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 钙离子信号在空间和时间上的非凡精确度可以精细地控制大量的细胞功能。 钙信号反映了高度协调的钙感受器和钙通道的功能。两国之间的相互作用 钙离子感受器STIM蛋白位于内质网和质膜中的钙通道，在钙信号的产生中起着关键作用。这个 两种蛋白质在内质网-质膜连接中物理偶联，介导“存储操作”的钙信号，在 控制基因的表达、分泌、运动和生长。它们在钙信号转导中的作用是至关重要的，而 Stim或Orai蛋白会引起一系列疾病，包括严重的联合免疫缺陷、肌肉 低眼压、自身免疫、皮肤发育不良和外分泌缺陷。STIM/Orai基因表达失调密切相关 与心血管和呼吸道重塑、神经退行性疾病、免疫力改变和癌症有关。我们 对STIM和ORAI蛋白的分子性质和细胞组织有很大的定义。我们现在 转向他们工作时所处的纳米级ER-PM连接环境。结合型钙离子在细胞周期中的作用 产生“局部”钙离子信号和维持贯穿细胞质的“全局”钙振荡 并穿透原子核。利用分子探针，成像技术，基因缺失的细胞 我们的工作针对三个主要领域：(1)确定关键的 疾病相关的STIM和ORAI亚型的差异，侧重于广泛表达的操作 STIM2.1缺少关键的Orai结合结构域的STIM2剪接变体；使用超分辨率STED 显微镜和TIRF/FRET成像，我们探索了一种模型，通过该模型STIM2.1破坏了Orai的交联 连接内的通道亚型，并确定集群如何组织连接的钙信号 机械设备。(2)利用遗传编码，确定钙离子进入连接的微生理环境 并将标记在STIM和Orai蛋白上的光遗传钙探针应用于监控高度受限的 结合Ca~(2+)微环境；利用这种测量来探索STIM如何介导Orai的聚集 通道控制局部连接钙信号，改变InsP3Rs和SERCA泵的接近程度，以及 调节全球钙离子振荡的产生。(3)了解存储操作的钙信号是如何 转录转导，探索平滑肌和B细胞靶向的“STIM2表型” STIM1缺失的动物与STIM1/STIM2.1的表达水平和连接钙信号有关，STIM- 调节的ORAI聚集性控制特定的NFAT亚型激活，并决定转录组的变化 与STIM类型特异性钙信号的产生有关。我们的研究解决了我们在知识上的关键差距 连接钙如何调节局部和全局钙信号的产生。STIM/ORAI介导的核心作用 细胞生理学中的存储操作钙信号，STIM/Orai突变引起的广泛功能障碍， STIM/Orai亚型之间的深层功能差异和亚型表达的异常变化 在重大疾病状态下，所有这些都将STIM-Orai界面定义为关键的治疗目标。