Suppression of Immune Functions by a Peptide Blocking Function of CRAC Channels

描述(申请人提供)：T细胞受体(TCR)刺激通过CRAC(钙释放激活的钙通道)触发钙信号是T细胞功能的关键步骤。PI最近确认并命名Orai1为CRAC通道中长期寻找的孔隙成分。我们已经证明，Orai1基因错义突变的人类患者患有致命的严重联合免疫缺陷(SCID)，强调其在免疫细胞功能中的主导作用。此外，对Orai1缺失小鼠的分析表明，Orai1对B、T和肥大细胞的激活至关重要。Orai1的鉴定为开发能够阻断免疫反应的小分子提供了一个分子靶点。这种小分子可能被用作免疫抑制剂，以减轻移植过程中的免疫并发症或自身免疫性疾病。我们对Orai1的突变分析确定了这样一个分子，同时描述了一种新的失活机制，涉及位于跨膜(TM)片段II和III之间的Orai1细胞内环。环残基N153VHNL157的突变两侧有两个脯氨酸，消除了通道失活。用串联的Orai1四聚体进行的实验表明，CRAC通道被四个细胞内环中的一个强烈抑制，这表明主要是由一个环阻断的。最后，来自TM II-TM III环的一种肽的外源表达阻断了T细胞中的CRAC通道。灭活颗粒对CRAC通道活性的强显性阻断作用，必将有利于多肽作为免疫抑制剂的开发。在目前的提案中，我们将研究抑制肽是否可以用作免疫抑制剂。具体目的如下：(1)确定Orai1灭活环衍生的抑制肽的结构要求。我们将确定阻断CRAC通道活性所需的最小多肽大小。将一个短的蛋白质转导序列连接到封闭肽上，以产生膜穿透形式，并测试其对T细胞功能的封堵效果。(2)探讨Orai1短抑制肽在临床相关动物模型中作为免疫抑制剂的可能性。首先，我们将确定该多肽在细胞因子产生和原代免疫细胞增殖方面的封闭效果。然后，我们将确定抑制肽在小鼠移植物抗宿主病模型发生/发展中的作用。CRAC通道抑制肽的开发将代表一种新的、改进的治疗方法来抑制免疫反应。这种多肽的副作用可能比环孢菌素A和他克莫司小得多，因为这种抑制多肽专门针对CRAC通道活性，而CRAC通道活性主要是在免疫细胞中占主导地位的。 公共卫生相关性：我们建议开发一种多肽阻滞剂，它可以抑制免疫细胞中的钙离子进入，从而抑制免疫反应。我们对CRAC通道的一种新成分Orai1的结构/功能分析发现了一种新型多肽，它可以灭活免疫受体刺激特异性触发的钙内流，并将使用移植物抗宿主病的动物模型来测试阻断免疫激活的可能性。我们的研究有助于开发能够抑制免疫功能的药物，用于治疗与免疫系统相关的问题，如移植物排斥反应或自身免疫性疾病。

DESCRIPTION (provided by applicant): Triggering of Ca2+ signaling though CRAC (Ca2+-release-activated Ca2+) channels by T cell receptor (TCR) stimulation is a key step for T cell functions. The PI has recently identified and named Orai1 as a long-sought pore component of the CRAC channel. We has shown that human patients with a missense mutation in the Orai1 gene have lethal, severe combined immune deficiency (SCID) emphasizing its predominant role in the function of immune cells. In addition, analyses of Orai1-null mice have shown that Orai1 is critical for B, T, and mast cell activation. Identification of Orai1 provides a molecular target to develop small molecules that can block immune responses. Such small molecules can be potentially used as immunosuppressants to alleviate immune complications during transplantation or autoimmune disorders. Our mutational analysis of Orai1 identified such a molecule while characterizing a novel inactivation mechanism that involves the Orai1 intracellular loop located between transmembrane (TM) segments II and III. Mutation of the loop residues N153VHNL157 flanked by two prolines, removed channel inactivation. Experiments carried out with concatenated Orai1 tetramer indicate that CRAC channels are strongly inhibited by one of four intracellular loops, suggesting dominant block by a single loop. Finally, exogenous expression of a peptide derived from the TM II-TM III loop blocked CRAC channels in T cells. The strong dominant blocking effect of the inactivation particle on the CRAC channel activity will definitely benefit development of the peptide as an immunosuppressant. In the current proposal, we will investigate whether the inhibitory peptide may be used as an immunosuppressant. The specific aims are as follows: (1) To determine the structural requirement of the inhibitory peptide derived from the Orai1 inactivation loop. We will determine the minimum size of the peptide required to block the CRAC channel activity. A short protein transduction sequence will be attached onto the blocking peptide to generate a membrane-permeant form and its blocking effect on the T cell function will be tested. (2) To test the possibility that the short inhibitory peptide of Orai1 can be used as immunosuppressant in a clinically relevant animal model. First, we will determine the blocking efficacy of the peptide in cytokine production and proliferation of primary immune cells. Then, we will determine the effect of the inhibitory peptide in the onset/progress of a mouse graft-versus- host disease model. Development of a CRAC channel inhibitory peptide would represent a novel and improved therapeutic approach to suppress immune responses. Such a peptide is likely to have much less side effects than cyclosporin A and tacrolimus since the inhibitory peptide is specifically targeting the CRAC channel activity that is predominant mostly in immune cells. PUBLIC HEALTH RELEVANCE: We propose to develop a peptide blocker that can suppress calcium entry in immune cells, thereby immune responses. Our structure/function analysis of a newly described component of the CRAC channel, Orai1 identified a novel peptide that can inactivate calcium entry specifically triggered by immune receptor stimulation, and the possibility of development of the blocking immune activation will be tested using an animal model of graft-versus-host disease. Our study can benefit development of drugs that can repress immune functions as therapy for immune system-related problems such as graft rejection or autoimmune disease.