In Situ Staining for Poly(ADP-Ribose) Polymerase Activity Using an NAD Analogue

Abstract

SUMMARY Poly(ADP-ribose) polymerase (PARP) is a highly abundant nuclear enzyme which metabolizes NAD, in response to DNA strand breakage, to produce chains of poly(ADP-ribose) attached to nuclear proteins. PARP activation has been implicated in ischemia/reperfusion injury, but its biological significance is not fully understood. We have modified an existing in situ method for detection of PARP activity by using an NAD analogue in which adenine is modified by an "etheno" (vinyl) bridge. Etheno-NAD serves as a PARP substrate in an initial enzymatic reaction; a specific antibody to ethenoadenosine is then used in an immunohistochemical reaction to detect the production of modified poly(ADP-ribose). The method produces strong and specific labeling of nuclei in which PARP has been activated, i.e., those in which DNA strand breaks have been produced, and the results can be analyzed by microscopy, flow cytometry, or colorimetry. The method is applicable to cultured cells in several formats and to frozen tissue sections. The particular characteristics of the new method may assist in future in situ studies of PARP activation.

Application of a Fluorometric Assay for Characterization of the Catalytic Competency of a Domain III Fragment of Pseudomonas aeruginosa Exotoxin A

“…They were able to probe the nucleotide binary and ternary complex formation using ⑀-NAD ϩ . The high sensitivity of fluorescent derivatives of NAD ϩ in measuring ADP-ribosylation reactions has also been shown by Davis and co-workers (22). They used ⑀-NAD ϩ to detect poly-ADP-ribosyltransferase (PARP) activity in an immunohistochemical assay in which a specific antibody to ethenoadenosine was used to detect the production of modified poly(ADP-ribose) in cell extracts.…”

Proteomics approaches to identify mono‐(ADP‐ribosyl)ated and poly(ADP‐ribosyl)ated proteins

“…Albeit with lower efficiency, these analogues can all serve as PARylation substrates. In particular in combination with a mouse monoclonal antibody 1G4 specific to ethenoadenosine , 1,N 6 ‐etheno NAD + (ε‐NAD + ) has been used since the 1990s to detect PARP activity in permeabilized cells , as well as MARylation activity on the cell surface . Recently, Guetg and colleagues (2012) have incubated ε‐NAD + with isolated nucleoli and identified that PARP1 and histones are ADP‐ribosylated in this membrane‐less organelle .…”

Endogenous ADP-Ribosylation

“…The NAD + -analog 1,N6-etheno-NAD + (etheno-NAD + ) is readily accepted as a substrate for ADPribosylation by a broad spectrum of bacterial and eukaryotic ADP-ribosyltransferases (Armstrong and Merrill 2001;Davis et al 1998;Giovane et al 1985;Hingorani and Ho 1988;Klebl and Pette 1996;Krebs et al 2003;). An antibody (1G4) is available that specifically recognizes 1,N6-ethenoadenosine.…”

“…This originally had been developed to detect ethenoadenosine as an adduct in DNA resulting from the exposure of workers to vinyl chloride (Young and Santella 1988). The 1G4 antibody detects etheno-ADP-ribose incorporated into both poly-and mono-ADP-ribosylated proteins, and can be used in a variety of assay systems including Western blot and immunofluorescence (Bannas et al 2005;Davis et al 1998;Krebs et al 2003;Liao and Puro 2006). Importantly, fluorochrome-conjugated 1G4 antibody has been used as a flow cytometric assay to monitor cell surface ADPribosyltransferase activity (Grahnert et al 2002;Krebs et al 2003;.…”