Molecular Studies of Noncanonical NF-kb Signaling

项目摘要/摘要 核因子-κB家族转录因子调节多种生物学过程，包括免疫和 炎症反应。核因子-κB激活涉及两条主要途径：规范途径和非规范途径 小路。非正则途径的中心步骤是泛素依赖的核因子-κB2的处理 前体蛋白P100，它产生成熟的NF-κB2 P52，并导致P100- 隔离的非规范的NF-κB成员，P52和RelB。在之前的资金周期中，PI的 实验室为这一相对较新的领域的发展做出了关键贡献，包括发现了 NIK作为中央信号转导元件和TRAF3被确定为主要的负调控因子 尼克的命运。现在已知TRAF3作为NIK特异性E3的底物结合亚单位起作用 泛素连接酶复合体，TRAF3-TRAF2-CIAP。尽管取得了这些进展，但我们对非正则核因子的了解- κB信号仍然相当有限。特别是，只有几个调控因子的这一途径被表征，并且 非规范的NF-κB在不同类型的免疫细胞中的功能尚不清楚。 本继续申请的总体目标是表征新的调节器和功能 非规范的NF-κB途径。拟议的研究基于创新的初步数据，来自 派的实验室。使用新生的NIK条件KO小鼠，我们证明了一种树突状细胞(DC)- 非典型性核因子-κB在调节粘膜免疫中的特定功能 潜在的机制。为了确定非典范的NF-κB途径的新调节因子，我们采用了一种 最近开发的方法，BioID，以筛选与这一途径的主要组成部分相互作用的蛋白质。 经过广泛的鉴定，我们已经确定了一个TRAF3结合蛋白TFG和一个P100结合蛋白 蛋白Otub1，它们在非典型性的NF-κB调控中起关键作用。TFG对于预防 异常的NK堆积，而Otub1是一种去泛素酶(DUB)，它抑制P100泛素化和 正在处理。我们的基因敲除和KO小鼠研究表明TFG和Otub1在 免疫调节。这些创新的发现为这种继续应用奠定了坚实的基础。至 为了完成我们的总体目标，我们将(1)阐明非规范的NF-κB功能的机制 树突状细胞中调节粘膜免疫的作用；(2)表征调节非规范的NF-κB的新因子 (3)研究新的非规范的NF-κB调节因子的免疫调节功能。 PI的实验室率先发现了非规范的NF-κB途径，并一直处于领先的 在这一地区的位置。在之前的资金周期中，我们取得了开创性的发现， 对该领域的进步起到了重要作用。我们相信，拟议的研究将再次导致 极具影响力的发现，大大推动了该领域的发展。凭借我们丰富的经验和创新的 根据初步数据，我们处于开展拟议研究的独特地位。

Project Summary/Abstract The NF-κB family of transcription factors regulates diverse biological processes, including immune and inflammatory responses. NF-κB activation involves two major pathways: the canonical and noncanonical pathways. A central step in the noncanonical pathway is ubiquitin-dependent processing of the NF-κB2 precursor protein p100, which generates mature NF-κB2 p52 and cause nuclear translocation of the p100- sequestered noncanonical NF-κB members, p52 and RelB. During the previous funding cycles, the PI's laboratory has made pivotal contributions to the advancement of this relatively new field, including discovery of NIK as a central signaling component and identification of TRAF3 as a primary negative regulator that controls the fate of NIK. It is now known that TRAF3 functions as the substrate binding subunit of a NIK-specific E3 ubiquitin ligase complex, TRAF3-TRAF2-cIAP. Despite these progresses, our knowledge on noncanonical NF- κB signaling is still quite limited. In particular, only few regulators of this pathway have been characterized, and the function of noncanonical NF-κB in different types of immune cells is poorly defined. The overall objective of this continuation application is to characterize novel regulators and functions of the noncanonical NF-κB pathway. The proposed studies are based on innovative preliminary data from the PI's laboratory. Using newly generated NIK-conditional KO mice, we demonstrated a dendritic cell (DC)- specific function of noncanonical NF-κB in regulating mucosal immunity and obtained important clues to the underlying mechanism. To identify novel regulators of the noncanonical NF-κB pathway, we employed a recently developed approach, BioID, to screen for proteins interacting with major components of this pathway. After extensive characterizations, we have identified a TRAF3-binding protein, TFG, and a p100-binding protein, Otub1, which are critically involved in noncanonical NF-κB regulation. TFG is crucial for preventing abnormal NIK accumulation, whereas Otub1 is a deubiquitinase (DUB) that inhibits p100 ubiquitination and processing. Our gene knockdown and KO mouse studies suggest important functions of TFG and Otub1 in immune regulation. These innovative findings form a solid foundation for this continuation application. To accomplish our overall objective, we will (1) elucidate the mechanism by which noncanonical NF-κB functions in DCs to regulate mucosal immunity; (2) characterize novel factors that regulate noncanonical NF-κB signaling; and (3) investigate the immunoregulatory functions of novel noncanonical NF-κB regulators. The PI's laboratory pioneered the discovery of noncanonical NF-κB pathway and has been in a leading position in this area. During the previous funding cycles, we have made seminal discoveries that have been instrumental for the advancement of the filed. We believe that the proposed studies will once again lead to high-impact findings that substantially advance the field. With our extensive experience and the innovative preliminary data, we are in a unique position to carry out the proposed studies.