Novel regulators of store-operated Ca2+ entry in immune systems

描述（由申请方提供）：通过CRAC（Ca 2+释放激活的Ca 2+）通道的Ca 2+内流对于免疫细胞功能至关重要。通过受体的刺激耗尽细胞内Ca 2+储存，随后导致质膜上CRAC通道的开放。最近，Orai 1（CRACM 1）和STIM 1被确定为CRAC通道的孔组分和Ca 2+库耗尽和CRAC通道开放之间的介质，分别。然而，关于CRAC通道调节的分子组成和机制仍有许多有待了解。我们已经在储存耗尽的条件下使用大规模亲和蛋白纯化来鉴定CRAC通道的调节剂。我们已经确定CRACR 2A（Ca 2+释放激活的Ca 2+通道调节器2A）作为一种新的CRAC通道的调节器使用这种方法。具体目的是：（1）验证Orai 1-CRACR 2A相互作用对CRAC通道功能至关重要的假设。将在Orai 1-null原代细胞中分析不能结合CRACR 2A的Orai 1突变体的活性。(2)检验CRACR 2A稳定Orai 1-STIM 1复合物的假设。我们的初步数据表明，CRACR 2A可以直接与Orai 1和STIM 1相互作用。Orai 1和STIM 1的功能将使用CRACR 2A敲低细胞进行检查。将确定Orai 1、STIM 1和CRACR 2A相互作用的功能重要性。(3)为了检验CRACR 2A对T细胞功能至关重要的假设。Jurkat T细胞中CRACR 2A的敲低减少了Ca 2+内流。我们将有条件地靶向CRACR 2A基因，以确定CRACR 2A在外周T细胞中钙池操作的Ca 2+内流中的作用。由于Orai 1和STIM 1的最新发现，对CRAC通道的新型调节剂的实验是及时的。在短期内，拟议的实验应该提供新的分子探针，研究哺乳动物细胞，特别是免疫细胞中的钙库操作的Ca 2+进入的调节机制。从长远来看，我们将测试开发药物来调节CRAC通道功能的可能性;从而免疫激活。公共卫生相关性：我们建议通过蛋白质组学分析、进一步的功能分析和基因操作来理解免疫细胞活化的关键步骤的机制，即通过质膜钙通道进入钙。我们使用这些方法成功地鉴定了一种新的分子CRACR 2A，我们将专注于阐明这种分子在免疫系统中的新功能。我们的研究有助于开发能够激活或抑制免疫功能的药物，用于治疗免疫系统相关问题，如自身免疫疾病或移植排斥反应。

DESCRIPTION (provided by applicant): Ca2+ influx though CRAC (Ca2+ release activated Ca2+) channels is critical for immune cell functions. Stimulation through receptors depletes intracellular Ca2+ stores and subsequently leads to opening of CRAC channels on the plasma membrane. Recently, Orai1 (CRACM1) and STIM1 were identified as a pore component of the CRAC channel and a mediator between Ca2+ store depletion and CRAC channel opening, respectively. However, much remains to be understood about the molecular composition and the mechanism of CRAC channel regulation. We have used large scale affinity protein purification under store depleted conditions to identify regulators of the CRAC channel. We have identified CRACR2A (Ca2+ release activated Ca2+ channel Regulator 2A) as a novel regulator of CRAC channels using this approach. The Specific Aims are: (1) To test the hypothesis that Orai1-CRACR2A interaction is critical for the CRAC channel function. The activities of Orai1 mutants incapable of binding to CRACR2A will be analyzed in Orai1-null primary cells. (2) To test the hypothesis that CRACR2A stabilizes the Orai1-STIM1 complex. Our preliminary data suggest that CRACR2A can directly interact with both Orai1 and STIM1. The functions of Orai1 and STIM1 will be examined using CRACR2A knock-down cells. The functional importance of reciprocal interaction of Orai1, STIM1, and CRACR2A will be determined. (3) To test the hypothesis that CRACR2A is critical for the T cell function. Knock-down of CRACR2A in Jurkat T cells decreased Ca2+ entry. We will conditionally target the CRACR2A gene to determine the role of CRACR2A in store-operated Ca2+ entry in peripheral T cells. The experiments on the novel regulator of the CRAC channel are timely because of the very recent discovery of Orai1 and STIM1. In the short term, the proposed experiments should provide new molecular probes for investigating the regulatory mechanism of store-operated Ca2+ entry in mammalian cells, particularly in immune cells. In the long term, we will test the possibility of development of drugs to modulate the function of the CRAC channel; thereby immune activation. Public Health Relevance: We propose to understand the mechanism of a critical step of immune cell activation, calcium entry via plasma membrane calcium channels through proteomic analysis, further functional analysis, and gene manipulation. We successfully indentified a novel molecule, CRACR2A using these approaches and we will focus to elucidate the novel function of this molecule in the immune system. Our study can benefit development of drugs that can activate or repress immune functions as therapy for immune system related problems such as auto-immune diseases or graft rejection.