The Role of Apoptosis Repressor with Caspase Recruitment Domain (ARC) in Amyloid-Induced Beta-cell Apoptosis

 描述(由申请人提供)：胰岛淀粉样蛋白形成是2型糖尿病的一个标志，与β细胞凋亡和β细胞质量丧失有关。已有研究表明，c-jun氨基末端激酶、Bc1-2相关死亡启动子和Bcl2相关X蛋白在淀粉样蛋白诱导的β细胞凋亡中具有重要的促凋亡作用。相反，最近在β细胞中发现的一种新的抗凋亡蛋白-带有半胱氨酸天冬氨酸酶招募结构域的凋亡抑制蛋白(ARC)的作用尚不清楚。因此，我建议研究ARC在淀粉样蛋白诱导的β细胞凋亡中的作用。这项工作的目标是获得有关ARC的知识，这将导致新的策略来防止β细胞丢失，这是2型糖尿病发展的关键组成部分。我提出了一个模型，在这个模型中，Arc拮抗β细胞中三种著名的促凋亡蛋白的作用，即Jnk、Bad和bax。我推测在正常情况下，ARC抑制了JNK的激活，并封存了Bad和Bax。在淀粉样蛋白形成过程中，ARC不再抑制JNK的激活，不能隔离Bad和BAX，导致β细胞的凋亡。在本申请中，我提出了解决这一假设的三个具体目标。具体目的1：确定ARC在β-细胞中的抗凋亡作用是否部分是通过抑制JNK途径的激活来实现的。具体目标2：确定ARC封存Bad和BAX是否有助于其在β-细胞中的抗凋亡作用。特异性目标3：确定ARC过表达是否能提高胰岛移植后转hIAPP基因胰岛移植物的存活率。我将使用胰岛淀粉样蛋白转基因小鼠模型的胰岛、野生型小鼠胰岛和永生化的β细胞系来进行拟议的研究。这项三年计划是一项着眼于科学和专业发展目标的综合培训方案。研究将扩大目前对β细胞凋亡的理解，同时促进新技术的获得，如腺病毒胰岛转导、免疫组织化学、定量显微镜、邻近结扎试验和胰岛移植。卡恩博士的科学研究 指导、实验室能力和对卓越指导的承诺将有助于这个项目的成功完成，并有助于我的职业发展。参加由华盛顿大学糖尿病研究中心(DRC)赞助的研讨会、实验室会议、期刊俱乐部、地区性和全国性会议以及研究活动将有助于获得全面的培训和职业发展体验。总而言之，这些活动将使我能够成功竞争职业发展(K)奖，并朝着成为一名独立的胰岛生物学研究者的目标迈进。

 DESCRIPTION (provided by applicant): Islet amyloid formation is a hallmark of type 2 diabetes that is associated with β-cell apoptosis and loss of β-cell mass. Prior studies have determined that c-Jun N-terminal kinase (JNK), Bcl-2-associated death promoter (Bad), and Bcl-2-associated X protein (Bax) have important pro-apoptotic roles in amyloid-induced β- cell apoptosis. Conversely, the role of a novel anti-apoptotic protein that was recently identified in the β-cell, apoptosis repressor with caspase recruitment domain (ARC), is not well understood. Therefore, I propose to study ARC's role in amyloid-induced β-cell apoptosis. The goal of this work is to gain knowledge of ARC which will lead to novel strategies to prevent β-cell loss, a critical component of the development of type 2 diabetes. I propose a model whereby ARC antagonizes the actions of three well-known pro-apoptotic proteins in the β-cell, namely JNK, Bad and Bax. I posit that under normal conditions, ARC represses JNK activation and sequesters Bad and Bax. In the setting of amyloid formation, ARC no longer represses JNK activation, and is unable to sequester Bad and Bax, leading to β-cell apoptosis. In this application I propose three specific aims to address this hypothesis. Specific Aim 1: Determine whether the anti-apoptotic effect of ARC in the β-cell is mediated in part by inhibition of JNK pathway activation. Specific Aim 2: Determine whether ARC sequestration of Bad and Bax contributes to its anti-apoptotic effect in the β-cell. Specific Aim 3: Determine whether ARC overexpression improves hIAPP transgenic islet graft survival following islet transplantation. I will use islets from a transgenic mouse model of islet amyloid, wild type mouse islets, and an immortalized β-cell line to perform the proposed studies. This three-year plan is a comprehensive training proposal focused on scientific and professional development objectives. Studies will expand the current understanding of β-cell apoptosis, while facilitating the acquisition of new skills such as adenoviral transduction of islets, immunohistochemistry, quantitative microscopy, proximity ligation assay, and islet transplantation. Dr. Kahn's scientific direction, laboratory capabilities, and commitment to exceptional mentoring will contribute to the successful completion of this project, and to my career development. Participation in seminars, lab meetings, journal club, regional and national meetings, and research events sponsored by the University of Washington Diabetes Research Center (DRC) will contribute to a comprehensive training and career development experience. Together, these activities will enable me to successfully compete for a Career Development (K) Award and progress towards my goal of becoming an independent investigator of islet biology.