The Ubiquitin Editing Enzyme A20 Preserves Intestinal Epithelial Cell Homeostasis

描述（由申请人提供）：肠上皮功能障碍是炎症性肠病（IBD）的标志。维持肠上皮细胞（IEC）的完整性需要 分化、增殖、存活和细胞死亡的平衡。A20是一种泛素编辑酶，与人类IBD有关。与人IBD中的潜在作用一致，IEC中A20的特异性缺失使小鼠对化学诱导形式的结肠炎敏感，而A20的转基因过表达通过支持IEC屏障功能来保护上皮。然而，由于A20被报道抑制NF-κ B依赖的存活信号以及TNF-κ B诱导的凋亡细胞死亡，因此尚不清楚A20如何调节IEC稳态。我们假设A20的功能是由其相关蛋白的作用调节的。 我们的初步数据表明，在调节细胞命运的一个重要的辅因子是A20结合抑制剂NF B家族（ABIN-1）的成员。与仅具有A20上皮缺陷的小鼠形成鲜明对比，IEC中A20和ABIN-1的急性遗传缺失导致猖獗的自发上皮细胞死亡和快速致死。该提议试图通过以下具体目标来阐明A20和ABIN-1如何维持IEC稳态的分子机制：1）确定A20和ABIN-1如何在体内调节IEC稳态;以及2）确定A20和ABIN-1如何合作以限制上皮细胞内的细胞死亡。在目标1中，我们提出了一套体内实验，以检查上皮细胞死亡，屏障功能，粘膜免疫激活响应IEC特异性A20和ABIN-1缺失。我们还建议通过遗传和药理学方法研究TNF-β和TLR依赖性信号传导的作用。目的二：研究A20和ABIN-1在体外对细胞死亡信号的调控机制。我们计划通过分析A20和ABIN-1在内源性和外源性凋亡途径中的作用来定位这两种蛋白的作用位点。然后，我们将通过经典的生物化学技术，如免疫沉淀和蛋白质印迹，以确定由A20和ABIN-1调控的特定复合物的候选方法。了解IEC稳态是如何维持或调节的，将为炎症性肠病的新疗法的开发提供潜在的新线索。 这些研究将为在UCSF结肠炎和克罗恩病中心主任Averil Ma博士指导下的R 01申请提供基础。职业发展计划，包括教学课程，研究研讨会，和期刊俱乐部已经制定了沿着与拟议的研究出版物和职业时间轴。由国际公认的导师，合作者和顾问组成的职业发展委员会在IBD，免疫学，炎症和凋亡生物学领域具有专业知识，将有助于确保邵博士达到拟议的里程碑，成功申请R 01资助，并成为炎症性肠病的独立研究者。

DESCRIPTION (provided by applicant): Intestinal epithelial dysfunction is a hallmark of inflammatory bowel disease (IBD). Maintaining intestinal epithelial cell (IEC) integrity requires a balance of differentiation, proliferation, survival, and cell death. A20 is a ubiquitin-editing enzyme which has been linked to IBD in humans. Consistent with a potential role in human IBD, specific deletion of A20 in IECs renders mice susceptible to chemically induced forms of colitis while transgenic overexpression of A20 protects the epithelium by supporting IEC barrier function. However, as A20 has been reported to both dampen both NF�B-dependent survival signals as well as TNF-� induced apoptotic cell death, it is not clear exactly how A20 regulates IEC homeostasis. We hypothesize that the function of A20 is modulated by the action of its associated proteins. Our preliminary data suggests that one essential cofactor in regulating cell fate is a member of the A20- binding inhibitors of NF�B family (ABIN-1). In stark contrast to mice with epithelial deficiency of A20 alone, acute genetic deletion of both A20 and ABIN-1 in IECs leads to rampant spontaneous epithelial cell death and rapid lethality. This proposal attempts to unravel the molecular mechanisms of how A20 and ABIN-1 maintain IEC homeostasis through the following specific aims: 1) Determine how A20 and ABIN-1 regulate IEC homeostasis in vivo; and 2) determine how A20 and ABIN-1 cooperate to restrict cell death within epithelial cells. In Aim 1, we propose a set of in vivo experiments to examine epithelial cell death, barrier function, and mucosal immune activation in response to IEC-specific deletion of A20 and ABIN-1. We also propose to study the role of TNF-� and TLR-dependent signaling through genetic and pharmacologic approaches. In Aim 2, we will study the molecular mechanism underlying cell death signaling regulation by A20 and ABIN-1 in vitro. We plan to localize the site of action of A20 and ABIN-1 by dissecting the contribution of these two proteins in both the intrinsic and extrinsic apoptotic pathway. We will then use a candidate approach through classical biochemical techniques such as immunoprecipitation and western blot to identify specific complexes regulated by A20 and ABIN-1. Understanding how IEC homeostasis is maintained or modulated will provide potential new leads for the development of novel therapeutics for inflammatory bowel disease. These studies will provide the foundation for an R01 application under the mentorship of Dr. Averil Ma, Director of the Center for Colitis and Crohn's Disease at UCSF. A Career Development Plan including didactic courses, research seminars, and journal clubs has been developed along with a proposed research publication and career timeline. A career development committee of internationally recognized mentors, collaborators, and advisors with expertise in the fields of IBD, immunology, inflammation and apoptosis biology will help ensure that Dr. Shao meets the proposed milestones, successfully applies for an R01 grant, and becomes an independent investigator in inflammatory bowel disease.