Li Peng, Zhang Yajie, Yu Yonghao
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
Department of Molecular Pharmacology and Therapeutics, Columbia University Vagelos College of Physicians and Surgeons, New York, NY 10032, USA.
bioRxiv. 2025 Mar 27:2025.03.27.645734. doi: 10.1101/2025.03.27.645734.
Poly-ADP-ribosylation (PARylation) is a reversible posttranslational modification that occurs in higher eukaryotes. While thousands of PARylated substrates have been identified, the specific biological functions of most PARylated proteins remain elusive. PARylation stoichiometry is a critical parameter to assess the potential functions of a PARylated protein. Here, we developed a large-scale strategy to measure the absolute stoichiometries of protein PARylation. By integrating mild cell lysis, boronate enrichment and carefully designed titration experiments, we were able to determine the PARylation stoichiometries for a total of 235 proteins. This approach enables the capture of all PARylation events on various amino acid acceptors. We revealed that PARylation occupancy spans over three orders of magnitude. However, most PARylation events occur at low stoichiometric values (median 0.578%). Notably, we observed that high stoichiometry PARylation (>1%) predominantly targets proteins involved in transcription regulation and chromatin remodeling. Thus, our study provides a systems-scale, quantitative view of PARylation stoichiometries under genotoxic conditions, which serves as invaluable resources for future functional studies of this important protein posttranslational modification.
多聚ADP核糖基化（PARylation）是一种发生在高等真核生物中的可逆翻译后修饰。虽然已经鉴定出数千种被PAR化的底物，但大多数被PAR化蛋白质的具体生物学功能仍然不清楚。PARylation化学计量是评估被PAR化蛋白质潜在功能的关键参数。在这里，我们开发了一种大规模策略来测量蛋白质PARylation的绝对化学计量。通过整合温和的细胞裂解、硼酸酯富集和精心设计的滴定实验，我们能够确定总共235种蛋白质的PARylation化学计量。这种方法能够捕获各种氨基酸受体上的所有PARylation事件。我们发现PARylation占有率跨越三个数量级。然而，大多数PARylation事件发生在低化学计量值（中位数为0.578%）。值得注意的是，我们观察到高化学计量PARylation（>1%）主要靶向参与转录调控和染色质重塑的蛋白质。因此，我们的研究提供了在基因毒性条件下PARylation化学计量的系统规模定量视图，这为这种重要的蛋白质翻译后修饰的未来功能研究提供了宝贵资源。
