CRAC CHANNEL COMPONENTS AND MOLECULAR BASIS OF STORE-OPERATED CALCIUM ENTRY

CRAC 通道成分和存储钙输入的分子基础

• 批准号: 8760704
• 负责人: Monika Vig
• 金额: $ 38.13万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8760704
• 关键词: 3-Dimensional; Affect; Agonist; Allergic; Anaphylaxis; Architecture; Autoimmune Diseases; Autoimmune Process; Binding; Biochemical; CD4 Positive T Lymphocytes; Calcium; Calcium Binding; Calcium Channel; Calcium Channel Agonists; Cell Degranulation; Cell membrane; Cell surface; Cells; Collaborations; Complex; Cytokine Gene; Defect; Disease; Drug Design; Endoplasmic Reticulum; Gene Expression; Generations; Genetic; Health; Human; Image; Immune response; Immunologic Deficiency Syndromes; Inositol; Investigation; Lead; Life; Lymphocyte; Lymphocyte Activation; Macromolecular Complexes; Maps; Mediating; Microscopy; Modeling; Molecular; Molecular Conformation; Molecular Structure; Mus; Optics; Patients; Peripheral; Pharmacotherapy; Physiological; Point Mutation; Proteins; RNA Interference; Regulation; Relative (related person); Resolution; Role; S-nitro-N-acetylpenicillamine; T-Cell Activation; T-Lymphocyte; Testing; Work; base; congenital immunodeficiency; design; genome-wide; insight; interdisciplinary approach; macromolecular assembly; mast cell; nanoscale; novel; nuclear factors of activated T-cells; porin; reconstruction; response; sensor; soluble NSF attachment protein; tool; tripolyphosphate

DESCRIPTION (provided by applicant): Store-operated calcium entry, or SOCE, via calcium release activated calcium (CRAC) channels regulates NFAT dependent gene expression required for lymphocyte activation and generation of immune response against antigenic insults. Orai (or CRACM) proteins form the pore subunit of CRAC channels in the plasma membrane (PM), and endoplasmic reticulum (ER) resident stromal interaction proteins (Stims) act as store calcium sensors that oligomerize and translocate to ER-PM junctions to activate CRAC channels. However, the final steps involved in CRAC channel activation inside live cells have remained elusive. For instance, until recently it was believed that Stim1-Orai1 clustering in ER-PM junctions is sufficient to activate SOCE. However, in the absence of alpha-soluble NSF attachment protein (α-SNAP), a novel essential component of active CRAC channel complex that we have recently identified, Stim1-Orai1 clusters show a strong defect in activating SOCE. α-SNAP directly binds Stim1 as well as Orai1 and regulates the ratio of Stim1:Orai1 molecules within CRAC channel clusters; a known determinant of CRAC channel activity and calcium selectivity. Furthermore, α-SNAP deficient mouse primary CD4 T lymphocytes show a specific defect in SOCE, NFAT activation and cytokine gene expression. In Aim1, we propose to test the hypothesis that α-SNAP facilitates a structural change within Stim1 that enables the Stim1-Orai1 clusters to acquire optimal Stim1:Orai1 ratios required for the functionality of CRAC channel clusters in live cells. To test our hypothesis, we will use a variety of cutting edge biochemical, structural and imaging approaches along with genetic tools. In Aim 2, we will use a combination of novel super resolution microscopy approaches to construct the 3 dimensional molecular architecture of CRAC channel macromolecular assembly in ER-PM junctional space of wildtype and α-SNAP depleted cells. In summary, we will employ novel and highly interdisciplinary approaches to gain much needed insights into the molecular mechanism of SOCE activation via CRAC channels. Identification of new non-redundant components of the CRAC channel complex and detailed insights into their mechanism of action will be highly beneficial for drug design against autoimmune and allergic disorders.

描述（由适用提供）：通过钙释放激活钙（CRAC）通道的商店经营的钙进入或SOCE调节淋巴细胞激活和针对抗原侮辱的免疫响应所需的NFAT依赖基因表达。 ORAI（或CRACM）蛋白质形成质膜（PM）中CRAC通道的孔亚基，内质网（ER）居民基质相互作用蛋白（Stim）充当储存的钙传感器，可将钙化和转化为ER-PM交接，以激活Crac crac通道。但是，活细胞内部CRAC通道激活涉及的最后一步仍然难以捉摸。例如，直到最近，人们认为ER-PM连接中的im1-orai1聚类足以激活SOCE。但是，在没有α-溶解的NSF附着蛋白（α-SNAP）的情况下，这是我们最近确定的活性CRAC通道复合物的新型基本要素，STIM1-ORAI1簇在激活SOCE中表现出很大的缺陷。 α-SNAP直接结合STIM1和ORAI1，并调节CRAC通道簇内的oti oRAI1分子的比率；已知确定CRAC通道活性和钙选择性的已知。此外，α-SNAP缺乏小鼠初级CD4 T淋巴细胞在SOCE，NFAT激活和细胞因子基因表达中表现出特定的缺陷。在AIM1中，我们建议检验以下假设：α-SNAP在STIM1中构成结构变化，使STIM1-ORAI1群集能够获得最佳的stim1：ORAI1：ORAI1比率在活细胞中CRAC通道簇的功能所需的比率。为了检验我们的假设，我们将使用各种尖端生化，结构和成像方法以及遗传工具。在AIM 2中，我们将使用新型的超级分辨率显微镜方法的组合来构建在野生型和α-SNAP耗尽细胞的ER-PM连接空间中CRAC通道大分子组装的3个维度分子结构。总而言之，我们将采用新型和高度的跨学科方法来通过CRAC通道对SOCE激活的分子机制获得急需的见解。 CRAC通道复合物的新非冗余成分以及对其作用机理的详细见解对针对自身免疫性和过敏性疾病的药物设计非常有益。