Metabolite of SIR2 Reaction Modulates TRPM2 Ion Channel

Abstract

The transient receptor potential melastatin-related channel 2 (TRPM2) is a nonselective cation channel, whose prolonged activation by oxidative and nitrative agents leads to cell death. Here, we show that the drug puromycin selectively targets TRPM2-expressing cells, leading to cell death. Our data suggest that the silent information regulator 2 (Sir2 or sirtuin) family of enzymes mediates this susceptibility to cell death. Sirtuins (2). This influx is thought to occur as a consequence of TRPM2 channel activation via ADP-ribose (ADPr) binding to the cytoplasmic, C-terminal domain of the channel. This domain, termed NudT9-H (NudT9 homology), contains an apparent Nudix enzymatic motif and shares significant homology to the mitochondrial NudT9 enzyme (4, 5), a member of the Nudix ADPr hydrolase family. Indeed, electrophysiology experiments have shown that low micromolar levels of ADPr activate the TRPM2 channel (4, 6), and that this activation is mediated through the NudT9-H domain (7, 8), implicating a direct ADPr and NudT9-H interaction.The NudT9-H domain has been reported to have hydrolytic activity toward ADPr, albeit at a ϳ100-fold lower rate than NudT9 (4, 9). Although it was initially thought that the ADPr hydrolase activity of TRPM2 might have a role in regulating TRPM2 gating via hydrolysis of bound ADPr, recent data have demonstrated that mutations in putative catalytic residues of NudT9-H do not affect TRPM2 channel gating, leaving the function of this activity unclear (8). Some studies suggest that hydrogen peroxide directly activates the TRPM2 channel (3, 10); however, it has been reported recently that this oxidative agent acts indirectly by triggering the release of ADPr from an intracellular compartment (8, 11). The mechanism of TRPM2 activation by hydrogen peroxide may prove to be more complex, as hydrogen peroxide and ADPr have also been suggested to act cooperatively in TRPM2 channel activation (12).In this article, we have investigated the ability of TRPM2 to sensitize cells to cellular insults other than those inducing oxidative stress. Here, we demonstrate that puromycin, a well known pleiotropic cell stress agent, selectively targets TRPM2-expressing cells, leading to cell death. Our studies suggest that Sir2 enzymes play an important role in mediating this response. We found that TRPM2 has the capacity to sense OAADPr, the unique metabolite of the Sir2 protein deacetylase reaction, and induce cell death in response to puromycin. Furthermore, we report the first direct evidence that OAADPr binds to the NudT9-H domain and modulates TRPM2 channel activity. Our data have important implications for the potential physiological roles of ADPr-related molecules as secondary messengers. This report links Sir2 enzymes (sirtuins) with the TRPM2 channel through the Sir2 metabolite OAADPr. This newly discovered association provides a plausible mechanism for previously described sirtuin functions, which include control of stress response pathways and metabolic regulation. EXPERIMENTAL PROCEDURESCell Cul...

Hydrolysis of O-Acetyl-ADP-ribose Isomers by ADP-ribosylhydrolase 3

“…8B). As expected, a 30-min reaction of heatinactivated ARH3 displayed some nonenzymatic 18 O incorporation (14% of total 558.15 ϩ 560.15 m/z) (Fig. 8C).…”

“…8A). Reactions with heat-inactivated ARH3 yielded nonenzymatically hydrolyzed ADPr (m/z ϭ 558.15) with no significant 18 O incorporation above natural abundance (Fig. 8B).…”

“…Then the mixture was diluted with methanol and subjected to mass spectrometry as described under "Experimental Procedures." B and C, m/z ϭ 558.15 corresponds to ADPr, and m/z ϭ 560.15 to ADPr containing 18 O atom. 18 O incorporation was measured by the relative peak heights at each m/z.…”

“…OAADPr is the product of NAD ϩ -dependent protein deacetylation reactions catalyzed by Silent information regulator 2 (Sir2) proteins that contribute to diverse operations such as gene silencing, DNA repair, chromosomal stability, and lifespan extension (12,13). OAADPr may act independently of protein deacetylation in the stabilization of chromatin, formation of silencing complexes, ion-channel gating, and energy metabolism (15)(16)(17)(18)(19). Thus, ARH3 can participate in distinct signal transduction pathways that involve poly(ADPr) and OAADPr.…”

“…The reaction product, ADPr, was analyzed by mass spectrometry to quantify 18 O incorporation into ADPr. The major ADPr product (85% of total 558.15 ϩ 560.15 m/z) of ARH3-catalyzed hydrolysis incorporated one 18 O (560.15 m/z) (Fig.…”