Novel biochemical and functional targets of PARP

项目概要 T 辅助细胞 17 子集在抵御微生物方面发挥着重要作用，但在以下方面也具有致病性： 炎症性疾病，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) 的线索。靶向特定细胞因子产品（例如 IL-17、IL-22） 各种形式的 Th17 细胞，或细胞因子 - 受体相互作用对于分化或分化效率至关重要 Th17 库 (IL-23) 的扩展可能被证明有助于 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的防御。因此，发现具有巨大的内在价值 Th17 子集的调节和功能的新机制。 信号机制为免疫治疗的新治疗方法提供了机会 介导的疾病。我们发现哺乳动物细胞内 ADP-核糖基转移酶 (ART) PARP14，促进多个 T 辅助亚群的分化。重要的是，它影响 Th17 的能力 分化取决于内在的 ART 活性。这些发现对于一些人来说尤其值得注意 原因。首先，该蛋白影响 T 辅助细胞分化的方式与 PARP1 有很大不同，PARP1 是 研究最多的哺乳动物 ART。事实上，虽然 PARP1 及其几个亲戚可以催化分支 在将初始加合物置于目标蛋白上后，PARP14 似乎无法形成 ADP-核糖 (ADPr) 聚合物 作为聚合酶发挥作用，而是 ADP-核糖基单转移酶 (mART)。因此，调查结果 为理解翻译后如何影响生理调节开辟了全新的视野 迄今为止，在正常哺乳动物生物学中对这种修饰的研究还很少。二、微生物病原体 经常利用或破坏宿主细胞中的信号机制。事实上，许多细菌外毒素的作用是通过 引入 ADP-核糖后，通过将 ADP-核糖添加到细胞内蛋白质中，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性途径。 以这些要点为背景，我们将确定 EAE 的疾病严重程度是否受到 ART 的影响 通过骨髓移植和将活性或非活性 PARP14 转导至细胞中来检测 PARP14 的活性 （目标 1），并确定对 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 ADP 核糖基化的 PARP14 依赖性分子靶标 CD4 T 细胞并确定这些靶标与百日咳毒素 (PT) 靶标的重叠（目标 3）。的 这项工作的综合结果将最终得出关于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.