Mass spectrometric approaches to protein ADP-ribosylation

项目摘要 聚腺苷二磷酸核糖基化（PAR化）是一种蛋白质翻译后修饰（PTM）， 1963年PAR化由称为聚ADP-核糖聚合酶（PARP）的酶家族催化。在 特别地，PARP 1是作为感应DNA链断裂的结果而被激活的核蛋白。的 静止细胞中的PAR化水平通常非常低。在对遗传毒性应激的反应中，PARP 1被募集， 切割DNA并迅速活化，导致大量PAR化蛋白质的合成， 启动DNA损伤修复机制。PARP 1在介导DNA修复中的关键作用， 细胞死亡提供了开发PARP 1抑制剂以治疗许多人类疾病的基本原理，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BRCA 1/2突变的细胞，依赖于PARP 1活性来保持基因组的完整性。这些细胞经历 对PARP 1抑制的不可持续的遗传损伤。事实上，后期临床研究表明，PARP 1 抑制剂治疗显著延长BRCA缺陷卵巢癌患者的无进展生存期。 这使得它最近获得了FDA的批准。与描绘上游的富有成效的努力相反， PARP 1的监管机构，其真正的下游目标定义不清，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， 它的不稳定性和异质性。为了解决这些紧迫的问题，我们开发了一个大规模的质量 光谱方法对全面表征的Asp和Glu-PAR化的蛋白质组。 我们在340种蛋白质上总共鉴定了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损伤的修复，而且还参与一系列令人惊讶的其他核蛋白的修复。 功能协调发展的使用定量质谱实验，我们还发现了许多以前未知的 PARP 1下游靶点，其PAR化对临床相关PARP 1抑制剂敏感。在这 建议，我们将利用这些初步结果继续表征蛋白质ADP-核糖基化， 长期目标是全面了解PARP和ADP-核糖基化在各种 病理生理过程我们建议的工作的具体目标是：（1）开发一个大规模的 D/E-单-ADP-核糖基化蛋白质组的位点特异性表征方法;（2）开发一个大的 规模的方法来测量绝对蛋白质PAR化化学计量;和（3）开发一种化学蛋白质组学方法， 系统研究临床相关PARP 1抑制剂特异性的方法。要全面完成 我们的目标与多学科的方法，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。我们在这份提案中获得的知识也将产生深远的影响， 如何进一步探索PARP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.