Novel biochemical and functional targets of PARP

项目摘要 辅助性T细胞17亚群在抵抗微生物中发挥重要作用，但也是致病的 炎症性疾病，如实验性自身免疫性脑脊髓炎(EAE)，一种用于 为多发性硬化症(MS)分析提供线索。靶向特定的细胞因子产物(如IL-17、IL-22) 各种形式的Th17细胞，或对分化效率至关重要的细胞因子-受体相互作用 Th17池(IL-23)的扩大可能会证明对MS有帮助，但这些干预可能会证明要么缺乏 足够的效力或过于有效地削弱宿主防御。因此，发现有很大的内在价值 Th17亚群被调节和发挥作用的新机制。 信号机制为免疫治疗提供了新的治疗方法。 介导性障碍。我们发现哺乳动物细胞内的ADP-核糖基转移酶(ART)， PARP14促进几个T辅助细胞亚群的分化。重要的是，它影响Th17的能力 分化依赖于内在的艺术活动。这些发现在以下几个方面尤其值得注意 理由。首先，这种蛋白影响T辅助细胞分化的方式与PARP1非常不同，PARP1是 研究最多的哺乳动物艺术。事实上，尽管PARP1及其几个近亲可以催化分支 ADP-核糖聚合物(ADPr)在将初始加合物放置在靶蛋白上后，PARP14似乎不能 作为聚合酶发挥作用，而不是ADP-核糖基单一转移酶(MART)。因此，这些发现 为理解翻译后如何影响生理调节开辟了全新的视野 到目前为止，在正常哺乳动物生物学中对这种修饰的研究很少。第二，微生物病原体 经常利用或颠覆宿主细胞中的信号机制。事实上，许多细菌外毒素的作用方式是 ADP-核糖引入细胞内蛋白后对哺乳动物细胞的毒性作用 在体内，事实上这些毒素会引发Th17反应。因此，拟议的工作可能会对 例如，百日咳毒素所利用的内源途径。 以这些点为背景，我们将确定EAE的疾病严重程度是否受到ART的影响 利用骨髓移植和将活性或失活的PARP14转导入细胞中的PARP14活性 (目标1)，并确定对EAE依赖PARP14至关重要的淋巴细胞类型(目标2)。此外，我们还将 利用加合物标签和蛋白质组学鉴定依赖于PARP14的ADP核糖化的分子靶点 CD4T细胞，并确定这些靶点与百日咳毒素(PT)的靶点重叠(目标3)。这个 这项工作的综合结果将最终敲定对分子调控的令人兴奋的新见解，并为 为更全面和持续地阐明这些过程奠定了基础。

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.