Understanding how TRAF6 controls T lymphocyte activation

TRAF 6（TNF受体相关因子6）的表达对于许多生物过程是必需的，所述生物过程包括先天免疫系统的信号网络、破骨细胞和骨形成的产生、淋巴结器官发生以及毛囊、汗液和皮脂腺的发育。TRAF 6也是树突状细胞、B细胞和T细胞功能所必需的。先天免疫领域的一个原则是TRAF 6（一种E3泛素连接酶活性）的基本作用是产生Lys 63连接的泛素链，然后激活系统的“主激酶”TAK 1。最近，我们的一个实验室（PC）发现，这种长期持有的观点是不正确的，因为先天免疫信号可以通过在TRAF 6敲除（KO）细胞中重新表达TRAF 6的E3连接酶失活突变体来恢复。这些研究还表明TRAF 6的E3连接酶活性不是必需的，因为另外两种E3连接酶Pellino 1和Pellino 2也能够产生信号传导所需的Lys 63连接的泛素链。这些研究的一个重要结论是，TRAF 6在先天免疫中的重要作用不依赖于其E3连接酶活性（1）。PC实验室还产生了敲入小鼠，其中TRAF 6被E3连接酶失活的TRAF 6 [L74 H]突变体取代。相比之下，TRAF 6 KO小鼠骨骼变形且无牙齿，而TRAF 6 [L74 H]小鼠骨骼和牙齿正常，因为TRAF 6 [L74 H]细胞中破骨细胞形成所需的RANKL信号通路未受损（1）。这些令人瞩目的发现提出了一个问题，即TRAF 6 E3连接酶的生理作用可能是什么？先前的研究表明，TRAF 6在抑制T细胞效应功能方面具有关键作用（2）。因此，TRAF 6的T细胞选择性缺失导致T细胞功能的失调和T细胞介导的自身免疫的发展。PC实验室对TRAF 6 [L74 H]小鼠的分析表明，它们在淋巴结和脾脏中具有过度活跃的T细胞。此外，这些小鼠发展自身免疫性炎性疾病，这表明TRAF 6的E3泛素连接酶活性在T细胞中具有抑制细胞效应子功能的关键作用。由于DC的实验室在T细胞信号传导领域非常突出，PC和DC实验室建议共同监督博士生，他们将阐明TRAF 6限制T细胞活化的分子机制。该项目的第一部分将探索TRAF 6 [L74 H]小鼠次级淋巴组织、肠道和肺中效应和调节性T细胞的发育。它还将探索TRAF 6 [L74 H] T细胞介导对病原体和癌症的体内免疫应答的能力及其在自身免疫中的作用。第二部分将研究TRAF 6激活抑制T细胞功能的机制，原则上，这可能发生在T细胞信号通路的任何步骤。Pellino同种型可能与TRAF 6冗余地起作用，并且该项目可能受益于PC实验室开发的Pellino敲入小鼠的可用性，其中所有三种Pellino都被E3连接酶失活突变体单独和组合取代。已经描述了TRAF 6表达是CD 4 T细胞中IL-18信号传导所需的，并抑制TGF介导的IL-2抑制（3）。因此，我们将首先关注TRAF 6如何有助于这些细胞因子控制T细胞生物学的信号通路。一个基本的策略将是使用高分辨率质谱法来精确地绘制IL-18如何调节野生型和TRAF 6 [L74 H] CD 4 T细胞的蛋白质组、磷酸化蛋白质组和泛素组。由这些细胞因子控制的信号通路如何整合以控制CD 4 T细胞的细胞反应在分子水平上知之甚少，DC和PC实验室的互补专业知识可能使这个问题的重要方面得以破解。

The expression of TRAF6 (TNF Receptor-Associated Factor 6) is essential for many biological processes, which include signaling networks of the innate immune system, the production of osteoclasts and bone formation, lymph node organogenesis, and the development of hair follicles, sweat and sebaceous glands. TRAF6 is also needed for dendritic cell, B cell and T-cell function. It is a tenet of the field of innate immunity that the essential role of TRAF6, an E3 ubiquitin ligase activity, is to generate Lys63-linked ubiquitin chains, which then activate TAK1, the "master kinase" of the system. Recently, one of our laboratories (PC) found that this long-held view is incorrect, since innate immune signaling could be restored by the re-expression of E3 ligase-inactive mutants of TRAF6 in TRAF6 knock-out (KO) cells. These studies also revealed that the E3 ligase activity of TRAF6 was not essential because two other E3 ligases, Pellino1 and Pellino2, are also able to generate the Lys63-linked ubiquitin chains required for signaling. An important conclusion from these studies was that the essential roles of TRAF6 in innate immunity are independent of its E3 ligase activity (1). The PC lab also generated knock-in mice in which TRAF6 was replaced by the E3 ligase-inactive TRAF6[L74H] mutant. In contrast, to TRAF6 KO mice that have deformed bones and no teeth, the TRAF6[L74H] mice have normal bones and teeth, because the RANKL signaling pathway required for osteoclast formation is unimpaired in TRAF6[L74H] cells (1). These remarkable findings have raised the question of what the physiological role of the TRAF6 E3 ligase might be? Previous studies showed that TRAF6 has a crucial role to restrain T cell effector function (2). Hence T cell selective deletion of TRAF6 results in deregulation of T cell function and the development of T cell mediated autoimmunity. Analysis of TRAF6[L74H] mice by the PC laboratory indicates that they have hyperactive T cells in lymph nodes and spleens. Moreover, these mice develop autoimmune inflammatory diseases indicating that the E3 ubiquitin ligase activity of TRAF6 has a critical role in T cells to restraint cell effector function. Since DC's lab is pre-eminent in the field of T cell signaling, the PC and DC labs propose to jointly supervise the PhD student who will elucidate the molecular mechanism by which TRAF6 restricts T cell activation. The first part of the project will explore the development of effector and regulatory T cells in the secondary lymphoid tissues, gut and lung of TRAF6[L74H] mice. It will also explore the ability of TRAF6[L74H] T cells to mediate in vivo immune responses to pathogens and cancer and their role in autoimmunity. The second part will investigate the mechanisms by which TRAF6 activation suppresses T cell function which, in principle, could occur at any step in T cell signaling pathways. Pellino isoforms may function redundantly with TRAF6 and the project may benefit from the availability of Pellino knock-in mice developed in PC's laboratory, in which all three Pellinos have been replaced by E3 ligase-inactive mutants, alone and in combination. It has been described that the TRAF6 expression is required for IL-18 signaling in CD4 T cells and inhibits TGF-mediated suppression of IL-2 (3). We will therefore focus initially on how TRAF6 contributes to the signalling pathways used by these cytokines to control T cell biology. A fundamental strategy will be to use high resolution mass spectrometry to precisely map how IL-18 regulates the proteome, phosphoproteome and ubiquitome of wild type and TRAF6[L74H] CD4 T cells. How the signaling pathways controlled by these cytokines are integrated to control the cellular response of CD4 T cells is poorly understood at the molecular level and the complementary expertise of the DC and PC labs may enable important aspects of this problem to be cracked.