Department of Chemical Physiology and Biochemistry, Oregon Health and Science University, 3181 SW Sam Jackson Park Rd., L334, Portland, Oregon 97239, United States.
ACS Sens. 2024 Oct 25;9(10):5246-5252. doi: 10.1021/acssensors.4c01406. Epub 2024 Oct 1.
ADP-ribosylation, the transfer of ADP-ribose (ADPr) from nicotinamide adenine dinucleotide (NAD) groups to proteins, is a conserved post-translational modification (PTM) that occurs most prominently in response to DNA damage. ADP-ribosylation is a dynamic PTM regulated by writers (PARPs), erasers (ADPr hydrolases), and readers (ADPR binders). PARP1 is the primary DNA damage-response writer responsible for adding a polymer of ADPR to proteins (PARylation). Real-time monitoring of PARP1-mediated PARylation, especially in live cells, is critical for understanding the spatial and temporal regulation of this unique PTM. Here, we describe a genetically encoded FRET probe (pARS) for semiquantitative monitoring of PARylation dynamics. pARS feature a PAR-binding WWE domain flanked with turquoise and Venus. With a ratiometric readout and excellent signal-to-noise characteristics, we show that pARS can monitor PARP1-dependent PARylation temporally and spatially in real-time. pARS provided unique insights into PARP1-mediated PARylation kinetics in vitro and high-sensitivity detection of PARylation in live cells, even under mild DNA damage. We also show that pARS can be used to determine the potency of PARP inhibitors in vitro and, for the first time, in live cells in response to DNA damage. The robustness and ease of use of pARS make it an important tool for the PARP field.
ADP-核糖基化，即将烟酰胺腺嘌呤二核苷酸（NAD）基团中的 ADP-核糖（ADPr）转移到蛋白质上，是一种保守的翻译后修饰（PTM），最常发生在 DNA 损伤时。ADP-核糖基化是一种受作家（PARPs）、橡皮擦（ADPr 水解酶）和读者（ADPR 结合物）调节的动态 PTM。PARP1 是主要的 DNA 损伤反应作家，负责将 ADPR 的聚合物添加到蛋白质上（PARylation）。实时监测 PARP1 介导的 PARylation，特别是在活细胞中，对于理解这种独特的 PTM 的时空调节至关重要。在这里，我们描述了一种遗传编码的 FRET 探针（pARS），用于半定量监测 PARylation 动力学。pARS 的特征是 PAR 结合的 WWE 结构域两侧是绿松石和 Venus。通过比率读数和出色的信噪比特性，我们表明 pARS 可以实时、时空监测 PARP1 依赖性 PARylation。pARS 提供了 PARP1 介导的 PARylation 动力学的独特见解，以及在活细胞中对 PARylation 的高灵敏度检测，即使在轻度 DNA 损伤下也是如此。我们还表明，pARS 可用于体外测定 PARP 抑制剂的效力，并首次在 DNA 损伤时在活细胞中测定。pARS 的稳健性和易用性使其成为 PARP 领域的重要工具。
