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 相关。与人类 IBD 中的潜在作用一致，IEC 中 A20 的特异性缺失使小鼠易患化学诱导的结肠炎，而 A20 的转基因过度表达通过支持 IEC 屏障功能来保护上皮。然而，据报道，A20 既能抑制 NFκB 依赖性生存信号，又能抑制 TNF-α 诱导的细胞凋亡，因此目前尚不清楚 A20 到底如何调节 IEC 稳态。我们假设 A20 的功能是通过其相关蛋白的作用来调节的。我们的初步数据表明，调节细胞命运的一个重要辅助因子是 NFκB 家族 A20 结合抑制剂 (ABIN-1) 的成员。与仅缺乏 A20 上皮细胞的小鼠形成鲜明对比的是，IEC 中 A20 和 ABIN-1 的急性基因缺失会导致上皮细胞自发性大量死亡和快速致死。该提案试图通过以下具体目标来揭示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； 2) 确定 A20 和 ABIN-1 如何合作限制上皮细胞内的细胞死亡。在目标 1 中，我们提出了一组体内实验来检查上皮细胞死亡、屏障功能和粘膜免疫激活，以响应 IEC 特异性删除 A20 和 ABIN-1。我们还建议通过遗传和药理学方法研究 TNF-α 和 TLR 依赖性信号传导的作用。在目标 2 中，我们将在体外研究 A20 和 ABIN-1 调节细胞死亡信号传导的分子机制。我们计划通过剖析 A20 和 ABIN-1 在内源性和外源性细胞凋亡途径中这两种蛋白的贡献来定位这两种蛋白的作用位点。然后，我们将通过免疫沉淀和蛋白质印迹等经典生化技术使用候选方法来鉴定受 A20 和 ABIN-1 调节的特定复合物。了解 IEC 稳态如何维持或调节将为炎症性肠病新型疗法的开发提供潜在的新线索。这些研究将为在加州大学旧金山分校结肠炎和克罗恩病中心主任 Averil Ma 博士的指导下的 R01 申请奠定基础。职业发展计划包括教学课程、研究研讨会和期刊俱乐部，以及拟议的研究出版物和职业时间表。由国际公认的导师、合作者和顾问组成的职业发展委员会将帮助确保邵博士达到拟议的里程碑，成功申请 R01 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 NFκB-dependent survival signals as well as TNF-α 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 NFκB 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-α 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.