The 39-kDa poly(ADP-ribose) glycohydrolase ARH3 hydrolyzes O-acetyl-ADP-ribose, a product of the Sir2 family of acetyl-histone deacetylases - PubMed (original) (raw)

The 39-kDa poly(ADP-ribose) glycohydrolase ARH3 hydrolyzes O-acetyl-ADP-ribose, a product of the Sir2 family of acetyl-histone deacetylases

Tohru Ono et al. Proc Natl Acad Sci U S A. 2006.

Abstract

The silent information regulator 2 (Sir2) family of NAD-dependent N-acetyl-protein deacetylases participates in the regulation of gene silencing, chromatin structure, and longevity. In the Sir2-catalyzed reaction, the acetyl moiety of N-acetyl-histone is transferred to the ADP-ribose of NAD, yielding O-acetyl-ADP-ribose and nicotinamide. We hypothesized that, if O-acetyl-ADP-ribose were an important signaling molecule, a specific hydrolase would cleave the (O-acetyl)-(ADP-ribose) linkage. We report here that the poly(ADP-ribose) glycohydrolase ARH3 hydrolyzed O-acetyl-ADP-ribose to produce ADP-ribose in a time- and Mg(2+)-dependent reaction and thus could participate in two signaling pathways. This O-acetyl-ADP-ribose hydrolase belongs to a family of three structurally related 39-kDa ADP-ribose-binding proteins (ARH1-ARH3). ARH1 was reported to hydrolyze ADP-ribosylarginine, whereas ARH3 degraded poly(ADP-ribose). ARH3-catalyzed generation of ADP-ribose from O-acetyl-ADP-ribose was significantly faster than from poly(ADP-ribose). Like the degradation of poly(ADP-ribose) by ARH3, hydrolysis of O-acetyl-ADP-ribose was abolished by replacement of the vicinal aspartates at positions 77 and 78 of ARH3 with asparagine. The rate of O-acetyl-ADP-ribose hydrolysis by recombinant ARH3 was 250-fold that observed with ARH1; ARH2 and poly(ADP-ribose) glycohydrolase were inactive. All data support the conclusion that the Sir2 reaction product O-acetyl-ADP-ribose is degraded by ARH3.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Identification of products of SIRT1- and ARH3-catalyzed reactions. (A) Synthesis of _O-_acetyl-[14C]ADP-ribose catalyzed by SIRT1. Data are expressed as picomoles of 14C per fraction. Peaks: 1, ADP-ribose; 2, _O-_acetyl-ADP-ribose; 3, β-NAD. The results of duplicate assays in three experiments were averaged (n = 6; average ± SD.). (B) Hydrolysis of _O-_acetyl-[14C]ADP-ribose catalyzed by ARH3. Peaks: 4, ADP-ribose; 5, _O-_acetyl-ADP-ribose. The results of duplicate assays in three experiments were averaged (n = 6; average ± SD). (C) High-resolution polyacrylamide gel electrophoresis of substrates and products in reactions involving of _O-_acetyl-[32P]ADP-ribose. Lane 1, [32P]NAD. Lane 2, [32P]AMP produced by pyrophosphatase cleavage of [32P]NAD (see Materials and Methods for details). Lane 3, [32P]ADP-ribose produced from [32P]NAD by CTA glycohydrolase activity (see Materials and Methods for details). Lane 4, _O-_acetyl-[32P]ADP-ribose synthesized by SIRT1 as in Materials and Methods. Lane 5, [32P]ADP-ribose produced by ARH3 from _O-_acetyl-[32P]ADP-ribose as in Materials and Methods. All assays were run in duplicate. Results were similar in three experiments. (D) Identification of _O-_acetyl-ADP-ribose by MALDI-TOF mass spectrometry analysis (see Materials and Methods for details). MALDI-TOF mass spectrometry was used to identify the RP-HPLC products; a molecule of 600 m/z was identified, consistent with the formation of _O-_acetyl-ADP-ribose. (E) Identification of the reaction products formed by ARH3 from _O-_acetyl-ADP-ribose. We identified a product of 558 m/z, consistent with the predicted mass of ADP-ribose.

