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）先天免疫的关键调节因子。刺激CNS 先天免疫由神经炎性激活剂，如病原体和脑损伤，以及在响应 神经变性导致星形胶质细胞转变为称为星形胶质细胞增生的反应性表型。虽然 星形胶质细胞增生可以作为一种保护机制，以尽量减少中枢神经系统的损害，这种机制是公认的， 调节星形胶质细胞增生的机制还不清楚。我们的初步结果和文献数据支持 我们的一般假设是PARP 7控制的MARylation关键地塑造了先天性免疫应答， CNS。我们的长期目标是了解PARP 7在星形胶质细胞增生中的作用，以及PARP 7是否代表星形胶质细胞增生。 作为CNS先天免疫激活的结果而出现的CNS病理的可作用靶标。的 这项工作的目的是阐明PARP 7调节先天免疫的机制， 星形胶质细胞PARP 7已经成为称为PARP的大酶家族中至关重要的成员， 尤其是先天免疫反应。与其他PARP家族成员相似，PARP 7催化后 翻译修饰称为单ADP核糖基化（MARylation），它涉及转移 ADP-核糖从NAD+到靶蛋白上的氨基酸。星形胶质细胞中PARP 7的MARylation靶标是 未知为了解码PARP 7调节星形胶质细胞先天免疫的机制，我们需要 识别星形胶质细胞中PARP 7的直接靶点。识别PARP 7的直接靶点一直具有挑战性， 然而，由于PARP共享相同底物NAD+的事实。为了克服这一限制，我们描述 开发工程化PARP 7-正交NAD+类似物对，用于鉴定 星形胶质细胞裂解物中的PARP 7（Aim I）。我们还描述了我们的膜渗透变体的产生。 正交NAD+类似物，其对于使用刺激物鉴定完整星形胶质细胞中的PARP 7靶标至关重要 激活星形胶质细胞中的先天免疫反应（Aim II）。最后，我们描述了一种改进的策略， PARP 7抑制剂的选择性（目的III）。PARP 7的选择性抑制剂是用于治疗糖尿病的重要化学探针。 评估PARP 7介导的MARylation在星形胶质细胞先天免疫应答中的功能。我们 预计这些研究不仅将澄清我们对PARP 7介导的功能的理解， MARylation在星形胶质细胞先天免疫中的作用，但也可能导致CNS疾病的新治疗策略。 病理学，特别是神经炎性（例如多发性硬化）和神经变性疾病（例如， 阿尔茨海默病）。更广泛地说，这些研究的结果将对我们的 了解细胞信号中的MARylation。

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.