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

Abstract

The 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 Mg2+‐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 (ARH1‐3). 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 alanine. 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.

Selective Small Molecule Inhibition of Poly(ADP-Ribose) Glycohydrolase (PARG)

“…The specificity of the RBPIs and ADP-HPD for PARG was further evaluated by testing these compounds against the other known PAR glycohydrolase enzyme, ADP-ribosylhydrolase 3 (ARH3), and against PARP-1. ARH3 is a 39 kDa human protein known to have two enzymatic functions: deacetylation of O -acetyl ADP-ribose and PAR degradation, though deacetylation has been suggested as its main function. − Although both enzymes can process PAR, ARH3 does not have a sequence or structure similar to that of PARG . To achieve a comparable rate of PAR degradation in these experiments, 1000-fold more ARH3 (256 nM) compared to PARG (0.24 nM) was required in the in vitro assay, along with the addition of 4 mM MgCl 2 .…”

ADP-Ribosylation, a Multifaceted Posttranslational Modification Involved in the Control of Cell Physiology in Health and Disease

“…205,206 Moreover, ARH3 hydrolyzes O-acetyl-ADPr, the product of the deacetylation reaction catalyzed by sirtuins. 207 Thus, so far these are the two only enzymes that can degrade PAR chains (Figure 3).…”

The ARH and Macrodomain Families of α-ADP-ribose-acceptor Hydrolases Catalyze α-NAD+ Hydrolysis

“…33 The PAR hydrolytic reaction is shared with ARH1, although ARH3 has about 50 times the specific activity of ARH1. 34 In addition to PAR, ARH3 and ARH1 cleave O-acetyl-ADPribose (OAADPr), 35 a Sirtuin deacetylase product. 36 Macrodomains are approximately 190 amino acid modules, first identified in Af1521 from Archaeoglobus f ulgidus, which bind mono-and poly-ADP-ribose.…”