体内检测和定量聚（ADP-核糖）

Detecting and Quantifying pADPr In Vivo.

作者信息

Lai Yi-Chen, Aneja Rajesh K, Satchell Margaret A, Clark Robert S B

机构信息

Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA.

Section of Pediatric Critical Care Medicine, Texas Children's Hospital, 6621 Fannin Street, WT6-006, Houston, TX, 77030, USA.

出版信息

Methods Mol Biol. 2017;1608:27-43. doi: 10.1007/978-1-4939-6993-7_3.

DOI:10.1007/978-1-4939-6993-7_3

PMID:28695501

Abstract

Poly(ADP-ribose) polymerases (PARP) participate in diverse biological processes contributing to cellular homeostasis or exacerbating injury. PARP catalyzes the addition of ADP-ribose molecules (pADPr) to the target proteins, a process termed poly-ADP-ribosylation. Overactivation of PARP, as reflected by increased poly-ADP-ribosylation, accumulation of pADPr-modified proteins or free pADPr, contributes to depletion of NAD and mitochondrial dysfunction, potentially leading to cell death. Since PARP overactivation and increases in free pADPr have been identified as key contributors to the pathobiology of many diseases, monitoring PARP-1 activation by detecting and quantifying pADPr may provide valuable mechanistic insights as well as facilitating therapeutic drug monitoring for PARP inhibitors.Several non-isotopic immunodetection methods for quantifying pADPr are discussed: western blotting of poly-ADP-ribosylated proteins, cellular localization of pADPr by immunohistochemistry, quantification of pADPr by enzyme-linked immunoassay and small scale two-dimensional gel electrophoresis.

摘要

聚（ADP-核糖）聚合酶（PARP）参与多种生物过程，这些过程有助于细胞内稳态或加剧损伤。PARP催化将ADP-核糖分子（pADPr）添加到靶蛋白上，这一过程称为多聚ADP-核糖基化。PARP的过度激活，表现为多聚ADP-核糖基化增加、pADPr修饰蛋白或游离pADPr的积累，会导致NAD耗竭和线粒体功能障碍，可能导致细胞死亡。由于PARP过度激活和游离pADPr增加已被确定为许多疾病病理生物学的关键因素，通过检测和定量pADPr来监测PARP-1激活可能会提供有价值的机制见解，并有助于对PARP抑制剂进行治疗药物监测。本文讨论了几种用于定量pADPr的非同位素免疫检测方法：多聚ADP-核糖基化蛋白的蛋白质印迹法、通过免疫组织化学对pADPr进行细胞定位、通过酶联免疫测定法对pADPr进行定量以及小规模二维凝胶电泳法。