the cytoplasm and penetrate the nucleus. Using a compendium of molecular probes, imaging technology, gene-deleted cellular systems, and animal knockout models, our work is directed toward three major areas: (1) Identifying critical differences in the disease-related STIM and Orai isoforms, focusing on operation of the widely expressed STIM2.1 splice variant of STIM2 that lacks the critical Orai-binding domain; using super-resolution STED microcopy and TIRF/FRET imaging we explore a model by which STIM2.1 disrupts the cross-linking of Orai channel subtypes within junctions, and determine how clustering organizes the junctional Ca2+ signaling machinery. (2) Defining the micro-physiological environment of Ca2+ entry junctions, utilizing genetically encoded and optogenetically applied Ca2+ probes tagged onto STIM and Orai proteins to monitor the highly restricted junctional Ca2+ micro-environment; using such measurements to explore how STIM-mediated clustering of Orai channels controls local junctional Ca2+ signals, modifies the proximity of InsP3Rs and SERCA pumps, and modulates the generation of global Ca2+ oscillations. (3) Understanding how store-operated Ca2+ signals are transcriptionally transduced, exploring how the “STIM2 phenotype” in smooth muscle- and B cell-targeted STIM1-deleted animals is related to STIM1/STIM2.1 expression levels and junctional Ca2+ signals, how STIM- modulated Orai clustering controls specific NFAT subtype activation, and determining transcriptome changes related to STIM type-specific Ca2+ signal generation. Our studies address the critical gap in our knowledge of how junctional Ca2+ tunes the generation of local and global Ca2+ signals. The central role of STIM/Orai-mediated store-operated Ca2+ signals in cell physiology, the widespread dysfunction caused by STIM/Orai mutations, the deep functional distinctions between STIM/Orai subtypes, and the extraordinary alteration of subtype expression in major disease states, all define the STIM-Orai interface as a pivotal therapeutic target.

项目摘要 Ca2+信号的显着空间和临时精度可很好地控制大量的细胞函数。 CA2+信号反映了高度协调的Ca2+传感器和Ca2+通道的功能。之间的相互作用 PM中的ER和ORAI Ca2+通道中的Ca2+传感器刺激蛋白对于Ca2+信号产生至关重要。这 在ER-PM连接中进行物理上的两种蛋白质，介导“商店操作” CA2+信号，必不可少的 控制基因表达，分泌，运动和生长。它们在Ca2+信号传导中的作用至关重要，缺陷在 刺激蛋白或ORAI蛋白会引起多种疾病，包括严重的联合免疫缺陷，肌肉 低调，自身免疫性，皮肤发育不良和外分泌缺陷。 Stim/Orai表达的失调紧密 与心血管和气道重塑，神经退行性疾病，免疫学改变和癌症有关。我们 在刺激和ORAI蛋白的分子特性和细胞组织上已经定义了很多定义。我们现在 转向其操作的纳米级ER-PM连接环境。连接CA2+至关重要 同时生成“局部” CA2+信号，并在维持跨细胞质的“全局” CA2+振荡中 并穿透细胞核。使用分子问题，成像技术，基因删除细胞的纲要 系统和动物淘汰模型，我们的工作针对三个主要领域：（1）确定关键 与疾病相关的刺激和ORAI同工型的差异，重点是广泛表达的操作 STIM2.1 STIM2的剪接变体缺乏关键的Orai结合域；使用超分辨率sted 微拷贝和TIRF/FRET成像我们探索了一个模型，该模型刺激了刺激 连接内的频道亚型，并确定聚类如何组织连接CA2+信号传导 机械。 （2）使用遗传编码的CA2+进入连接的微观生理环境 并在上源应用的Ca2+问题上标记在Stim和Orai蛋白上，以监视高度限制的 连接CA2+微环境；使用此类测量探索刺激介导的ORAI的聚类 频道控制本地连接CA2+信号，修改INSP3R和SERCA泵的接近性，以及 调节全局Ca2+振荡的产生。 （3）了解商店经营的CA2+信号是如何的 转录翻译，探索平滑肌和B细胞靶向平滑肌肉中的“ STIM2表型” 刺激动物与stim1/stim2.1表达水平和连接ca2+信号有关，刺激如何 调制Orai聚类控制特定的NFAT亚型激活，并确定转录组变化 与刺激类型特异性CA2+信号产生有关。我们的研究解决了我们了解的关键差距 连接CA2+如何调整本地和全局Ca2+信号的产生。 Stim/Orai介导的核心作用 细胞生理学中的商店经营的Ca2+信号，刺激/ORAI突变引起的宽度功能障碍， Stim/Orai亚型之间的深度功能区别，以及亚型表达的特殊变化 在主要疾病状态下，所有疾病都将刺激界面定义为关键的治疗靶点。