Fig. 2.

Fig. 2.

Hydrolysis of _O-_acetyl-ADP-ribose by ARH3. (A) Hydrolysis of _O-_acetyl-ADP-ribose by ARH3. Assays containing 1.5 pmol of mouse ARH3 and 2.5 μM _O-_acetyl-[14C]ADP-ribose, 50 mM potassium phosphate (pH 7.0), 10 mM MgCl2, and 5 mM DTT (total volume 200 μl) were incubated at 30°C for the indicated time before separation of substrate and products by using RP-HPLC as described in Materials and Methods. Data are means ± 1/2 the range of values from duplicate assays. Results were similar in two experiments. (B) Assays containing 2.5 μM _O-_acetyl-[14C]ADP-ribose and the indicated amount of mouse ARH3 in 200 μl of buffer were incubated for 1 h at 30°C before separation of substrate and products by using RP-HPLC as described in Materials and Methods. Data are means ± 1/2 the range of values from duplicate assays. Results were similar in two experiments.

Fig. 3.

Fig. 3.

Effect of DTT and Mg2+ on _O-_acetyl-ADP-ribose hydrolase activity. Assays with 1.5 pmol of mouse ARH3 and 2.5 μM _O-_acetyl-[14C]ADP-ribose, 50 mM potassium phosphate (pH 7.0), and with or without 5 mM DTT and/or 10 mM MgCl2 (total volume 200 μl) were incubated for 1 h at 30°C before separation of substrate and products by using RP-HPLC as described in Materials and Methods. Data are means ± 1/2 the range of values from duplicate assays. Results were similar in two experiments.

Fig. 4.

Fig. 4.

Hydrolysis of _O-_acetyl-ADP-ribose by ARH1, ARH2, and ARH3. (A) Hydrolysis of _O-_acetyl-[14C]ADP-ribose by ARH1, ARH2, and ARH3. Assays with the indicated amount of mouse ARH1(●), ARH2 (■), and ARH3 (○) and 2.5 μM _O-_acetyl-[14C]ADP-ribose, 50 mM potassium phosphate (pH 7.0), 10 mM MgCl2, and 5 mM DTT (total volume, 200 μl) were incubated for 2 h at 30°C before separation of substrate and products by using RP-HPLC as described in Materials and Methods. Data are means ± 1/2 the range of values from duplicate assays. Results were similar in two experiments. (B) Assays containing 230 pmol of mouse ARH1 in 200 μl of buffer were incubated at 30°C for the indicated time before separation of substrate and products by using RP-HPLC as described in Materials and Methods. Data are means ± 1/2 the range of values from duplicate assays. Results were similar in two experiments.

Fig. 5.

Fig. 5.

Inhibition assay of ARH3 hydrolase activity by ADP-ribose and β-NAD. Assays containing 2 pmol of mouse ARH3 and 2.5 μM _O-_acetyl-[14C]ADP-ribose and the indicated amount of β-NAD (●) or ADP-ribose (○), 50 mM potassium phosphate (pH 7.0), 10 mM MgCl2, and 5 mM DTT (total volume 200 μl) were incubated for 2 h at 30°C before separation of substrate and products by using RP-HPLC as described in Materials and Methods. Data are means ± 1/2 the range of values from duplicate assays. Results were similar in three experiments.

Fig. 6.

Fig. 6.

Hydrolysis of _O-_acetyl-ADP-ribose by wild-type and mutant forms of ARH3 or PARG. Assays with 1.5 pmol of wild-type or mutant human (D77/78N) ARH3 or 20 pmol of PARG and 2.5 μM _O-_acetyl-[14C]ADP-ribose, 50 mM potassium phosphate (pH 7.0), 10 mM MgCl2, and 5 mM DTT (total volume, 200 μl) were incubated for 2 h at 30°C before separation of substrate and products by using RP-HPLC as described in Materials and Methods. All assays were run in duplicate. Data are means ± 1/2 the range of values from duplicate assays. Results were similar in two experiments. *D77/78N, assays incubated with 15 pmol of mutant human ARH3 (D77/78N).

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