Zhang Lu, Lin Hening
Department of Chemistry and Chemical Biology, Howard Hughes Medical Institute, Cornell University, Ithaca, NY, 14853, USA.
Methods Mol Biol. 2017;1608:95-109. doi: 10.1007/978-1-4939-6993-7_8.
ADP-ribosylation has been well known as an important posttranslational modification, which is catalyzed by a family of enzymes called poly(ADP-ribose) polymerases (PARPs). PARPs transfer of a single or multiple adenine diphosphate ribose (ADP-ribose) units from nicotinamide adenine dinucleotide (NAD) to specific amino acids on substrate proteins. Through mono- or poly-ADP-ribosylation enzymatic activities, PARPs regulate various biological processes, including DNA damage repair, chromatin remodeling, transcriptional regulation, RNA processing and metabolism. Notably, PARP inhibitors are in clinical trials to treat human diseases, in particular cancer. To further investigate the biological function of PARPs, and to achieve better therapeutic effect of PARP inhibitors, it is important to identify the physiological substrates of PARPs. Here we describe a protocol to use clickable analog of nicotinamide adenine dinucleotide (NAD) that can be applied for the detection, affinity purification and identification of substrate proteins of PARPs.
ADP核糖基化作为一种重要的翻译后修饰已广为人知，它由一类称为聚（ADP-核糖）聚合酶（PARP）的酶催化。PARP将单个或多个腺嘌呤二磷酸核糖（ADP-核糖）单元从烟酰胺腺嘌呤二核苷酸（NAD）转移到底物蛋白上的特定氨基酸。通过单ADP核糖基化或多ADP核糖基化酶活性，PARP调节各种生物学过程，包括DNA损伤修复、染色质重塑、转录调控、RNA加工和代谢。值得注意的是，PARP抑制剂正在进行治疗人类疾病，尤其是癌症的临床试验。为了进一步研究PARP的生物学功能，并实现PARP抑制剂更好的治疗效果，识别PARP的生理底物很重要。在这里，我们描述了一种使用烟酰胺腺嘌呤二核苷酸（NAD）可点击类似物的方案，该方案可用于检测、亲和纯化和鉴定PARP的底物蛋白。
