Understanding how TRAF6 controls T lymphocyte activation

TRAF6(肿瘤坏死因子受体相关因子6)的表达在许多生物学过程中是必不可少的，这些过程包括天然免疫系统的信号网络、破骨细胞和骨形成的产生、淋巴器官的形成以及毛囊、汗腺和皮脂腺的发育。树突状细胞、B细胞和T细胞的功能也需要TRAF6。TRAF6是一种E3泛素连接酶，它的基本作用是产生Lys63连接的泛素链，然后激活系统的“主激酶”TAK1，这是天然免疫领域的一个宗旨。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TRAF6基因敲除(KO)细胞中TRAF6的E3连接酶失活突变体的重新表达来恢复。这些研究还表明，TRAF6的E3连接酶活性不是必需的，因为另外两种E3连接酶Pellino1和Pellino2也能够产生信号所需的Lys63连接的泛素链。这些研究的一个重要结论是，TRAF6在先天性免疫中的重要作用不依赖于其E3连接酶的活性(1)。PC实验室还产生了敲入小鼠，其中TRAF6被E3连接酶失活的TRAF6[L74H]突变体取代。相比之下，对于骨骼变形且没有牙齿的TRAF6 KO小鼠，TRAF6[L74H]小鼠的骨骼和牙齿正常，因为破骨细胞形成所需的RANKL信号通路在TRAF6[L74H]细胞中没有受到损害(1)。这些引人注目的发现提出了TRAF6 E3连接酶的生理作用可能是什么的问题。以往的研究表明，TRAF6在抑制T细胞效应器功能方面具有重要作用(2)。因此，T细胞选择性缺失TRAF6导致T细胞功能的紊乱和T细胞介导的自身免疫的发展。PC实验室对TRAF6[L74H]小鼠的分析表明，它们在淋巴结和脾中有过度活跃的T细胞。此外，这些小鼠发生了自身免疫性炎症性疾病，表明TRAF6的E3泛素连接酶活性在T细胞抑制细胞效应功能中起着关键作用。由于DC的实验室在T细胞信号领域处于领先地位，PC和DC实验室建议联合监督博士生，后者将阐明TRAF6限制T细胞激活的分子机制。该项目的第一部分将探索TRAF6[L74H]小鼠次级淋巴组织、肠道和肺中效应性和调节性T细胞的发育。它还将探索TRAF6[L74H]T细胞在体内介导对病原体和癌症的免疫反应的能力及其在自身免疫中的作用。第二部分将研究TRAF6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一步。Pellino异构体可能与TRAF6一起发挥多余的功能，该项目可能受益于PC实验室开发的Pellino敲入小鼠的可用性，在PC的实验室中，所有三个Pellino都被E3连接酶失活突变所取代，无论是单独还是组合。据报道，TRAF6的表达是CD4T细胞中IL-18信号传递所必需的，并抑制了转化生长因子介导的对IL-2的抑制。因此，我们首先将重点放在TRAF6如何参与这些细胞因子控制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学来精确地绘制IL-18如何调节野生型和TRAF6[L74H]CD4T细胞的蛋白质组、磷蛋白质组和泛素组。这些细胞因子控制的信号通路如何整合来控制CD4T细胞的细胞反应在分子水平上还知之甚少，DC和PC实验室的互补专业知识可能会使这个问题的重要方面得到破解。

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.