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

描述(由申请人提供)：免疫受体介导的转录因子核因子？B和MAPKs(MAPKs)的激活对于正确调节参与先天性和获得性免疫反应的多个基因是必不可少的。这些信号通路的不适当的慢性激活可能导致细胞因子的过度产生和炎症驱动的疾病，如关节炎、狼疮和癌症。先前的无细胞系统和转化细胞系的体外实验表明，Lys-63(K63)连接的肿瘤坏死因子受体相关因子6(TRAF6)、受体相互作用蛋白1(RIP)和I？B激酶Nemo亚单位(IKK)在免疫受体信号转导中起着关键的调节作用。目前尚不清楚这些在体外确定的修饰步骤是否是体内治疗干预的有吸引力的靶点。例如，体外研究表明，K63连接的NEMO泛素化对淋巴细胞中的抗原受体(AGR)信号至关重要。然而，申请人实验室的初步结果表明，AGR信号在携带NEMO泛素化缺陷突变(NEMO-KR)的“敲入”小鼠中完全起作用。相反，依赖Toll样受体(TLR)的巨噬细胞和树突状细胞分泌的细胞因子在Nemo-KR小鼠中显著受损。这些体内结果强调了在生理环境中测试蛋白质修饰的下游后果的必要性。为了达到这一基本目标，我们描述了新的体内研究，以探讨最近提出的NEMO作为K63连接的多泛素链的传感器的作用，这可能有助于激活的免疫受体复合体的形成和下游信号传递。将在编码NEMO的小鼠基因中设计生殖线点突变，破坏其与K63连接链的结合能力，使我们能够在原代细胞而不是转化细胞中研究NEMO泛素结合(NUB)结构域的生化功能(目标1)。对相应的敲入小鼠的表型分析将揭示NUB结构域在TLR、AGR和细胞因子受体信号转导(AIM 2)介导的先天性免疫反应和获得性免疫反应中的生理作用。结合我们对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相关泛素化的靶点和传感器的全部工作范围提供一个强有力的体内框架。与公共健康相关：免疫系统细胞内的信号传输协调宿主对微生物病原体的防御，必须严格监管，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。新的体内研究被提出，以调查特定的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疾病的可能性。

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.