STIM-ORAI signaling and other calcium influx pathways in lymphocytes

 说明(申请人提供)：钙-钙调神经磷酸酶-NFAT通路对于适应性免疫反应是必不可少的，钙调神经磷酸酶抑制剂环孢素A(CsA)和FK506的临床疗效强调了这一点。在T细胞中，转录因子NFAT在钙离子通过“CRAC”通道进入细胞核时从细胞质转位到细胞核，而该通道是由钙传感器STIM1和STIM2打开的，以响应内质网(ER)钙储存的耗尽。在该项目的前两个资助期，我们使用了在果蝇中进行的全基因组RNAi筛选以及在人和小鼠T细胞中的功能研究，以证明长期寻找的CRAC通道是由ORAI家族蛋白组装的；表明ORAI1的突变与一种遗传性人类免疫缺陷疾病有关；并且通过突变分析确定ORAI1是CRAC通道的孔亚基。我们产生了完全缺失ORAI1或带有ER钙传感器STIM1或STIM2的条件等位基因的基因紊乱的小鼠，并分析了它们的免疫表型。我们还在人类细胞(HeLa细胞)中进行了全基因组RNAi筛选，发现了多种调节NFAT激活的蛋白质。这一筛选的后续研究表明，Septins是细胞内STIM-ORAI钙离子进入途径的重要调节器。通过对STIM1和ORAI1及其蛋白-蛋白质相互作用的生化研究，我们发现STIM1的细胞质区域本身就足以开启ORAI1通道；绘制了ORAI1通道的跨膜螺旋；并揭示了STIM1的ER-腔结构域二聚引发STIM1胞质结构域的构象变化，从而激活STIM打开ORAI通道的机制。在这项提案中，我们将以这些发现为基础，扩展我们对STIM-ORAI信号和T细胞中钙离子进入途径的理解。在目标1中，我们将使用纯化的STIM1、ORAI1和蛋白质调节剂来研究ORAI1通道门控。为此，我们正在开发和利用最先进的技术来监测蛋白质相互作用和蛋白质构象变化。在目标2中，我们将研究在分化的小鼠和人类T细胞中，除了STIM-ORAI钙内流途径之外，还有一种新的替代钙内流途径。目的1与许多生物学过程和癌症有关，因为STIM-ORAI蛋白不仅介导免疫和造血细胞(T和B淋巴细胞、肥大细胞、血小板)，而且还介导骨骼肌、平滑肌、外分泌器官、某些癌症和几乎可以肯定的大多数其他细胞类型的储存操作的钙离子进入。目的2旨在建立新发现的钙离子进入免疫细胞途径的分子同一性，这一信息可能对免疫疾病和哮喘的治疗具有重要意义。这两个目标相吻合，旨在更全面地了解T细胞中的生理钙信号。

 DESCRIPTION (provided by applicant): The Ca2+-calcineurin-NFAT pathway is essential for the adaptive immune response, a point underscored by the clinical efficacy of the calcineurin inhibitors cyclosporin A (CsA) and FK506. In T cells, the transcription factor NFAT translocates from the cytoplasm into the nucleus in response to Ca2+ entry through `CRAC' channels, which are opened by the calcium sensors STIM1 and STIM2 in response to depletion of endoplasmic reticulum (ER) Ca2+ stores. During the previous two funding periods of this project, we used genome-wide RNAi screening in Drosophila and functional studies in human and mouse T cells to demonstrate that the long-sought CRAC channel is assembled from ORAI-family proteins; showed that a mutation in ORAI1 is responsible for a hereditary human immunodeficiency disease; and established by mutational analysis that ORAI1 is the pore subunit of the CRAC channel. We generated gene-disrupted mice with a complete deletion of ORAI1 or with conditional (`floxed') alleles of the ER Ca2+ sensors STIM1 or STIM2 and analyzed their immune phenotypes. We also performed a genome-wide RNAi screen in human cells (HeLa cells) that identified multiple proteins that modulate NFAT activation. Followup to this screen demonstrated that septins are essential modulators of the store-operated STIM-ORAI Ca2+ entry pathway in cells. Through biochemical studies of STIM1, ORAI1, and their protein-protein interactions, we showed that the cytoplasmic region of STIM1 by itself suffices to gate the ORAI1 channel; mapped the pore-lining transmembrane helices of the ORAI1 channel; and unraveled the mechanism through which dimerization of the ER-luminal domains of STIM1 triggers a conformational change in the cytoplasmic domains of STIM1, and thereby activates STIM to open the ORAI channel. In this proposal, we will build on these findings to extend our understanding of STIM-ORAI signalling and Ca2+ entry pathways in T cells. In Aim 1, we will investigate ORAI1 channel gating using purified STIM1, ORAI1, and protein modulators. For this, we are developing and utilizing state-of-art-techniques for monitoring protein- protein interactions and protein conformational changes. In Aim 2, we will investigate a novel alternative Ca2+ influx pathway that operates in addition to the STIM-ORAI Ca2+ entry pathway in differentiated mouse and human T cells. Aim 1 is broadly relevant to many biological processes and to cancer, since STIM-ORAI proteins mediate store-operated Ca2+ entry not only in immune and haematopoietic cells (T and B lymphocytes, mast cells, platelets) but also in skeletal muscle, smooth muscle, exocrine organs, certain cancers, and almost certainly most other cell types. Aim 2 is designed to establish the molecular identity of the newly documented pathway for Ca2+ entry into immune cells, information that could have major relevance for the treatment of immune disorders and asthma. The two aims dovetail in being directed toward a more complete understanding of physiological Ca2+ signalling in T cells.