Karpova Yaroslava, Piatz Sara, Bordet Guillaume, Tulin Alexei V
Department of Biomedical Sciences, School of Medicine and Health Sciences, University of North Dakota, 501 North Columbia Road, Grand Forks, ND 58202, USA.
Cells. 2025 Jul 9;14(14):1049. doi: 10.3390/cells14141049.
Poly(ADP-ribosyl)ation is a crucial posttranslational modification that governs gene expression, chromatin remodeling, and cellular homeostasis. This dynamic process is mediated by the opposing activities of poly(ADP-ribose) polymerases (PARPs), which synthesize poly(ADP-ribose) (pADPr), and poly(ADP-ribose) glycohydrolase (PARG), which degrades it. While PARP function has been extensively studied, the structural and mechanistic basis of PARG-mediated pADPr degradation remain incompletely understood. To investigate the role of PARG in pADPr metabolism, we employed CRISPR/Cas9-based genome editing to generate a novel mutant mouse embryonic stem cell (ESC) line carrying a precise deletion within the PARG catalytic domain. This deletion completely abolished pADPr hydrolytic activity, resulting in massive nuclear pADPr accumulation, yet ESC viability, proliferation, and cell cycle progression remained unaffected. Using as a model system, we demonstrated that this mutation completely disrupted the pADPr pathway and halted developmental progression, highlighting the essential role of PARG and pADPr turnover in organismal development. Our results define a critical structural determinant of PARG catalytic function, underscore the distinct requirements for pADPr metabolism in cellular versus developmental contexts, and provide a genetically tractable model for studying the regulation of poly(ADP-ribose) dynamics and therapeutic responses to PARP inhibition in vivo.
聚（ADP-核糖）化是一种关键的翻译后修饰，它调控基因表达、染色质重塑和细胞内稳态。这个动态过程由聚（ADP-核糖）聚合酶（PARP）和聚（ADP-核糖）糖苷水解酶（PARG）的相反活性介导，PARP合成聚（ADP-核糖）（pADPr），而PARG降解它。虽然PARP的功能已被广泛研究，但PARG介导的pADPr降解的结构和机制基础仍未完全了解。为了研究PARG在pADPr代谢中的作用，我们采用基于CRISPR/Cas9的基因组编辑技术，生成了一种新型突变小鼠胚胎干细胞（ESC）系，该细胞系在PARG催化结构域内有一个精确的缺失。这种缺失完全消除了pADPr水解活性，导致大量的核pADPr积累，但ESC的活力、增殖和细胞周期进程仍未受影响。以该细胞系作为模型系统，我们证明这种突变完全破坏了pADPr途径并阻止了发育进程，突出了PARG和pADPr周转在机体发育中的重要作用。我们的结果确定了PARG催化功能的一个关键结构决定因素，强调了细胞环境与发育环境中pADPr代谢的不同要求，并为研究体内聚（ADP-核糖）动态调控和对PARP抑制的治疗反应提供了一个易于遗传操作的模型。
