TRAF6 E3 LIGASE FUNCTION IN OSTEOCLASTOGENESIS

TRAF6 E3 连接酶在破骨细胞生成中的功能

• 批准号: 7462392
• 负责人: BRYANT G DARNAY
• 金额: $ 21.24万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7462392
• 关键词: Address; Albers-Schonberg disease; Binding; Chimeric Proteins; Complex; Data; Disease; Dominant-Negative Mutation; Event; Homeostasis; Homologous Gene; Immunity; In Vitro; Inflammation; Interleukin-1; Knock-out; Knockout Mice; Ligase; Link; Lysine; MAP Kinase Gene; MAP3K7 gene; MAPK14 gene; MAPK8 gene; Maintenance; Maps; Mediating; Modification; Molecular; Multienzyme Complexes; Mus; Mutation; N-terminal; Osteoclasts; Pathway interactions; Phosphotransferases; Physiological; Play; Polyubiquitin; Proteins; Proteomics; Recruitment Activity; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Site; TNF gene; TNF receptor-associated factor 6; TNFSF11 gene; TRAF6 gene; Techniques; Therapeutic; Ubiquitin; Ubiquitin-Conjugating Enzymes; Ubiquitination; bone; in vivo; insight; macrophage; monocyte; novel; osteoclastogenesis; progenitor; receptor; research study; scaffold; ubiquitin ligase; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): RANK plays a central role in the maintenance of the bone homeostasis by regulating the differentiation and activation of osteoclasts. The binding of RANKL to its receptor results in the recruitment of TRAF6, which activates the NF-(B, JNK, p38, and MAPK pathways. TRAF6 is the critical RANK adaptor molecule since TRAF6-deficient mice develop severe osteopetrosis. Recent evidence indicates that the N-terminal RING domain of TRAF6 is required for its ability to signal, most likely by functioning as an E3 ubiquitin ligase, which together with the ubiquitin conjugating enzyme complex Ubc13/Uev1A catalyzes the synthesis of a unique polyubiquitin chain linked through lysine-63 (K63). However, this unique ubiquitin modification does not target TRAF6 for degradation, but rather the auto-ubiquitination of TRAF6 serves as a scaffold to recruit molecules required for the activation of kinase complexes including TAK1 and IKK. In this application, we provide preliminary data to address the functional role of TRAF6 auto-ubiquitination in RANK signaling. We show that upon RANKL stimulation endogenous TRAF6 is auto-ubiquitinated via K63 linkages, which is critical for NF-(B activation, induction of NFATc1 and subsequent osteoclast differentiation. Endogenous TAB2 and NEMO, which are potential TRAF6 downstream signaling molecules, are also ubiquitinated upon RANK activation. TRAF6 auto-ubiquitination is dependent on its RING domain and Ubc13 and mutations in either of these proteins disrupt RANK signaling. Strikingly, we identified a single critical Ub acceptor site residue (K124) in TRAF6 that is required for TRAF6 auto-ubiquitination. Mutation of this site abolished TRAF6-mediated TAB2 recruitment, TAB2 and NEMO ubiquitination, TAK1 and IKK activation, NF-(B activation, and osteoclast differentiation. Furthermore, we present evidence that the kinase activity of TAK1 is required for the efficient activation of IKK and JNK, induction of NFATc1, and osteoclast differentiation by RANKL. Through structural mapping studies, we have identified a unique domain in the C-terminus of TAK1 that is required to interact with TAB2 and its homologue TAB3. Expression of a GFP-fusion protein consisting of the last 100 residues of TAK1 acts as a dominant negative mutant that suppresses RANKL signaling and osteoclast differentiation. These results support a novel Ub-dependent TRAF6 signaling pathway that is required for efficient osteoclast differentiation. We hypothesize that upon RANKL stimulation of osteoclast progenitors, TRAF6 functions through its ubiquitin ligase activity to coordinate the recruitment (via site-specific ubiquitination of TAB2 and NEMO) and activation of the TAB2/TAK1 and IKK complexes to activate the NF-(B pathway, which is required for terminal osteoclast differentiation. To address this hypothesis, we propose three integrated specific aims. 1) Characterize non-proteasomal ubiquitination events in primary BMM after RANKL treatment. 2) Characterize the role TRAF6 auto-ubiquitination for its ability to recruit and ubiquitinate downstream targets. 3) Determine the physiological significance of TAK1 in osteoclastogenesis by generating and characterizing conditional knock-out mice deficient in TAK1 in the monocyte/macrophage linage. Taken together, these studies will reveal the molecular mechanism by which TRAF6 through ubiquitin modification regulates osteoclast differentiation. In particular, the data gathered from these experiments may provide insights to developing therapeutic strategies for a variety of bone related disease and possibly other diseases associated with immunity and inflammation

