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

描述（由申请人提供）：T细胞受体（TCR）刺激通过CRAC （Ca2+释放激活的Ca2+）通道触发Ca2+信号是T细胞功能的关键步骤。PI最近确定并命名了Orai1作为CRAC通道的一个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具有致命的，严重的联合免疫缺陷（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和肥大细胞活化至关重要。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标。这种小分子可以潜在地用作免疫抑制剂，以减轻移植或自身免疫性疾病期间的免疫并发症。我们对Orai1的突变分析确定了这样一个分子，同时表征了一种新的失活机制，该机制涉及位于跨膜（TM）区段II和III之间的Orai1细胞内环。两个脯氨酸的环残基N153VHNL157突变，消除了通道失活。用连接的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，表明单个环占主导地位。最后，来自TM II-TM III环的肽的外源表达阻断了T细胞中的CRAC通道。失活颗粒对CRAC通道活性的强大的显性阻断作用必将有利于该肽作为免疫抑制剂的开发。在目前的建议中，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失活环衍生的抑制肽的结构要求。我们将确定阻断CRAC通道活性所需肽的最小尺寸。阻断肽上附着一个短的蛋白转导序列，形成膜渗透形式，并测试其对T细胞功能的阻断作用。(2)在临床相关动物模型中检验Orai1短抑制肽作为免疫抑制剂的可能性。首先，我们将确定多肽在细胞因子产生和原代免疫细胞增殖中的阻断功效。然后，我们将确定抑制肽在小鼠移植物抗宿主病模型的发病/进展中的作用。CRAC通道抑制肽的开发将代表一种新的和改进的治疗方法来抑制免疫反应。这种肽的副作用可能比环孢素a和他克莫司小得多，因为这种抑制肽专门针对主要在免疫细胞中占主导地位的CRAC通道活性。

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.