Molecular Studies of Noncanonical NF-kb Signaling

项目总结/摘要 转录因子NF-κB家族调节多种生物学过程，包括免疫和 炎症反应。NF-κB的激活主要涉及两条途径：经典途径和非经典途径 途径。非经典途径的一个中心步骤是NF-κB2的泛素依赖性加工 前体蛋白p100，其产生成熟的NF-κB2 p52并引起p100- 隔离的非典型NF-κB成员、p52和RelB。在过去的融资周期中，PI 该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 NIK作为中枢信号传导组分和TRAF 3作为控制细胞凋亡的主要负调节因子的鉴定 NIK的命运现在已知TRAF 3作为NIK特异性E3的底物结合亚基起作用 泛素连接酶复合物，TRAF 3-TRAF 2-cIAP。尽管有这些进展，我们对非经典NF-κ B的认识， κB信号传导仍然相当有限。特别是，只有少数调节剂的这一途径已被确定， 非典型NF-κB在不同类型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剂和功能 非经典NF-κB通路的影响。拟议的研究是基于创新的初步数据， PI的实验室使用新产生的NIK条件性KO小鼠，我们证明了树突状细胞（DC）- 非经典NF-κB在调节粘膜免疫中的特异性作用，为进一步阐明NF-κB在粘膜免疫中的作用机制提供了重要线索。 潜在机制为了鉴定非经典NF-κB通路的新调节因子，我们采用了一种 最近开发的方法，BioID，以筛选与该途径的主要成分相互作用的蛋白质。 经过广泛的表征，我们已经确定了一个TRAF 3结合蛋白，TFG，和一个p100结合蛋白。 蛋白Otub 1，其在非经典NF-κB调节中起关键作用。过渡联邦政府对于防止 异常NIK积累，而Otub 1是一种去泛素化酶（DUB），抑制p100泛素化， 处理.我们的基因敲除和KO小鼠研究表明TFG和Otub 1在肿瘤发生中的重要功能。 免疫调节这些创新的发现为继续申请奠定了坚实的基础。到 为了实现我们的总体目标，我们将（1）阐明非经典NF-κB的功能机制 在DC中调节粘膜免疫;（2）表征调节非经典NF-κB新因子 （3）研究新型非经典NF-κB调节因子的免疫调节功能。 PI的实验室率先发现了非经典NF-κB通路，并一直处于领先地位。 在这个领域的位置。在之前的融资周期中，我们已经取得了开创性的发现， 为推动本领域的发展而努力。我们认为，拟议的研究将再次导致 高影响力的发现，大大推进该领域。凭借我们丰富的经验和创新的 根据初步数据，我们处于进行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.