Novel biochemical and functional targets of PARP

描述（由申请人提供）：T 辅助细胞 17 子集在抵御微生物方面发挥着重要作用，但在炎症性疾病中也具有致病性，例如实验性自身免疫性脑脊髓炎 (EAE)，这是一种用于生成用于分析多发性硬化症 (MS) 的引线的小鼠模型。针对各种形式的 Th17 细胞的特定细胞因子产物（例如 IL-17、IL-22），或对 Th17 库（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，但这些干预措施可能被证明要么缺乏足够的功效，要么过于有效地损害宿主防御。因此，发现 Th17 子集的调节和功能新机制具有巨大的内在价值。 信号传导机制为治疗免疫介导疾病的新治疗方法提供了机会。我们发现哺乳动物细胞内 ADP-核糖基转移酶 (ART) PARP14 可促进多种 T 辅助细胞亚群的分化。重要的是，其影响 Th17 分化的能力取决于内在的 ART 活性。由于几个原因，这些发现特别值得注意。首先，可以采取哪些方式 这种影响T辅助细胞分化的蛋白质与研究最多的哺乳动物ART PARP1有很大不同。事实上，虽然 PARP1 及其几个相关蛋白在将初始加合物置于目标蛋白上后可以催化 ADP-核糖 (ADPr) 的分支聚合物，但 PARP14 似乎无法充当聚合酶，而是 ADP-核糖基单转移酶 (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，而迄今为止，在正常哺乳动物生物学中对翻译后修饰的研究还很少。其次，微生物病原体经常利用或破坏宿主细胞中的信号传导机制。事实上，许多细菌外毒素通过在引入哺乳动物细胞后将ADP-核糖添加到细胞内蛋白质而使哺乳动物细胞中毒而起作用，并且事实上这些毒素引发Th17反应。因此，拟议的工作可能有助于阐明百日咳毒素等内源性途径。 以这些要点为背景，我们将通过骨髓移植和将活性或非活性 PARP14 转导到细胞中来确定 EAE 疾病的严重程度是否受到 PARP14 ART 活性的影响（目标 1），并确定对于 EAE 对 PARP14 依赖性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识别 CD4 T 细胞中 ADP 核糖基化的 PARP14 依赖性分子靶标，并确定这些靶标与百日咳毒素 (PT) 的重叠（目标 3）。这项工作的综合结果将最终对分子调控产生令人兴奋的新见解，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.