In Vivo Function of NEMO As a Sensor of K63-Linked Polyubiquitination

描述（由申请方提供）：免疫受体介导的转录因子NF-？B和MAP激酶（MAPK）对于参与先天性和适应性免疫应答的多个基因的适当调节是必需的。这些信号通路的不适当的慢性激活可能导致过度的细胞因子产生和炎症驱动的疾病，如关节炎，狼疮和癌症。先前在体外实验与无细胞系统和转化细胞系表明，赖氨酸-63（K63）连接的多泛素化的TNF受体相关因子6（TRAF 6），受体相互作用蛋白1（RIP），和NEMO亚基的I？B激酶（IKK）在免疫受体信号传导中起着重要的调节作用。体外鉴定的这些修饰步骤是否是体内治疗干预的有吸引力的靶点仍然未知。例如，体外研究表明NEMO的K63连接的泛素化对于淋巴细胞中的抗原受体（AgR）信号传导至关重要。然而，来自申请人实验室的初步结果表明，AgR信号传导在携带NEMO的泛素化缺陷突变体（NEMO-KR）的“敲入”小鼠中是完全有效的。相反，在NEMO-KR小鼠中，巨噬细胞和树突状细胞的Toll样受体（TLR）依赖性细胞因子分泌显著受损。这些在体内的结果强调，需要测试在生理环境中的蛋白质修饰的下游后果。为了满足这一基本目标，新的体内研究描述调查最近提出的作用NEMO作为传感器的K63连接的多聚泛素链，这可能有助于形成激活的免疫受体复合物和下游信号传递。将在编码NEMO的小鼠基因中进行种系点突变，破坏其结合K63连接链的能力，使我们能够研究NEMO泛素结合（NUB）结构域在原代细胞而不是转化细胞中的生物化学功能（Aim 1）。相应敲入小鼠的表型分析将揭示NUB结构域在由TLR、AgR和细胞因子受体信号传导介导的先天性与适应性免疫应答中的生理作用（Aim 2）。连同我们对NEMO-KR小鼠的研究，拟议项目的结果将为理解NEMO作为哺乳动物免疫系统中K63连接的泛素化的靶点和传感器的全部工作范围提供强有力的体内框架。公共卫生相关性：免疫系统细胞内的信号传递协调宿主对微生物病原体的防御，必须严格调节以避免慢性炎症。最近的体外实验表明，信号传递涉及细胞内蛋白质与泛素链的相互作用。提出了新的体内研究来研究特定泛素结合蛋白的生理功能，这种泛素传感机制与人类健康的相关性，以及在泛素化水平上治疗炎症性疾病的潜力。

DESCRIPTION (provided by applicant): Immunoreceptor-mediated activation of transcription factor NF-?B and MAP kinases (MAPKs) is essential for proper regulation of multiple genes involved in innate and adaptive immune responses. Inappropriate, chronic activation of these signaling pathways may lead to excessive cytokine production and inflammation-driven diseases such as arthritis, lupus, and cancer. Prior in vitro experiments with cell-free systems and transformed cell lines suggest that Lys-63 (K63)-linked polyubiquitination of TNF receptor-associated factor 6 (TRAF6), receptor-interacting protein 1 (RIP), and the NEMO subunit of I?B kinase (IKK) plays a crucial regulatory role in immunoreceptor signaling. It remains unknown whether these modification steps identified in vitro are attractive targets for therapeutic intervention in vivo. For example, in vitro studies suggested that K63-linked ubiquitination of NEMO is crucial for antigen receptor (AgR) signaling in lymphocytes. However, preliminary results from the applicant's laboratory indicate that AgR signaling is fully operative in "knock-in" mice harboring a ubiquitination-defective mutant of NEMO (NEMO-KR). Instead, Toll-like receptor (TLR)-dependent secretion of cytokines by macrophages and dendritic cells is significantly impaired in NEMO-KR mice. These in vivo results underscore the need to test the downstream consequences of protein modifications in a physiologic setting. To meet this fundamental objective, new in vivo studies are described to investigate the recently proposed role of NEMO as a sensor of K63-linked polyubiquitin chains, which may facilitate the formation of activated immunoreceptor complexes and downstream signal transmission. A germline point mutation will be engineered in the mouse gene encoding NEMO that disrupts its capacity to bind K63-linked chains, enabling us to investigate biochemical functions of the NEMO ubiquitin-binding (NUB) domain in primary rather than transformed cells (Aim 1). Phenotypic analyses of the corresponding knock-in mice will reveal the physiologic role of the NUB domain in innate versus adaptive immune responses mediated by TLR, AgR, and cytokine receptor signaling (Aim 2). Together with our studies of NEMO-KR mice, results from the proposed project will provide a strong in vivo framework for understanding the full workscope of NEMO as both a target and a sensor of K63-linked ubiquitination in the mammalian immune system. PUBLIC HEALTH RELEVANCE: Signal transmission within cells of the immune system coordinates the host defense against microbial pathogens and must be tightly regulated to avoid chronic inflammation. Recent in vitro experiments suggest that signal transmission involves the interaction of intracellular proteins with ubiquitin chains. New in vivo studies are proposed to investigate the physiologic function of a specific ubiquitin-binding protein, the relevance of this ubiquitin sensing mechanism to human health, and the potential for treating inflammation-based disease at the level of ubiquitination.