In Vivo Function of TRAF6 As a Target of K63-Linked Polyubiquitination

描述（由申请人提供）：白细胞介素-1受体/ toll样受体（IL-1R/TLR）、肿瘤坏死因子受体（TNFR）和免疫球蛋白（Ig）超家族细胞表面成员的信号转导协调宿主对微生物感染的防御。tnfr相关因子6 （TRAF6）是一种泛素（Ub）蛋白连接酶（E3），支持许多tlr依赖性免疫应答，这些免疫应答是由细菌和病毒表达的病原体相关分子模式（PAMPs）。如果不加以控制，这些依赖traf6的反应会导致过度的细胞因子产生、组织损伤和其他炎症的病理后果。TRAF6也介导NF-?受体激活剂的信号传导。TNFR家族成员B （RANK）和CD40分别参与破骨细胞和B淋巴细胞的分化和效应功能。根据最近的体外数据，Lys-63 （K63）连接的TRAF6泛素化在其作用机制中发挥关键作用，通过为细胞内蛋白激酶创建一个对接位点，将免疫受体信号传递给转录因子NF-？B和AP-1。尽管使用体外模型取得了这些进展，但TRAF6泛素化的生理功能和临床相关性仍未得到验证。与TRAF6类似，NF-？B必需调节剂（NEMO）受K63连接泛素化的影响。先前转化T细胞的实验表明，这种Ub修饰NEMO可以调节抗原受体（AgR）向NF-:B的信号传导。然而，本文报道的体内实验显示，在含有去除NEMO的Ub受体位点的点突变的“敲入”小鼠中，TLR而不是AgR信号通路受损。这一发现强调了在生理环境中测试Ub偶联的下游后果的必要性，并建立了一个补充项目的可行性，以确定TRAF6泛素化在体内的意义。使用类似的敲入方法，小鼠将被改造成具有选择性阻断k63连接的TRAF6泛素化但不阻断其ub连接酶活性的突变（Aim 1）。随后的表型研究将重点关注与TRAF6缺乏相关的缺陷，这些缺陷会影响造血和破骨细胞生成（Aim 1）、先天免疫与病理性炎症（Aim 2）和适应性免疫（Aim 3）。免疫受体对NF-?信号的生化研究B和AP-1将在受种系突变影响的原代细胞中进行。重要的是，该分析将在确保其正确的空间、时间和定量表达的条件下，在TRAF6水平上定义K63-linked泛素化的功能，这是通过常规转染方法无法实现的。从拟议的项目中积累的体内数据将进一步了解TRAF6-Ub偶联物的功能工作范围，远远超出目前体外系统的限制，这是评估k63连接多泛素化作为炎症性疾病治疗靶点的潜在价值的先决条件。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，信号传递涉及非典型泛素链附着在细胞内蛋白TRAF6上，从而刺激宿主防御基因的表达。新的体内研究被提出来研究这种特异性蛋白修饰的生理功能，它与人类健康的相关性，以及在TRAF6泛素化水平上治疗炎症性疾病的潜力。

DESCRIPTION (provided by applicant): Signal transduction from cell-surface members of the interleukin-1 receptor/Toll-like receptor (IL-1R/TLR), tumor necrosis factor receptor (TNFR), and immunoglobulin (Ig) superfamilies coordinates the host defense against microbial infections. TNFR-associated factor 6 (TRAF6), a ubiquitin (Ub)-protein ligase (E3), underpins many of the TLR-dependent immune responses to pathogen-associated molecular patterns (PAMPs) expressed by bacteria and viruses. If left unchecked, these TRAF6-dependent responses can lead to excessive cytokine production, tissue injury, and other pathological consequences of inflammation. TRAF6 also mediates signaling from receptor activator of NF-?B (RANK) and CD40, TNFR family members involved in the differentiation and effector functions of osteoclasts and B lymphocytes, respectively. According to recent in vitro data, Lys-63 (K63)-linked ubiquitination of TRAF6 plays a key role in its mechanism of action by creating a docking site for cytosolic protein kinases that relay immunoreceptor signals to transcription factors NF-?B and AP-1. Despite these advances using in vitro models, the physiologic function and clinical relevance of TRAF6 ubiquitination remains untested. Similar to TRAF6, NF-?B essential modulator (NEMO) is subject to K63- linked ubiquitination. Prior experiments with transformed T cells suggested that this Ub modification to NEMO regulates antigen receptor (AgR) signaling to NF-:B. However, in vivo experiments reported here reveal impaired TLR rather than AgR signaling in "knock-in" mice harboring a point mutation that removes the Ub acceptor site of NEMO. This discovery underscores the need to test downstream consequences of Ub conjugation in a physiologic setting and establishes the feasibility of a complementary project to determine the in vivo significance of TRAF6 ubiquitination. Using a similar knock-in approach, mice will be engineered to harbor a mutation that selectively blocks K63-linked ubiquitination of TRAF6 but not its Ub-ligase activity (Aim 1). Subsequent phenotypic studies will focus on defects associated with TRAF6 deficiency that impinge on hematopoiesis and osteoclastogenesis (Aim 1), innate immunity versus pathologic inflammation (Aim 2), and adaptive immunity (Aim 3). Biochemical studies of immunoreceptor signaling to NF-?B and AP-1 will be conducted with primary cells affected by the germline mutation. Importantly, this analysis will define the function of K63-linked ubiquitination at the level of TRAF6 under conditions that ensure its correct spatial, temporal, and quantitative expression, which cannot be achieved via conventional transfection approaches. In vivo data accrued from the proposed project will advance knowledge about the functional workscope of TRAF6-Ub conjugates well beyond the current limitations of in vitro systems, a prerequisite for assessing the potential value of K63-linked polyubiquitination as a therapeutic target in inflammation-based disease. 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 attachment of atypical ubiquitin chains to the intracellular protein TRAF6, which in turn stimulates the expression of host defense genes. New in vivo studies are proposed to investigate the physiologic function of this specific protein modification, its relevance to human health, and the potential for treating inflammation-based disease at the level of TRAF6 ubiquitination.