Detecting and Quantifying pADPr In Vivo.

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 - reflected by increased poly-ADP-ribosylation and accumulation of pADPr-modified proteins or free pADPr - contributes to depletion of NAD+ and mitochondrial dysfunction, potentially leading to cell death. Thus, PARP overactivation and increases in free pADPr have been identified as key contributors to the pathobiology of many diseases. In stark contrast, PARP inhibitors are in clinical use in cancer patients where they potentiate cell death induced by chemotherapeutic agents. Accordingly, monitoring PARP-1 activation - responsible for up to 80-90% of cellular pADPr synthesis - 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.