Per Instructions of Dr. Richard Cannon, we do not list human specimens in this report, even though they are used in our research. The required survey is given as part of the LAM annual report. OAADPr may act independently of protein deacetylation in the stabilization of chromatin, formation of silencing complexes, ion-channel gating, and energy metabolism. Thus, ARH3 can participate in distinct signal transduction pathways that involve poly(ADPr) and OAADPr. During the Sir2-catalyzed reaction, 2-OAADPr is described as the product released from the enzyme. After release, 2-OAADPr rapidly interconverts at pH 7.5 with 3-OAADPr, with which it exists in equilibrium, via acetyl migration, in a ratio of about 1:1 (26,27). In contrast, 2- and 3-N-acetyl-ADPr analogs of OAADPr do not undergo acetyl migration, although they do mimic OAADPr binding to macro H2A1.1. We thought it unlikely that ARH3 would hydrolyze the 1-O-ADPr linkage in poly(ADPr), and act also on 2- or 3-linkage in OAADPr. We proposed that, analogous to its poly(ADPr) glycohydrolase activity, ARH3 uses 1-OAADPr, rather than the 2- or 3-isomer as substrate, and that 1, 2, and 3-OAADPr exist in equilibrium at neutral and basic pH values, thus generating 1-OAADPr for hydrolysis by ARH3. In agreement, we found at pH 9.0 not only the 2- and 3-isomers, but substantial quantities of a third isomer whose properties were consistent with 1-OAADPr. Further, ARH3 OAADPr hydrolase activity was greater at pH 9.0 than at pH 7.0, or pH 5.0 where 3-OAADPr predominated. Consistent with this hypothesis, IC50 values for ARH3 inhibition by 2- and 3-N-acetyl-ADPr analogs of OAADPr were significantly higher than the IC50 value for ADPr. ARH1, which catalyzed hydrolysis of -ADP-ribosyl(arginine), also hydrolyzed OAADPr, but at a much slower rate than ARH3, and with increased activity at pH 9.0. Other ARH1-catalyzed reactions that involve cleavage at C-1 were more rapid at pH 7.0. ARH3-catalyzed hydrolysis of OAADPr in H218O resulted in incorporation of one 18O into ADP-ribose by mass spectrometric analysis, consistent with cleavage at the C-1 position. Together, these data suggest that ARH3 catalyzes hydrolysis of the 1-O linkage in both poly(ADPr)and OAADPr.

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