Site-Specific Antibody for Protein Poly-ADP-Ribosylation

Poly-ADP-核糖化是一种蛋白质翻译后修饰(PTM)，由 称为聚-ADP-核糖聚合酶(PAPS)的一组酶。在各种PARP家族中 成员，PARP1是一种核酶，在细胞应激反应中起关键作用。根据检测到的DNA 链断裂时，PARP1被激活，然后利用辅因子NAD+合成大量的 PARylated蛋白质。这些蛋白质连接的PAR链作为招募DNA修复机制的平台， 并启动DNA损伤反应(DDR)。这些发现为开发PARP1提供了理论基础 治疗人类恶性肿瘤的抑制剂。事实上，FDA最近已经批准了四种PARP1抑制剂 用于治疗BRCA缺陷的卵巢癌和/或乳腺癌患者。尽管PARP1取得了令人兴奋的进展 临床上的抑制剂，PARP1的基本信号机制和相关的PARP酶仍然存在 人们对此知之甚少。蛋白质PAR化的分析是一项艰巨的挑战，因为 这种PTM的丰富性、不稳定性和异质性。因此，很少有真正的PARP1底物 (及其修饰位点)已经被确定，在大多数情况下，PAR化如何调节 DNA损伤反应蛋白的功能还不清楚。最近，我们开发了第一个大规模的 蛋白质Asp-/Glu-PAR化位点特异性表征的质谱学方法。 虽然这种策略能够对PARylated蛋白质组进行定量评估，但这是一个相当长的过程 需要同时使用质谱学设备和计算蛋白质组学的程序 软件。此外，目前的抗PAR抗体只允许分析总PAR化水平，但 而不是特定残基上的PAR化。在这项建议中，我们将开发一种化学生物学方法来解决 开发通用的位点特异性PAR化抗体。我们将选择一些符合以下条件的PAR化目标 与DDR密切相关，相应的抗体将使用细胞系统和 动物模型。我们设想，抗体的开发将允许简单、敏感、特异和 快速检测PAR化位点将大大有助于理解PAR化在DDR和 癌症。我们也期望这种方法将对生物标志物的未来发展非常有用。 以这些异常的“蛋白质修饰特征”为目标。

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”.