Mass spectrometric approaches to protein ADP-ribosylation

项目摘要 Poly-ADP-核糖化是一种最早被报道的蛋白质翻译后修饰(PTM) 1963年。PAR化是由一种称为聚-ADP-核糖聚合酶(PAPS)的酶家族催化的。在……里面 特别是，PARP1是一种核蛋白，由于检测到DNA链断裂而被激活。这个 在静止的细胞中，PAR化水平通常很低。为了应对遗传毒性应激，PARP1被招募到 并被快速激活，导致合成大量的PARylated蛋白质和 启动DNA损伤修复机制。PARP1在介导DNA修复中的关键作用 细胞死亡为开发PARP1抑制剂治疗许多人类疾病提供了理论基础，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，如 作为BRCA1/2突变的细胞，依赖于PARP1活性来确保基因组的完整性。这些细胞经历了 PARP1抑制造成的不可持续的遗传损害。事实上，晚期临床研究表明，PARP1 抑制物治疗显著延长BRCA缺陷卵巢癌患者的无进展生存期。 这导致它最近获得了FDA的批准。与刻画上游的卓有成效的努力相反 PARP1的监管机构，其真正的下游目标定义不明确，这大大阻碍了其 进一步的功能研究。特别是，蛋白质PAR化的位点定位仍然是一个艰巨的挑战， 由于其不稳定和异质的性质。为了解决这些紧迫的问题，我们开发了一种大规模的 天冬氨酸和谷氨酰化蛋白质组的综合表征的光谱方法。 我们在340个蛋白质上发现了1,048个独特的、内源性修饰的D/E-PAR化位点。这些 蛋白质不仅参与dna损伤修复，而且还参与其他核物质的广泛分布。 功能。利用定量质谱学实验，我们还发现了许多以前未知的 PARP1下游靶点，其PAR化对临床相关的PARP1抑制剂敏感。在这 提案中，我们将利用这些初步结果来继续表征蛋白质ADP-核糖化，具有 长期目标是全面了解PARPS和ADP-核糖化在各种 病理生理过程。我们提出的工作的具体目标是：(1)发展大规模的 D/E-单-ADP-核糖化蛋白质组的位点特异性鉴定方法 测定绝对蛋白质PAR化化学计量学的标度法；以及(3)建立化学蛋白质组 系统研究临床相关PARP1抑制剂的特异性的方法。我们将完成 我们的目标是采用多学科方法，利用包括蛋白质组学、化学生物学、生物化学、 生物信息学和分子生物学。我们在这项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PAPS作为治疗人类疾病的潜在治疗靶点。

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.