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

TRAF6 作为 K63 连接多泛素化靶点的体内功能

• 批准号: 7641802
• 负责人: DEAN BALLARD
• 金额: $ 19.34万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-22 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7641802
• 关键词: Acute; Affect; Agonist; Antibodies; Antigen Presentation; Antigen Receptors; Antigens; Area; B-Lymphocytes; Bacteria; Biochemical; Bone Marrow; CD40 Ligand; Cell Lineage; Cell Surface Receptors; Cell model; Cell surface; Cells; Chronic; Complement; Complex; Couples; Data; Defect; Dendritic Cells; Deubiquitinating Enzyme; Development; Disease; Docking; Dose; Endotoxic Shock; Engineering; Ensure; Family member; Genes; Germ-Line Mutation; Health; Hematopoiesis; Homeostasis; Host Defense; Human; Immune; Immune response; Immune system; Immunity; Immunoglobulins; In Vitro; Infection; Inflammation; Injury; Interleukin-1 Receptors; Intervention; Knock-in Mouse; Knowledge; Lead; Left; Link; Location; Lymphocyte; Lymphocyte Function; Lymphoid; Measures; Mediating; Modification; Molecular; Monitor; Mus; Mutation; Natural Immunity; Nuclear; Organ; Osteoclasts; Pathologic; Pathway interactions; Pattern; Peripheral; Phenotype; Phosphotransferases; Physiological; Play; Point Mutation; Polyubiquitination; Post-Translational Protein Processing; Process; Production; Protein Kinase; Proteins; Proteolysis; Receptor Signaling; Reporting; Research; Role; Serum; Signal Pathway; Signal Transduction; Site; Staging; System; T-Lymphocyte; TNF Receptor-Associated Factors; TNFRSF5 gene; Testing; Tissues; Toll-like receptors; Transcription Factor AP-1; Transfection; Tumor Necrosis Factor Receptor; Ubiquitin; Ubiquitination; Upper arm; Virus; activating transcription factor; antimicrobial; base; bone metabolism; clinically relevant; cytokine; drug discovery; in vitro Model; in vivo; insight; macrophage; meetings; member; microbial; mortality; mutant; novel therapeutics; osteoclastogenesis; pathogen; prevent; protein degradation; protein expression; public health relevance; receptor; receptor-mediated signaling; research study; response; therapeutic target; transcription factor; transmission process; ubiquitin ligase; ubiquitin-protein ligase

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.

描述（由申请方提供）：来自白细胞介素-1受体/Toll样受体（IL-1 R/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。肿瘤坏死因子相关因子6（TRAF 6），一种泛素（Ub）-蛋白连接酶（E3），支持细菌和病毒表达的病原体相关分子模式（PAMP）的许多TLR依赖性免疫应答。如果不加以控制，这些TRAF 6依赖性反应可能导致过度的细胞因子产生，组织损伤和炎症的其他病理后果。TRAF 6还介导NF-？B（RANK）和CD 40，TNFR家族成员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，赖氨酸-63（K63）连接的TRAF 6泛素化在其作用机制中起着关键作用，通过创建一个细胞溶质蛋白激酶的对接位点，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TRAF 6遍在蛋白化的生理功能和临床相关性仍未得到测试。与TRAF 6相似，NF-？B必需调节因子（NEMO）受到K63连接的泛素化。先前用转化的T细胞进行的实验表明，这种对NEMO的UB修饰调节抗原受体（AgR）向NF-：B的信号传导。然而，在体内实验报告显示受损的TLR，而不是AgR信号在“敲入”小鼠窝藏点突变，删除Ub受体位点的NEMO。这一发现强调了在生理环境中测试Ub缀合的下游后果的需要，并建立了确定TRAF 6泛素化的体内意义的互补项目的可行性。使用类似的敲入方法，小鼠将被工程化以携带选择性阻断TRAF 6的K63连接的泛素化但不阻断其Ub连接酶活性的突变（Aim 1）。后续表型研究将重点关注与TRAF 6缺乏相关的缺陷，这些缺陷影响造血和破骨细胞生成（目标1）、先天免疫与病理性炎症（目标2）和适应性免疫（目标3）。NF-κ B免疫受体信号转导的生物化学研究B和AP-1将使用受种系突变影响的原代细胞进行。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TRAF 6水平上定义K63连接的泛素化的功能，这是通过常规转染方法无法实现的。从拟议的项目中获得的体内数据将推进关于TRAF 6-Ub缀合物的功能工作范围的知识，远远超出目前体外系统的限制，这是评估K63连接的多聚泛素化作为炎症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与细胞内蛋白TRAF 6的连接，这反过来又刺激宿主防御基因的表达。提出了新的体内研究来研究这种特定蛋白质修饰的生理功能，其与人类健康的相关性，以及在TRAF 6泛素化水平上治疗炎症性疾病的潜力。