Mass spectrometric approaches to protein ADP-ribosylation

蛋白质 ADP 核糖基化的质谱方法

• 批准号: 9568790
• 负责人: Yonghao Yu
• 金额: $ 30.58万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-30 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9568790
• 关键词: ADP ribosylation; Address; Adenosine Diphosphate Ribose; Atlases; BRCA1 gene; Basic Science; Binding; Biochemical; Biochemistry; Bioinformatics; Biological; Biological Assay; Biology; Cancer Patient; Cell Death; Cell Line; Cells; Chemicals; Clinical Research; DNA; DNA Damage; DNA Repair; DNA strand break; Data; Defect; Dose-Limiting; Double Strand Break Repair; Enzymes; FDA approved; Family; Family member; Genetic; Genotoxic Stress; Goals; Ischemic Stroke; Knowledge; Malignant Neoplasms; Malignant neoplasm of ovary; Mass Spectrum Analysis; Measures; Mediating; Mediator of activation protein; Methods; Modification; Molecular; Molecular Biology; Mono-S; Mutate; Nature; Nuclear; Nuclear Protein; PTEN gene; Pathway interactions; Phosphorylation; Physiological; Poly(ADP-ribose) Polymerases; Post-Translational Protein Processing; Process; Progression-Free Survivals; Proteins; Proteome; Proteomics; Reaction; Reagent; Role; Signal Transduction; Site; Specificity; Substrate Specificity; Technology; Therapeutic Intervention; Time; Toxic effect; Validation; Work; analog; aspartylglutamate; cancer cell; chemoproteomics; clinical development; clinically relevant; delta protein; experimental study; follow-up; genome integrity; human disease; in vivo; inhibitor/antagonist; insight; interdisciplinary approach; malignant breast neoplasm; negative elongation factor; novel; phase III trial; recruit; response; stoichiometry; therapeutic target; tool

Project Summary Poly-ADP-ribosylation (PARylation) is a protein posttranslational modification (PTM) that was first documented in 1963. PARylation is catalyzed by a family of enzymes called Poly-ADP-ribose polymerases (PARPs). In particular, PARP1 is a nuclear protein that is activated as a result of sensing DNA strand breaks. The PARylation level in a quiescent cell is usually very low. In response to genotoxic stress, PARP1 is recruited to nicked DNA and is rapidly activated, resulting in the synthesis of a large number of PARylated proteins and initiation of the DNA damage repair mechanisms. The critical roles of PARP1 in mediating DNA repair and also cell death provide the rationale for developing PARP1 inhibitors to treat a number of human diseases, including cancer and ischemic stroke. In particular, cancer cells with defects in double-strand break (DSB) repair, such as BRCA1/2-mutated cells, are reliant on PARP1 activity for genome integrity. These cells undergo unsustainable genetic damage upon PARP1 inhibition. Indeed, late-stage clinical studies revealed that PARP1 inhibitor treatment significantly prolonged progression-free survival of BRCA-deficient ovarian cancer patients. This led to its recent approval by the FDA. Contrary to the fruitful efforts of characterizing the upstream regulators of PARP1, its genuine downstream targets are poorly defined, which has significantly hampered its further functional study. In particular, site-localization of protein PARylation remains a daunting challenge, due to its labile and heterogeneous nature. To address these pressing questions, we developed a large-scale mass spectrometric approach towards comprehensive characterization of the Asp- and Glu-PARylated proteome. We identified a total of 1,048 unique, endogenously modified D/E-PARylation sites on 340 proteins. These proteins are involved in not only DNA damage repair, but also a surprisingly wide array of other nuclear functions. Using a quantitative mass spectrometry experiment, we also identified many previously unknown PARP1 downstream targets, whose PARylation is sensitive to clinically relevant PARP1 inhibitors. In this proposal, we will leverage these preliminary results to continue to characterize protein ADP-ribosylation, with a long term goal of comprehensively understanding the role of PARPs and ADP-ribosylation in various pathophysiological processes. The specific aims of our proposed work are to: (1) develop a large-scale approach to site-specific characterization of the D/E-mono-ADP-ribosylated proteome; (2) develop a large- scale method to measure absolute protein PARylation stoichiometries; and (3) develop a chemoproteomic approach to systemically investigating the specificity of clinically relevant PARP1 inhibitors. We will accomplish our goals with a multi-disciplinary approach, utilizing tools including proteomics, chemical biology, biochemistry, bioinformatics and molecular biology. Knowledge we garner in this proposal will also have a profound impact on how to further explore PARPs as potential therapeutic targets for treating human diseases.

项目概要 聚 ADP 核糖基化 (PARylation) 是一种首次被记录的蛋白质翻译后修饰 (PTM) 1963 年。PARylation 由称为聚 ADP 核糖聚合酶 (PARP) 的酶家族催化。在 特别是，PARP1 是一种核蛋白，由于检测到 DNA 链断裂而被激活。这 静止细胞中的 PARylation 水平通常非常低。为了应对基因毒性应激，PARP1 被招募来 DNA 上有切口并被迅速激活，导致大量 PARylated 蛋白的合成 DNA损伤修复机制的启动。 PARP1 在介导 DNA 修复中的关键作用 细胞死亡为开发 PARP1 抑制剂来治疗许多人类疾病提供了理论依据，包括 癌症和缺血性中风。特别是具有双链断裂（DSB）修复缺陷的癌细胞，例如 作为 BRCA1/2 突变细胞，它们依赖于 PARP1 活性来保证基因组完整性。这些细胞经历 PARP1 抑制导致不可持续的遗传损伤。事实上，后期临床研究表明 PARP1 抑制剂治疗可显着延长 BRCA 缺陷型卵巢癌患者的无进展生存期。 这导致其最近获得 FDA 的批准。与表征上游的卓有成效的努力相反 PARP1 的监管者认为，其真正的下游目标定义不明确，这极大地阻碍了其 进一步的功能研究。特别是，蛋白质 PARylation 的位点定位仍然是一项艰巨的挑战，因为 其不稳定和异质性。为了解决这些紧迫的问题，我们开发了一个大规模的大规模 全面表征 Asp- 和 Glu-PARylated 蛋白质组的光谱分析方法。 我们在 340 种蛋白质上总共鉴定了 1,048 个独特的内源修饰 D/E-PARylation 位点。这些 蛋白质不仅参与 DNA 损伤修复，还参与一系列令人惊讶的其他核 功能。通过定量质谱实验，我们还鉴定了许多以前未知的物质 PARP1 下游靶标，其 PARylation 对临床相关的 PARP1 抑制剂敏感。在这个 提案中，我们将利用这些初步结果继续表征蛋白质 ADP-核糖基化，并 全面了解 PARP 和 ADP-核糖基化在各种疾病中的作用的长期目标 病理生理过程。我们提出的工作的具体目标是：（1）开发大规模的 D/E-单-ADP-核糖基化蛋白质组的位点特异性表征方法； (2) 发展大 测量绝对蛋白质PARylation化学计量的标度方法； (3) 开发化学蛋白质组学 系统研究临床相关 PARP1 抑制剂特异性的方法。我们将完成 我们的目标采用多学科方法，利用蛋白质组学、化学生物学、生物化学等工具， 生物信息学和分子生物学。我们在此提案中获得的知识也将产生深远的影响 关于如何进一步探索PARP作为治疗人类疾病的潜在治疗靶点。