Defining the Biochemical Function and Therapeutic Utility of Unique PARP14 and PARP15 ADP-Ribosylation Sites

摘要 ADP-核糖化是一种普遍存在的翻译后修饰，存在于所有生命王国中。 虽然这是第一个被描述的PTM，但ADP的生化选择性和细胞后果- 人们对核糖化反应知之甚少。该项目的目标是定义连接独特ADP的路径- 核糖化修饰它们在细胞中的下游功能。我们将利用这些信息开发出第一款- 针对故障的ADP-核糖信号网络的多肽类抑制剂。在人类身上，有17种不同的 多聚ADP核糖聚合酶(PARP)催化ADP核糖化。PARP酶被认为与一种 许多生理功能(如DNA修复、RNA调节、细胞发育)和广泛的 疾病，最明显的是病毒感染。虽然之前的努力已经验证了Parp 13、14和15的角色 在病毒调控方面，它们的作用机制尚不清楚。在很大程度上，这种不明确性是由于我们的 不能将ADP-核糖化的单一位点与其下游功能联系起来。在激酶领域的研究已经有了 展示了将酶特定的翻译后修饰位点(PTM)连接到其 特定的细胞功能，从根本上改变了我们对信号转导的理解，揭示了新的 疾病治疗干预的途径。通过利用我的实验室和我的 合作者，我们的工作将使用多学科方法来确定功能后果 细胞内独特的ADP-核糖化事件，并开发新型PARP选择性多肽抑制剂 要阻止特定修改，请执行以下操作。在第一个目标中，我们将定义PARP14和 PARP15自身(自动修改)和其他目标(反式修改)。我们一直在努力 广泛地与这些PARP家族成员一起定义他们的特定蛋白质靶点，我们有一个强大的病毒 模型来询问特定的位置改变如何改变路径行为。我们在这一目标上的工作将提供 在特定的ADP-核糖化位点和它们在细胞中的作用之间的第一个链接，它将适应另一个 帕普斯。在第二个目标中，我们开发的PARP14特异性靶肽序列将作为 用于鉴定选择性PARP14抑制剂的平台。我们将合成肽和类肽(多N-取代) 甘氨酸)衍生物和鉴定结构活性关系(SAR分析)与 生成的库。这项工作将扩大我们对目标选择的生化机制的了解 并将为靶向PAPS治疗疾病提供一种新的途径。这项提案的工作将广泛地 适用于其余PARP家族成员的研究，并将在医学、细胞 生物学、发育生物学和翻译后修饰蛋白质组的研究。

SUMMARY ADP-ribosylation is a widespread and ubiquitous post-translational modification across all kingdoms of life. Although it was the first PTM described, the biochemical selectivity and cellular consequences of ADP- ribosylation is very poorly understood. The objective of this project is to define the pathways linking unique ADP- ribosylation modifications to their downstream function in the cell. We will use this information to develop first-in- class peptide-based inhibitors to target malfunctioning ADP-ribose signaling networks. In humans, 17 different poly-ADP-ribose polymerases (PARPs) catalyze ADP-ribosylation. PARP enzymes have been implicated in a number of physiological functions (e.g. DNA repair, RNA regulation, cell development) and a broad array of diseases, most notably viral infection. While previous efforts have validated the roles for PARPs 13, 14, and 15 in viral regulation, their mechanism of action remains unclear. In large part, this lack of clarity arises from our inability to connect a single site of ADP-ribosylation to its downstream function. Work in the kinase field has demonstrated the power of connecting enzyme-specific post-translational modification sites (PTMs) to their specific cellular function, fundamentally altering our understanding of signal transduction and revealing new avenues for therapeutic intervention in disease. By leveraging the expertise from my lab and that of my collaborators, our work will use a multidisciplinary approach to determine the functional consequences of unique ADP-ribosylation events in the cell and develop novel PARP-selective peptide-based inhibitors to block specific modifications. In the first aim, we will define the sites of modification for both PARP14 and PARP15 on themselves (auto-modification) and on other targets (trans-modification). We have worked extensively with these PARP family members to define their specific protein targets and we have a robust viral model to interrogate how specific site alterations change pathway behavior. Our work in this aim will provide the first links between specific ADP-ribosylation sites and their effects in the cell and it will be adaptable to the other PARPs. In the second aim, a PARP14-specific target peptide sequence that we have developed will serve as a platform to identify selective PARP14 inhibitors. We will synthesize peptide and peptoid (poly-N-substituted glycine) derivatives of the initial target peptide and identify structure activity relationships (SAR analysis) with the resulting library. This work will expand our knowledge of the biochemical mechanisms governing target selection and will provide a novel avenue for targeting PARPs to treat disease. The work from this proposal will be broadly applicable to the study of the remaining PARP family members and will have applications in medicine, cell biology, developmental biology, and the study of the post-translationally modified proteome.