Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga, 526-0829, Japan.
Joint Research Center for Human Retrovirus Infection, Kagoshima University, Sakuragaoka 8-35-1, Kagoshima, 890-8544, Japan.
Exp Cell Res. 2022 Aug 1;417(1):113163. doi: 10.1016/j.yexcr.2022.113163. Epub 2022 Apr 18.
Protein targets of polyADP-ribosylation undergo covalent modification with high-molecular-weight, branched poly(ADP-ribose) (PAR) of lengths up to 200 or more ADP-ribose residues derived from NAD. PAR polymerase 1 (PARP1) is the most abundant and well-characterized enzyme involved in PAR biosynthesis. Extensive studies have been carried out to determine how polyADP-ribosylation (PARylation) regulates cell proliferation during cell cycle, with conflicting conclusions. Since significant activation of PARP1 occurs during cell lysis in vitro, we changed the standard method for cell lysis, and using our sensitive ELISA system, quantified without addition of a PAR glycohydrolase inhibitor and clarified that the PAR level is significantly higher in S phase than that in G1. Under normal condition in the absence of exogenous DNA-damaging agent, PAR turns over with a half-life of <40 s; consistent with significant decrease of NAD levels in S phase, which is rescued by PARP inhibitors, in line with the observed rapid turnover of PAR. PARP inhibitors delayed cell cycle in S phase and decreased cell proliferation. Our results underscore the importance of a suitable assay system to measure rapid PAR chain dynamics in living cells and aid our understanding of the function of PARylation during the cell cycle.
聚 ADP-核糖（PAR）的靶蛋白与来自 NAD 的长度高达 200 个或更多 ADP-核糖残基的高分子量、支链聚（ADP-核糖）（PAR）发生共价修饰。PAR 聚合酶 1（PARP1）是参与 PAR 生物合成的最丰富和研究最充分的酶。已经进行了广泛的研究来确定多 ADP-核糖基化（PARylation）如何在细胞周期中调节细胞增殖，得出的结论相互矛盾。由于 PARP1 在体外细胞裂解过程中会被显著激活，因此我们改变了细胞裂解的标准方法，并使用我们灵敏的 ELISA 系统进行定量，而无需添加 PAR 糖基水解酶抑制剂，并澄清 PAR 水平在 S 期明显高于 G1 期。在没有外源 DNA 损伤剂的正常条件下，PAR 的半衰期<40 s；与 S 期 NAD 水平的显著下降一致，PARP 抑制剂可挽救这一现象，与观察到的 PAR 快速周转一致。PARP 抑制剂延迟 S 期细胞周期并减少细胞增殖。我们的结果强调了合适的测定系统在测量活细胞中快速 PAR 链动力学方面的重要性，并有助于我们理解 PARylation 在细胞周期中的功能。
