Negative regulation of NF-kB and inflammation by TAX1BP1

描述(由申请人提供)：核因子-kB转录因子在免疫、炎症、发育和细胞生存相关基因的调控中发挥核心作用。由于多种负反馈抑制物如IkBA和A20(也称为TNFAIP3)的上调，促炎症细胞因子如肿瘤坏死因子(TNF)或白介素1(IL-1)以及细菌产物如脂多糖(LPS)对核因子-kB的激活通常是短暂的。A20是一种泛素编辑酶，通过靶向TRAF6和RIP1等关键信号分子使其失活，在终止由肿瘤坏死因子和内毒素触发的核因子-kB反应中发挥重要作用。持续的核因子-kB激活可能导致慢性炎症、自身免疫或恶性肿瘤等病理后果。这项提案的重点是一种细胞蛋白TAX1BP1(也称为T6BP或TXBP151)，它是一种已知的A20、TRAF6和HTLV-I Tax癌蛋白的相互作用蛋白。为了阐明TAX1BP1的生理功能，我们通过基因捕获策略产生了基因敲除小鼠。虽然纯合子缺失小鼠不能存活，但这些小鼠的胚胎成纤维细胞(MEF)在肿瘤坏死因子、IL-1和内毒素的刺激下，表现出高水平和持续的核因子-kB以及c-Jun氨基末端激酶(JNK)的激活。TAX1BP1作为RIP1和TRAF6泛素化的负调控因子，是A20作为适配分子发挥功能和特异性所必需的。此外，我们还发现TAX1BP1通过两个PPXY基序将Hect E3连接酶Ich招募到A20。瘙痒-/-MEF类似地表现出在对促炎症细胞因子刺激的反应中终止NF-kB和JNK信号的缺陷。因此，TAX1BP1和Itch都是限制炎症信号通路的A20泛素编辑蛋白复合体的重要亚基。最后，我们证明了TAX1BP1在致炎信号的反应中经历了IKKA依赖的磷酸化。在肿瘤坏死因子诱导的TAX1BP1、Itch、A20和RIP1之间的相互作用中，Ikka是必需的，而不是IKKB或Nemo。因此，TAX1BP1的Ikka磷酸化是促进A20泛素编辑复合体组装和功能的关键负反馈机制。在这项研究中，我们将进一步明确TAX1BP1在细胞因子诱导的NF-kB和JNK激活中的负调控作用的机制。该建议的具体目的是确定(1)TAX1BP1抑制持续的NF-kB激活的分子机制；(2)TAX1BP1抑制TRAF2泛素化的机制；(3)磷酸化在TAX1BP1功能中的作用。拟议研究的完成将提供对核因子-kB信号如何负调控的机械性洞察，并可能导致自身免疫和慢性炎症的新疗法。公共卫生相关性：转录因子NF-kB的持续激活与自身免疫、慢性炎症和恶性肿瘤有关。我们发现，细胞蛋白TAX1BP1和Itch通过作为A20泛素编辑酶复合体的亚单位发挥作用，是持续激活NF-kB的关键负调节因子。这项提议将提供对TAX1BP1、Itch和A20如何共同作用以灭活关键信号分子和限制炎症信号通路的机械性见解。

DESCRIPTION (provided by applicant): The NF-kB transcription factor plays a central role in the regulation of genes involved in immunity, inflammation, development and cell survival. NF-kB activation by proinflammatory cytokines such as tumor necrosis factor (TNF) or interleukin-1 (IL-1), and bacterial products such as lipopolysaccharide (LPS) is normally transient due to the upregulation of multiple negative feedback inhibitors, including IkBa and A20 (also known as TNFAIP3). A20 is a ubiquitin-editing enzyme that plays an essential role in the termination of NF-kB responses triggered by TNF and LPS by targeting key signaling molecules such as TRAF6 and RIP1 for inactivation. Persistent NF-kB activation may lead to pathologic consequences such as chronic inflammation, autoimmunity or malignancy. The focus of this proposal is centered on a cellular protein TAX1BP1 (also known as T6BP or TXBP151), a known interacting protein of A20, TRAF6 and the HTLV-I Tax oncoprotein. To elucidate the physiological function of TAX1BP1, we have generated knockout mice by a gene trapping strategy. Although homozygous null mice are not viable, murine embryonic fibroblasts (MEFs) from these mice exhibit elevated and persistent NF-kB as well as c-jun N-terminal kinase (JNK) activation in response to TNF, IL-1 and LPS stimulation. TAX1BP1 functions as a negative regulator of RIP1 and TRAF6 ubiquitination and is required for the function and specificity of A20 by acting as an adaptor molecule. Furthermore, we have found that TAX1BP1 recruits the HECT E3 ligase Itch to A20 via two PPXY motifs. Itch-/- MEFs similarly exhibit a defect in the termination of NF-kB and JNK signaling in response to stimulation with proinflammatory cytokines. Thus, both TAX1BP1 and Itch are essential subunits of an A20 ubiquitin- editing protein complex that restricts inflammatory signaling pathways. Finally, we have demonstrated that TAX1BP1 undergoes IKKa-dependent phosphorylation in response to proinflammatory signals. IKKa, but not IKKb or NEMO, is required for TNF-induced interactions among TAX1BP1, Itch, A20 and RIP1. Therefore, IKKa phosphorylation of TAX1BP1 is a key negative feedback mechanism that promotes the assembly and function of the A20 ubiquitin-editing complex. In this proposal, we will further define the mechanisms of TAX1BP1 negative regulatory function in cytokine-induced NF-kB and JNK activation. The specific aims of this proposal are to determine (1) the molecular mechanisms by which TAX1BP1 inhibits persistent NF-kB activation; (2) the mechanism by which TAX1BP1 inhibits TRAF2 ubiquitination; and (3) the role of phosphorylation in TAX1BP1 function. Completion of the proposed studies will provide mechanistic insight into how NF-kB signaling is negatively regulated, and may lead to novel therapies for autoimmunity and chronic inflammation. PUBLIC HEALTH RELEVANCE: Persistent activation of the transcription factor NF-kB is associated with autoimmunity, chronic inflammation and malignancy. We have found that the cellular proteins TAX1BP1 and Itch are pivotal negative regulators of persistent NF-kB activation by functioning as subunits of an A20 ubiquitin editing enzyme complex. This proposal will provide mechanistic insight into how TAX1BP1, Itch and A20 function together to inactivate key signaling molecules and restrict inflammatory signaling pathways.