Site-Specific Antibody for Protein Poly-ADP-Ribosylation

聚ADP核糖基化（PAR化）是一种蛋白质翻译后修饰（PTM）， 称为聚ADP核糖聚合酶（PARP）的酶家族。在各种PARP家族中， PARP 1是一种重要参与细胞应激反应的核酶。一旦感应到DNA 链断裂，PARP 1被激活，然后利用辅因子NAD+合成大量的 PAR化蛋白质。这些蛋白质连接的PAR链作为一个平台来招募DNA修复机制， 并启动DNA损伤反应（DDR）。这些发现为开发PARP 1提供了理论基础。 抑制剂来治疗人类恶性肿瘤。事实上，FDA最近批准了四种PARP 1抑制剂， 治疗BRCA缺陷的卵巢癌和/或乳腺癌患者。尽管PARP 1取得了令人兴奋的进展， 尽管PARP抑制剂在临床上的应用，但PARP 1和相关PARP酶的基本信号传导机制仍然存在， 不太了解。蛋白质PAR化的分析是一个艰巨的挑战，由于低- 这种PTM的丰度、不稳定性和异质性。因此，很少有真正的PARP 1底物 (and它们的修饰位点）已经被确定，并且在大多数情况下，PAR化如何调节 DNA损伤应答蛋白的功能还不清楚。最近，我们开发了第一个大规模的 质谱（MS）方法对蛋白质Asp-/Glu-PAR化的位点特异性表征。 虽然这种策略能够定量评估PAR化蛋白质组，但这是一个相当漫长的过程。 需要使用质谱仪和计算蛋白质组学的程序 软件此外，目前的抗PAR抗体仅允许分析总PAR化水平，但 而不是在特定残基上的PAR化。在本提案中，我们将开发一种化学生物学方法， 一般位点特异性PAR化抗体的开发。我们将选择一些PAR化靶点， 在DDR中起关键作用，相应的抗体将使用细胞系统进行评估， 动物模型我们设想，抗体的发展，允许简单，敏感，特异性和 PAR化位点的快速检测将极大地促进理解PAR化在DDR中的作用， 癌我们也期望这种方法对未来生物标志物的发展非常有用 针对这些异常的“蛋白质修饰特征”。

Poly-ADP-ribosylation (PARylation) is a protein posttranslational modification (PTM) that is catalyzed by a family of enzymes known as Poly-ADP-ribose polymerases (PARPs). Among the various PARP family members, PARP1 is a nuclear enzyme that is critically involved in cell stress responses. Upon sensing DNA strand breaks, PARP1 becomes activated, and then utilizes the cofactor, NAD+ to synthesize a large array of PARylated proteins. These protein-linked PAR chains serve as a platform to recruit the DNA repair machinery, and to initiate the DNA damage response (DDR). These findings provide the rationale to develop PARP1 inhibitors to treat human malignancies. Indeed, four PARP1 inhibitors have been recently approved by the FDA to treat BRCA-deficient ovarian and/or breast cancer patients. Despite the exciting progresses of PARP1 inhibitors in the clinic, the basic signaling mechanism of PARP1 and the related PARP enzymes remains poorly understood. The analysis of protein PARylation represents a daunting challenge, owing to the low- abundance, labile and heterogeneous nature of this PTM. As a result, very few genuine PARP1 substrates (and their modification sites) have been identified, and in most of the cases, how PARylation regulates the function of the DNA damage responsive proteins is poorly defined. Recently, we developed the first large-scale mass spectrometric (MS) approach towards site-specific characterization of protein Asp-/Glu-PARylation. Although this strategy enables the quantitative assessment of the PARylated proteome, it is a quite lengthy procedure that requires the access to both mass spectrometry equipment and computational proteomics software. Furthermore, current anti-PAR antibodies only allow the analysis of the level of total PARylation, but not PARylation at a specific residue. In this proposal, we will develop a chemical biology approach towards the development of general site-specific PARylation antibodies. We will select a number of PARylation targets that are critically involved in DDR, and the corresponding antibodies will be evaluated using cell systems and animal models. We envision that the development of antibodies that allow for the simple, sensitive, specific and rapid detection of PARylation sites would greatly facilitate the understanding the role of PARylation in DDR and cancer. We also expect that this approach will be highly useful for the future development of biomarkers targeting these aberrant “protein modification signatures”.