STIM-ORAI signaling and other calcium influx pathways in lymphocytes

 描述（由申请人提供）：Ca 2 +-钙调神经磷酸酶-NFAT通路对于适应性免疫应答至关重要，钙调神经磷酸酶抑制剂环孢菌素A（CsA）和FK 506的临床疗效强调了这一点。在T细胞中，转录因子NFAT响应于通过“CRAC”通道的Ca 2+进入而从细胞质易位到细胞核中，所述“CRAC”通道由钙传感器STIM 1和STIM 2响应于内质网（ER）Ca 2+储存的耗尽而打开。在该项目的前两个资助期内，我们在果蝇中进行了全基因组RNAi筛选，并在人类和小鼠T细胞中进行了功能研究，以证明长期寻找的CRAC通道是由ORAI家族蛋白组装而成的;表明ORAI 1中的突变是遗传性人类免疫缺陷疾病的原因;并通过突变分析确定ORAI 1是CRAC通道的孔亚基。我们产生了基因破坏的小鼠与一个完整的删除的ORAI 1或条件（“被破坏的”）等位基因的ER Ca 2+传感器STIM 1或STIM 2和分析他们的免疫表型。我们还在人类细胞（HeLa细胞）中进行了全基因组RNAi筛选，鉴定了调节NFAT激活的多种蛋白质。对该筛选的后续研究表明，septins是细胞中储存操作的STIM-ORAI Ca 2+进入途径的重要调节剂。通过对STIM 1、ORAI 1及其蛋白质-蛋白质相互作用的生物化学研究，我们发现STIM 1的胞质区域本身足以门控ORAI 1通道;并阐明了STIM 1的ER-管腔结构域的二聚化触发STIM 1的胞质结构域构象变化的机制，从而激活STIM以打开奥赖通道。在这项提案中，我们将在这些发现的基础上扩展我们对STIM-ORAI信号传导和T细胞中Ca 2+进入途径的理解。在目标1中，我们将研究使用纯化的STIM 1，ORAI 1和蛋白质调节剂的ORAI 1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-蛋白质相互作用和蛋白质构象变化。在目标2中，我们将研究一种新的替代Ca 2+内流途径，该途径除了在分化的小鼠和人T细胞中的STIM-ORAI Ca 2+进入途径之外还起作用。目的1与许多生物学过程和癌症广泛相关，因为STIM-ORAI蛋白不仅在免疫和造血细胞（T和B淋巴细胞、肥大细胞、血小板）中，而且在骨骼肌、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中介导钙池操作的Ca 2+进入。目的2旨在建立新记录的Ca 2+进入免疫细胞的途径的分子身份，这可能与免疫疾病和哮喘的治疗有关。这两个目标是相互吻合的，旨在更全面地了解T细胞中的生理Ca 2+信号传导。

 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.