Site-Specific Antibody for Protein Poly-ADP-Ribosylation

蛋白质聚 ADP 核糖基化位点特异性抗体

• 批准号: 10610163
• 负责人: Yonghao Yu
• 金额: $ 24.76万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10610163
• 关键词: Site; Specific; Antibody; Protein; Poly

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”.

聚 ADP 核糖基化 (PARylation) 是一种蛋白质翻译后修饰 (PTM)，由以下物质催化： 称为聚 ADP 核糖聚合酶 (PARP) 的酶家族。在各种 PARP 家族中 PARP1 是一种核酶，与细胞应激反应密切相关。感测 DNA 后 链断裂，PARP1 被激活，然后利用辅因子 NAD+ 合成大量 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 提供了理论依据 治疗人类恶性肿瘤的抑制剂。事实上，FDA 最近批准了四种 PARP1 抑制剂 治疗缺乏 BRCA 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和相关PARP酶的基本信号机制仍然存在 不太了解。蛋白质 PARylation 的分析是一项艰巨的挑战，因为 该 PTM 的丰富性、不稳定性和异质性。因此，真正的 PARP1 底物很少 （及其修饰位点）已被确定，并且在大多数情况下，PARylation 如何调节 DNA损伤反应蛋白的功能尚不清楚。最近，我们开发了第一个大型 蛋白质 Asp-/Glu-PARylation 的位点特异性表征的质谱 (MS) 方法。 尽管该策略能够对 PARylated 蛋白质组进行定量评估，但它是一个相当漫长的过程 需要使用质谱设备和计算蛋白质组学的程序 软件。此外，目前的抗 PAR 抗体只能分析总 PARylation 的水平，但 不是在特定残基上进行 PARylation。在本提案中，我们将开发一种化学生物学方法 通用位点特异性 PARylation 抗体的开发。我们将选择一些 PARylation 目标 与 DDR 密切相关，将使用细胞系统和相应的抗体进行评估 动物模型。我们设想开发出能够简单、灵敏、特异性和 快速检测 PARylation 位点将极大地促进了解 PARylation 在 DDR 和 癌症。我们还期望这种方法对于生物标志物的未来开发非常有用 针对这些异常的“蛋白质修饰特征”。