Novel biochemical and functional targets of PARP

项目摘要 辅助性T细胞17亚群在防御微生物方面起着重要作用，但在免疫系统中也是致病性的。 炎性疾病，如实验性自身免疫性脑脊髓炎（EAE），一种用于 生成用于分析多发性硬化症（MS）的线索。靶向特异性细胞因子产物（例如，IL-17、IL-22） 各种形式的Th17细胞，或对分化效率至关重要的精氨酸-受体相互作用，或 Th17池（IL-23）的扩增可能被证明有助于MS，但这些干预措施可能被证明缺乏 足够的功效或过于有效地削弱宿主防御。因此，在发现 Th17亚群调节和发挥作用的新机制。 信号传导机制为治疗免疫缺陷提供了新的治疗方法。 介导的疾病。我们已经发现哺乳动物细胞内ADP-核糖基转移酶（ART）， PARP14促进几种辅助性T细胞亚群的分化。重要的是，其影响Th17的能力 分化依赖于内在的ART活性。这些发现对一些人来说尤其值得注意。 原因首先，这种蛋白质影响T辅助细胞分化的方式与PARP1非常不同， 事实上，尽管PARP1和它的几个亲戚可以催化分支， 在将初始加合物置于靶蛋白上后，ADP-核糖（ADPr）的聚合物，PARP14似乎不能 作为聚合酶起作用，而是ADP-核糖基单转移酶（mART）。因此，调查结果 打开了一个全新的视野，了解生理调节是如何影响的翻译后 迄今为止，在正常哺乳动物生物学中几乎没有研究过这种修饰。二、微生物病原体 通常利用或破坏宿主细胞中的信号传导机制。事实上，许多细菌外毒素的功能， 通过在被引入后向细胞内蛋白质添加ADP-核糖来使哺乳动物细胞中毒 事实上这些毒素会引发Th17反应因此，拟议的工作可能会阐明 例如百日咳毒素利用的内源途径。 以这些观点为背景，我们将确定EAE的疾病严重程度是否受到ART的影响 通过使用骨髓转移和将活性或非活性PARP14转导到细胞中的PARP14活性 (Aim 1），并确定EAE对PARP14依赖性的关键淋巴细胞类型（目的2）。此外，我们将 使用加合物标签和蛋白质组学来鉴定PARP 14依赖的ADP核糖基化分子靶点， CD4 T细胞，并确定这些靶标与百日咳毒素（PT）的那些靶标的重叠（目的3）。的 这项工作的综合结果将最终确定对分子调控的令人兴奋的新见解，并奠定 为更全面和持续地阐明这些进程奠定了基础。

Project Summary The T helper 17 subset plays important roles in defense against microbes but also is pathogenic in inflammatory diseases such as experimental autoimmune encephalomyelitis (EAE), a mouse model used to generate leads for analysis of multiple sclerosis (MS). Targeting specific cytokine products (e.g., IL-17, IL-22) of the various forms of Th17 cells, or cytokine-receptor interactions vital for the efficiency of differentiation or expansion of a Th17 pool (IL-23), may prove to help MS but these interventions may prove either to lack sufficient efficacy or to impair host defenses too effectively. As such, there is great intrinsic value in discovering new mechanisms by which the Th17 subset is regulated and functions. Signaling mechanisms offer opportunities for new therapeutic approaches to treatments for immune- mediated disorders. We have discovered that a mammalian intracellular ADP-ribosyl transferase (ART), PARP14, promotes the differentiation of several T helper subsets. Importantly, its capacity to impact Th17 differentiation is dependent on the intrinsic ART activity. These findings are particularly notable for several reasons. First, the ways in which this protein affects T helper differentiation are quite different from PARP1, the most-studied mammalian ART. Indeed, whereas PARP1 and several of its relatives can catalyze branching polymers of ADP-ribose (ADPr) after placement of an initial adduct on target proteins, PARP14 appears unable to function as a polymerase and instead is an ADP-ribosyl mono-transferase (mART). As such, the findings open entirely new vistas for understanding how physiological regulation is effected by a post-translational modification that until now has been little studied in normal mammalian biology. Second, microbial pathogens often exploit or subvert signaling mechanisms in host cells. Indeed, a number of bacterial exotoxins function by intoxicating the mammalian cells through addition of ADP-ribose to intracellular proteins after being introduced inside, and in fact these toxins elicit Th17 responses. The proposed work may, therefore, shed light on endogenous pathways exploited by, for instance, pertussis toxin. With these points as backdrop, we will determine if disease severity in EAE is influenced by the ART activity of PARP14 by using bone marrow transfers and transduction of active or inactive PARP14 into cells (Aim 1), and identify lymphocyte types critical for the dependence of EAE on PARP14 (Aim 2). Further, we will use adduct tagging and proteomics to identify PARP14-dependent molecular targets for ADP-ribosylation in CD4 T cells and determine the overlap of these targets with those of pertussis toxin (PT) (Aim 3). The combined results from this work would finalize an exciting new insight into molecular regulation and lay the foundations for a more comprehensive and sustained elucidation of these processes.