Novel biochemical and functional targets of PARP

描述(申请人提供)：T辅助17亚群在抵御微生物方面发挥重要作用，但也是炎症性疾病的病原体，如实验性自身免疫性脑脊髓炎(EAE)，这是一种用于分析多发性硬化症(MS)的小鼠模型。针对各种形式的Th17细胞的特定细胞因子产物(如IL-17、IL-22)，或对Th17池(IL-23)的分化效率至关重要的细胞因子-受体相互作用，可能被证明对MS有帮助，但这些干预可能被证明缺乏足够的疗效或过于有效地损害宿主防御。因此，发现Th17亚群的调节和功能的新机制具有GRAT内在价值。信号机制为治疗免疫介导性疾病提供了新的治疗方法。我们发现哺乳动物细胞内ADP-核糖基转移酶(ART)PARP14促进几个辅助性T细胞亚群的分化。重要的是，其影响Th17分化的能力依赖于固有的ART活性。这些发现之所以特别值得注意，有几个原因。首先，以什么方式 这种影响T辅助细胞分化的蛋白与研究最多的哺乳动物ART蛋白PARP1有很大不同。事实上，虽然PARP1及其几个近亲可以催化ADP-核糖(ADPr)的分支聚合物，但PARP14似乎不能作为聚合酶发挥功能，而是一种ADP-核糖基单一转移酶(MART)。因此，这些发现为理解翻译后修饰如何影响生理调节开辟了全新的前景，到目前为止，在正常哺乳动物生物学中对翻译后修饰的研究很少。其次，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，许多细菌外毒素在进入细胞内后，通过将ADP-核糖添加到细胞内蛋白质中而使哺乳动物细胞中毒，事实上这些毒素可以激发Th17反应。因此，这项拟议的工作可能会揭示例如百日咳毒素所利用的内源途径。以这些点为背景，我们将通过骨髓移植和将活性或非活性的PARP14转导到细胞中来确定EAE的疾病严重程度是否受到PARP14的ART活性的影响(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，我们将使用加合物标签和蛋白质组学来确定CD4T细胞中依赖PARP14的ADP核糖化的分子靶点，并确定这些靶点与百日咳毒素(PT)的重叠(目标3)。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，并为更全面和持续地阐明这些过程奠定基础。

DESCRIPTION (provided by applicant): 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 r 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 grat 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 n 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.