MacroGreen, a simple tool for detection of ADP-ribosylated proteins

Abstract

Enzymes in the PARP family partake in the regulation of vital cellular signaling pathways by ADP-ribosylating their targets. The roles of these signaling pathways in disease development and the de-regulation of several PARP enzymes in cancer cells have motivated the pursuit of PARP inhibitors for therapeutic applications. In this rapidly expanding research area, availability of simple research tools will help assess the functions of ADP-ribosylation in a wider range of contexts. Here, we generated a mutant Af1521 macrodomain fused to green fluorescent protein (GFP) to generate a high-affinity ADP-ribosyl binding reagent. The resulting tool – which we call MacroGreen – is easily produced by expression in Escherichia coli , and can detect both mono-and poly-ADP-ribosylation of diverse proteins in vitro. Staining with MacroGreen allows detection of ADP-ribosylation at sites of DNA damage by fluorescence microscopy. MacroGreen can also be used to quantify modification of target proteins in overlay assays, and to screen for PARP inhibitors in high-throughput format with excellent assay statistics. We expect that this broadly applicable tool will facilitate ADP-ribosylation related discoveries, including by laboratories that do not specialize in this field.

Assay technologies facilitating drug discovery for ADP‐ribosyl writers, readers and erasers

“…Coupled enzyme assay measures generation of nicotinamide; nicotinamidase needed to generate ammonia; glutamate dehydrogenase (continuous) or ortho-phthaldehyde (sensitive) needed for detection [46] Fluorescence 384-well plates Detection based on fluorescent macrodomain protein produced in E. coli; some hydrolysis activity present despite inactivating mutations; non-homogeneous assay [23] Colorimetry Fluorescence Luminescence 96-well plates Binding-assay; similar to ELISA; HA-tagged macrodomain for detection; anti-HA HRP conjugate needed; non-homogeneous [47] Luminescence…”

“…Recently, an assay describing a fluorescently GFP-tagged macrodomain was reported, which also allowed assays using microplates. [23] The detection is based on high affinity protein reagent and this provided a convenient method for measuring inhibitor potencies but requires the use of a plate washer for automation.…”

“…[3] Later, an assay method to detect PARP protein and PARP activity directly from nitrocellulose blots using radiolabeled substrate [ 32 P]NAD + was developed by Simonin et al [15] The low-throughput Western blot or dot blot methods have been used with other detection methods including biotinylated NAD + analogs [16] and streptavidin coupled HRP, PAR H10, or MAR/PAR antibodies [17][18][19] and various ADP-ribosylation affinity reagents developed recently. [20][21][22][23] It is noteworthy that when using purified proteins, it is possible to see the disappearance of a protein band upon addition of NAD + with a simultaneous appearance of a high molecular weight smear on a coomassie stained SDS-PAGE. This is a simple and widely available qualitative method of assessing poly-ART activity of enzymes capable of forming PAR chains [24] and can similarly be used to measure PAR hydrolysis activity.…”

ADP-Ribosylation as Post-Translational Modification of Proteins: Use of Inhibitors in Cancer Control

“…Other domains may contain the RNA recognition motif (RRM), the serine arginine repeats, and the lysine arginine repeats (SR/KR) motifs, or the Forkhead-associated (FHA) domain (binding to iso-ADP-ribose) and the BRCA1 C-Terminal (BRCT) in the Breast Cancer 1 (BRCA1) antioncogene, binding to (ADPR) n units [ 32 , 34 ]. ADPR-binding macrodomains can behave as readers, as in the case of AF1521 from Archeglobus fulgidus , that has been applied to proteomic studies for the enrichment of ADP-ribosylated proteins [ 35 , 36 , 37 ]. A PAR-recognizing macrodomain is present in a histone H2A variant named H2A1.1 (mH2A1.1).…”

Adp-Ribosylation as Post-Translational Modification of Proteins: Use of Inhibitors in Cancer Control

“…Other domains may contain the RNA recognition Motif (RRM), the serine arginine repeats and lysine arginine repeats (SR/KR) motifs, or the Forkheadassociated (FHA) domain (binding to iso-ADP-ribose), or the BRCA1 C-Terminal (BRCT) in the Breast Cancer 1 (BRCA1) antioncogene, binding to (ADPR)n units [32,34]. ADPRbinding macrodomains can behave as readers, as in the case of AF1521 from Archeglobus fulgidus, that has been applied to proteomic studies for enrichment of ADP-ribosylated proteins [35][36][37]. PAR recognizing macrodomain is present in a histone H2A variant named H2A1.1 (mH2A1.1).…”