Fig 1.
A scheme for the NUDT5-coupled AMP-Glo (NCAG) assay.
NUDT5 has a unique substrate selectivity and can selectively cleave the protein-free mono-ADP-ribose into AMP and ribose phosphate, but not protein-bound ADP-ribosylations or protein-free poly(ADP-ribose) or NAD+. The cleaved AMP is subsequently detected by the AMP-Glo assay.
Fig 2.
NUDT5 selectively cleaves protein-free ADP-ribose, but not protein-bound poly- and mono-ADP-ribosylations.
A. NUDT5 is unable to bind to the protein-bound mono-ADP-ribosylation or poly(ADP-ribose). The sites for chain elongation or protein attachment are blocked in NUDT5 but are open in NUDT16. The substrate-binding pocket for NUDT5 and NUDT16 are superimposed and the orientations of ADP-ribose binding are shown. B. NUDT5 is unable to cleave the protein-bound PARylation. PARylated PARP1 was treated with recombinant human NUDT5 (5 μM) and NUDT16 (5 μM). While NUDT16 efficiently reverses the protein-bound PARylation, NUDT5 shows little-to-no PAR hydrolysis activity. C-D. NUDT5 is unable to cleave the protein-bound mono-ADP-ribosylations. Ser- and Asp/Glu-MARylated PARP1 substrates (see the “Materials and Methods”) were digested with 5 μM of each human ARH3 and NUDT5 (in panel C) and TARG1 and NUDT5 (in panel D), respectively. NUDT5 shows little-to-no MAR hydrolase activity, whereas ARH3 and TARG1 effectively reverse Ser- and Asp/Glu-MARylated PARP1 substrates, respectively.
Fig 3.
The NUDT5-coupled AMP-Glo (NCAG) assay effectively and selectively monitors the protein-free mono-ADP-ribose.
A. NUDT5 titration against 10 μM ADPR. The luminescence signals from NUDT5-mediated digestion of ADPR are normalized with respect to that from 10 μM AMP. B. Comparison of NCAG luminescence signals from different types of substrates. Experiments were done using 10 μM of all substrates and 400 nM NUDT5. C. A standard curve of ADPR and AMP in the NCAG assay using 400 nM NUDT5. Standard deviations from triplicates were calculated for all NCAG assays and are shown as error bars. D. NUDT5 is unable to cleave protein-free poly(ADP-ribose) chains. Isolated PAR chains (7 mer, 38 μM) were assayed using the NCAG assay after NUDT5 (2 μM) and PARG (2 μM) digestions. A control reaction with the basic NCAG reagents (AMP-Glo reagents and 400 nM NUDT5) did not show signal increase in comparison to that with only AMP-Glo reagents. The AMP-Glo-only readout was used for background subtraction in panel D.
Fig 4.
Monitoring of the reversal of residue-specific mono-ADP-ribosylations by ADP-ribosyl-acceptor hydrolases using the NCAG assay.
The Ser- and Asp/Glu-MARylated PARP1 substrates (see the “Materials and Methods”) were digested with 2 μM TARG1, ARH3, and PARG, and released ADP-ribose was measured using the NCAG assay. While PARG shows limited hydrolysis of MARylated substrates, ARH3 and TARG1 specifically reverse the Ser- and Asp/Glu-MARylation, respectively. The luminescence signals from different enzymes were normalized with respect to that from ARH3 (in Ser-MAR) and TARG1 (in Asp/Glu-MAR), respectively. Standard deviations from triplicates were calculated for all NCAG assays and are shown as error bars.
Fig 5.
A kinetic analysis of the exo-glycohydrolase activity of PARG using the NCAG assay.
A-B. A time-dependent and dose-dependent monitoring of the exo-glycohydrolase activity of PARG by the NCAG assay. 16.75 μM of ADP-ribose (cleavable ADP-ribose units in PARylated PARP1) was treated with increasing concentration of PARG. The initial velocities for each PARG concentration were calculated using the early and linear portions and plotted as a function of the PARG concentration in panel B. C-D. Michalis-Menton kinetics for the exo-glycohydrolase activity of PARG. The initial velocities were calculated at different concentrations of substrates (in panel C) and plotted as a function of substrate concentrations (in panel D). Standard deviations from triplicates were calculated for all NCAG assays and are shown as error bars.