Negative regulation of NF-kB and inflammation by TAX1BP1

描述（由申请人提供）：NF-kB转录因子在涉及免疫、炎症、发育和细胞存活的基因调控中起核心作用。NF-kB被促炎细胞因子如肿瘤坏死因子（TNF）或白细胞介素-1 （IL-1）和细菌产物如脂多糖（LPS）激活通常是短暂的，因为多种负反馈抑制剂上调，包括IkBa和A20（也称为TNFAIP3）。A20是一种泛素编辑酶，通过靶向TRAF6和RIP1等关键信号分子灭活，在TNF和LPS触发的NF-kB应答的终止过程中发挥重要作用。持续的NF-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。该提案的重点是细胞蛋白TAX1BP1（也称为T6BP或TXBP151），这是一种已知的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敲除小鼠。虽然纯合子缺失小鼠不能存活，但来自这些小鼠的小鼠胚胎成纤维细胞（mef）在TNF、IL-1和LPS刺激下表现出持续升高的NF-kB和c-jun n -末端激酶（JNK）激活。TAX1BP1作为RIP1和TRAF6泛素化的负调节因子，作为adaptor分子是A20功能和特异性所必需的。此外，我们发现TAX1BP1通过两个PPXY基序将HECT E3连接酶Itch募集到A20上。在促炎细胞因子刺激下，Itch-/- mef同样表现出NF-kB和JNK信号终止的缺陷。因此，TAX1BP1和Itch都是限制炎症信号通路的A20泛素编辑蛋白复合物的重要亚基。最后，我们证明了TAX1BP1在响应促炎信号时经历ikka依赖性磷酸化。tnf诱导的TAX1BP1、Itch、A20和RIP1之间的相互作用需要IKKa，但不需要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。在本提案中，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机制。本提案的具体目的是确定(1)TAX1BP1抑制持续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。上述研究的完成将提供NF-kB信号负调控的机制，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作为A20泛素编辑酶复合物的亚基，是NF-kB持续激活的关键负调控因子。该提案将提供关于TAX1BP1， Itch和A20如何共同作用以灭活关键信号分子并限制炎症信号通路的机制见解。

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.