Decoding protein MARylation networks in astrocytes using chemical biology approaches

项目摘要 星形胶质细胞是中枢神经系统(CNS)天然免疫的重要调节者。中枢神经系统的刺激 神经炎性激活物如病原体和脑损伤的先天免疫，以及对 神经变性，导致星形胶质细胞经历一种称为星形胶质细胞增生症的反应性表型的转变。当它 已被公认为星形胶质细胞增多症可以作为一种保护机制将中枢神经系统的损害降至最低 调节星形胶质细胞增多症的机制还不是很清楚。我们的初步结果和来自文献的数据支持 我们的一般假设是，PARP7控制的MAR化关键地塑造了先天免疫反应 中枢神经系统。我们的长期目标是了解PARP7在星形胶质细胞增生症中的作用，以及PARP7是否代表 作为中枢神经系统先天免疫激活的结果而产生的中枢神经系统病理的可操作靶点。这个 这项工作的目的是阐明PARP7调节先天性免疫的机制。 星形胶质细胞。PARP7已经成为一个被称为PAPS的大酶家族中至关重要的成员， 尤其是在先天免疫反应中。与其他PARP家族成员类似，PARP7催化POST- 翻译修饰称为单-ADP-核糖基化(MAR化)，它涉及到 ADP-核糖从NAD+转变为靶蛋白上的氨基酸。星形胶质细胞中PARP7的MAR化靶点是 未知。为了破译PARP7调节星形胶质细胞天然免疫的机制，我们需要 确定PARP7在星形胶质细胞中的直接靶点。确定PARP7的直接目标一直具有挑战性， 然而，由于PAP共享相同的底物NAD+。为了克服这一限制，我们描述了 PARP7-NAD+工程正交化模拟对的研制 星形胶质细胞裂解物中的PARP7(目标I)。我们还描述了我们的薄膜变种的产生 正交NAD+类似物，对于使用刺激识别完整星形胶质细胞中的PARP7靶点至关重要 激活星形胶质细胞的先天免疫反应(AIM II)。最后，我们描述了一种改进策略 PARP7抑制剂的选择性(目标III)。PARP7选择性抑制剂是重要的化学探针 评估PARP7介导的MAR化在星形胶质细胞天然免疫反应中的作用。我们 预计这些研究不仅将澄清我们对PARP7介导的功能的理解 星形胶质细胞先天免疫中的MAR化，但也可能导致中枢神经系统的新治疗策略 病理学，特别是神经炎性疾病(如多发性硬化症)和神经退行性疾病(如 阿尔茨海默病)。更广泛地说，这些研究的结果将对我们的 了解细胞信号转导中的MAR化。

Project Summary Astrocytes are critical regulators of innate immunity in the central nervous system (CNS). Stimulation of CNS innate immunity by neuroinflammatory activators such as pathogens and brain injury, as well as in response to neurodegeneration, cause astrocytes to undergo a transition to a reactive phenotype called astrogliosis. While it is well accepted that astrogliosis can act as a protective mechanism to minimize CNS damage, the mechanisms that regulate astrogliosis are not well understood. Our preliminary results and data from the literature support our general hypothesis that PARP7 controlled MARylation critically shapes the innate immune responses in the CNS. Our long-term goal is to understand the role of PARP7 in astrogliosis and whether PARP7 represents an actionable target for CNS pathologies that arise as a consequence of activation of CNS innate immunity. The objective of the proposed work is elucidate the mechanisms by which PARP7 regulates innate immunity in astrocytes. PARP7 has emerged as a critically important member of a large enzyme family known as PARPs, especially in the innate immune response. Similar to other PARP family members, PARP7 catalyzes the post- translational modification known as mono-ADP-ribosylation (MARylation), which involves the transfer of ADP-ribose from NAD+ to amino acids on target proteins. The MARylation targets of PARP7 in astrocytes are unknown. To decode the mechanisms by which PARP7 regulates innate immunity in astrocyte, we need to identify the direct targets of PARP7 in astrocytes. Identifying the direct targets of PARP7 has been challenging, however, due to the fact that PARPs share the same substrate NAD+. To overcome this limitation, we describe the development of engineered PARP7—orthogonal NAD+ analogue pairs for identifying the direct targets of PARP7 in astrocytes lysates (Aim I). We also describe the generation of membrane-permeant variants of our orthogonal NAD+ analogues, which are critical for identifying PARP7 targets in intact astrocytes using stimuli that activate the innate immune response in astrocytes (Aim II). Lastly, we describe a strategy for improving the selectivity of PARP7 inhibitors (Aim III). Selective inhibitors of PARP7 are essential chemical probes for evaluating the function of PARP7-mediated MARylation in the innate immune response in astrocytes. We anticipate that these studies will not only clarify our understanding of the function of PARP7-mediated MARylation in innate immunity in astrocytes, but could also lead to new therapeutic strategies for CNS pathologies, particularly neuroinflammatory (e.g. multiple sclerosis) and neurodegenerative diseases (e.g. Alzheimer's disease). More generally, the results obtained from these studies will have far-reaching impact on our understanding of MARylation in cell signaling.