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

CRAC 通道的肽阻断功能对免疫功能的抑制

• 批准号: 7873630
• 负责人: Yousang Gwack
• 金额: $ 19.25万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-15 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7873630
• 关键词: Adverse effects; Animal Model; Autoimmune Diseases; Calcium; Cell physiology; Cells; Cyclosporine; Development; Disease model; Genes; Graft Rejection; Human; Image; Immune; Immune response; Immune system; Immunologic Receptors; Immunosuppressive Agents; In Vitro; Knockout Mice; Length; Mediating; Membrane; Methods; Missense Mutation; Molecular Target; Mus; Mutation; Names; Organ Transplantation; Patients; Peptides; Pharmaceutical Preparations; Production; Proline; Proteins; Role; SCID Mice; Signal Transduction; Structure; Symptoms; T-Cell Proliferation; T-Cell Receptor; T-Lymphocyte; Tacrolimus; Testing; Therapeutic; Transplantation; Viral Proteins; clinically relevant; cytokine; drug development; efficacy testing; graft vs host disease; immune activation; immune function; improved; in vivo; intravenous injection; mast cell; novel; particle; public health relevance; research study; small molecule

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.

描述（由申请人提供）：T细胞受体（TCR）刺激通过CRAC（Ca 2+释放激活的Ca 2+）通道触发Ca 2+信号传导是T细胞功能的关键步骤。PI最近已确定并命名Orai 1为CRAC通道的长期寻找的孔组分。我们已经证明，Orai 1基因错义突变的人类患者具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Orai 1缺失小鼠的分析表明，Orai 1对B、T和肥大细胞活化至关重要。Orai 1的鉴定为开发可阻断免疫反应的小分子提供了分子靶点。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Orai 1的突变分析确定了这样一种分子，同时表征了一种新的失活机制，该机制涉及位于跨膜（TM）片段II和III之间的Orai 1细胞内环。突变侧翼为两个脯氨酸的环残基N153 VHNL 157，去除了通道失活。用串联的Orai 1四聚体进行的实验表明，CRAC通道被四个细胞内环之一强烈抑制，表明由单个环主导阻断。最后，外源性表达的肽来源于TM II-TM III环阻断CRAC通道的T细胞。灭活颗粒对CRAC通道活性的强的显性阻断作用将肯定有益于肽作为免疫抑制剂的开发。在目前的建议中，我们将研究是否抑制肽可用作免疫抑制剂。具体目的如下：（1）确定Orai 1失活环抑制肽的结构要求。我们将确定阻断CRAC通道活性所需的肽的最小大小。将短蛋白转导序列连接到封闭肽上以产生膜渗透形式，并测试其对T细胞功能的封闭作用。(2)在临床相关的动物模型中测试Orai 1的短抑制肽可用作免疫抑制剂的可能性。首先，我们将确定肽在原代免疫细胞的细胞因子产生和增殖中的阻断功效。然后，我们将确定抑制肽在小鼠移植物抗宿主病模型的发作/进展中的作用。CRAC通道抑制肽的开发将代表抑制免疫应答的新的和改进的治疗方法。这样的肽可能具有比环孢菌素A和他克莫司少得多的副作用，因为抑制性肽特异性靶向CRAC通道活性，该CRAC通道活性主要在免疫细胞中占主导地位。 公共卫生相关性：我们建议开发一种肽阻断剂，可以抑制免疫细胞中的钙离子进入，从而抑制免疫反应。我们的结构/功能分析的CRAC通道，Orai 1的新描述的组件，确定了一种新的肽，可以阻止钙离子进入特异性触发的免疫受体刺激，并将使用移植物抗宿主病的动物模型测试的可能性发展的阻断免疫激活。我们的研究有助于开发抑制免疫功能的药物，用于治疗免疫系统相关问题，如移植物排斥或自身免疫性疾病。