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. 2018 Dec 20;25(12):1562-1570.e19.
doi: 10.1016/j.chembiol.2018.09.008. Epub 2018 Oct 11.

Monitoring Poly(ADP-ribosyl)glycohydrolase Activity With a Continuous Fluorescent Substrate

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Free PMC article

Monitoring Poly(ADP-ribosyl)glycohydrolase Activity With a Continuous Fluorescent Substrate

Bryon S Drown et al. Cell Chem Biol. .
Free PMC article

Abstract

The post-translational modification (PTM) and signaling molecule poly(ADP-ribose) (PAR) has an impact on diverse biological processes. This PTM is regulated by a series of ADP-ribosyl glycohydrolases (PARG enzymes) that cleave polymers and/or liberate monomers from their protein targets. Existing methods for monitoring these hydrolases rely on detection of the natural substrate, PAR, commonly achieved via radioisotopic labeling. Here we disclose a general substrate for monitoring PARG activity, TFMU-ADPr, which directly reports on total PAR hydrolase activity via release of a fluorophore; this substrate has excellent reactivity, generality (processed by the major PARG enzymes), stability, and usability. A second substrate, TFMU-IDPr, selectively reports on PARG activity only from the enzyme ARH3. Use of these probes in whole-cell lysate experiments has revealed a mechanism by which ARH3 is inhibited by cholera toxin. TFMU-ADPr and TFMU-IDPr are versatile tools for assessing small-molecule inhibitors in vitro and probing the regulation of ADP-ribosyl catabolic enzymes.

Keywords: ARH3; PARG; cholera toxin; enzyme assay; fluorescent probe; poly(ADP-ribose).

Figures

Figure 1.
Figure 1.
Design of PARG/ARH3 substrate. A. PAR is cleaved by PARG and ARH3 via hydrolysis of the glycosyl bond (red). B. Synthetic PARG/ARH3 substrates mimic ADP-ribose and release a chromophore or fluorophore upon hydrolysis. Synthesis of these compounds requires a late-stage pyrophosphate formation and 1,2-cis selective glycosylation.
Figure 2.
Figure 2.
Synthesis of pNP-ADPr and TFMU-ADPr. Abbreviations: ADDP = 1,1’-(azodicarbonyl)dipiperidine, DIAD = diisopropyl azodicarboxylate, Fm = 9H-fluorenylmethyl, DCI = 4,5-dicyanoimidazole, NCS = N-chlorosuccinimide, DBU = 1,8-diazabicycloundec-7-ene, TBS = tert-butyldimethylsilyl.
Figure 3.
Figure 3.
Molecular docking sugar nucleotides with hPARG and LchARH3 using Glide XP. A. ADPr (blue) and IDPr (yellow) are docking into hPARG (PDB: 5A7R). B. ADPr (blue) and IDPr (yellow) are docking into LchARH3 (PDB: 6G1Q). C. Synthesis of TFMU-IDPr; for pNP-IDPr synthesis please see Figure S2.
Figure 4.
Figure 4.
Michaelis-Menton kinetics of recombinantly expressed human PARG and ARH3. A. Kinetics of human PARG processing TFMU-ADPr. B. Kinetics of human ARH3 processing TFMU-ADPr. C. Selectivity of TFMU-IDPr for processing by human ARH3 over human PARG. Error bars indicate SEM, n = 3.
Figure 5.
Figure 5.
Measurement of PARG activity in cell lysate and validation of TFMU-IDPr selectivity. A. Selectivity for ARH3 by TFMU-IDPr validated by CRISPR-Cas9 knockout of ARH3 in U2OS cells. PARG activity was measured in the presence and absence of PARG inhibitor PDD00017273 using TFMU-IDPr at 200 μM. Error bars indicated SEM, n=3. Significance levels are given by asterisks: p<0.05 (*), p<0.01 (**), p<0.001 (***), p>0.05 (n.s.). B. Same as A but with 200 μM TFMU-ADPr. C. Validation of ARH3 knockout in U2OS as measured by Western blotting. D. Western blotting to determine ARH3 expression levels in various indicated cell lines; representative blot of two independent replicates shown, see Figure S4 for all replicates. E. Correlation of ARH3 expression (measured by Western blotting) with ARH3 activity (measured by TFMU-IDPr hydrolysis). Data points reflect average values from different mammalian cell lines. Red indicates MCF10A. ARH3 activity was measured with 200 μM TFMU-IDPr.
Figure 6.
Figure 6.
Selective inhibition of ARH3 by ADP-ribosylated arginine. A. Kinetics of inhibition of ARH3 by ADPr-Arg. B. Lineweaver-Burke plot of ADPr-Arg inhibition of ARH3. C. Selectivity of ADPr-Arg for ARH3 over hPARG is indicated by a shift in dose-response curve. D. Structure of ADPr-Arg.

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