描述（由申请方提供）：RANK通过调节破骨细胞的分化和活化在维持骨稳态中发挥核心作用。RANKL与其受体的结合导致TRAF 6的募集，TRAF 6激活NF-β B、JNK、p38和MAPK通路。TRAF 6是关键的RANK衔接分子，因为TRAF 6缺陷小鼠发生重度骨硬化症。最近的证据表明，TRAF 6的N-末端RING结构域是其信号传导能力所必需的，最有可能是作为E3泛素连接酶发挥作用，其与泛素缀合酶复合物Ubc 13/Uev 1A一起催化通过赖氨酸-63（K63）连接的独特聚泛素链的合成。然而，这种独特的泛素修饰并不靶向TRAF 6进行降解，而是TRAF 6的自身泛素化作为一种支架来募集激活激酶复合物（包括TAK 1和IKK）所需的分子。 在本申请中，我们提供了初步数据来解决TRAF 6自身泛素化在RANK信号传导中的功能作用。我们发现，RANKL刺激后，内源性TRAF 6通过K63连接自动泛素化，这对NF-β B激活、NFATc 1诱导和随后的破骨细胞分化至关重要。内源性TAB 2和NEMO是潜在的TRAF 6下游信号分子，在RANK激活后也被泛素化。TRAF 6自身泛素化依赖于其RING结构域和Ubc 13，这些蛋白质中的任何一种突变都会破坏RANK信号传导。引人注目的是，我们在TRAF 6中鉴定了TRAF 6自身泛素化所需的单个关键Ub受体位点残基（K124）。该位点的突变消除了TRAF 6介导的TAB 2募集、TAB 2和NEMO泛素化、TAK 1和IKK激活、NF-β B激活和破骨细胞分化。此外，我们提出的证据表明，TAK 1的激酶活性是必需的IKK和JNK的有效激活，诱导NFATc 1，破骨细胞分化RANKL。通过结构图谱研究，我们已经确定了一个独特的结构域在C-末端的TAK 1，需要与TAB 2和它的同源TAB 3相互作用。由TAK 1的最后100个残基组成的GFP融合蛋白的表达作为抑制RANKL信号传导和破骨细胞分化的显性负突变体。这些结果支持了一种新的Ub依赖性TRAF 6信号通路，这是有效的破骨细胞分化所必需的。 我们假设，RANKL刺激破骨细胞祖细胞后，TRAF 6通过其泛素连接酶活性发挥作用，以协调TAB 2/TAK 1和IKK复合物的募集（通过TAB 2和NEMO的位点特异性泛素化）和激活，从而激活终末破骨细胞分化所需的NF-β B通路。为了解决这一假设，我们提出了三个综合的具体目标。1)描述RANKL治疗后原发性BMM中非蛋白酶体泛素化事件的特征。2)描述TRAF 6自身泛素化在募集和泛素化下游靶点方面的作用。3)通过产生和表征单核细胞/巨噬细胞谱系中TAK 1缺陷的条件性敲除小鼠，确定TAK 1在破骨细胞生成中的生理学意义。总之，这些研究将揭示TRAF 6通过泛素修饰调节破骨细胞分化的分子机制。特别是，从这些实验中收集的数据可以为开发各种骨相关疾病以及可能与免疫和炎症相关的其他疾病的治疗策略提供见解