Biochemical characterization of the ADP-ribosylation activity of sirtuins.

摘要

Endogenous mono-ADP-ribosylation in eukaryotes regulates several biological processes, though few cellular ADP-ribosyltransferases have been identified. Members of the sirtuin family, known for their protein deacetylase activity, have been reported to carry out protein ADP-ribosylation. However, it was generally thought that sirtuin ADP-ribosylation is a side reaction of the deacetylation mechanism, and not a true enzymatic reaction. In this work biochemical and biophysical studies were used to demonstrate that ADP-ribosylation activity of two sirtuin homologs is genuinely enzymatic. SIRT4, a mitochondrial sirtuin, ADP-ribosylates glutamate dehydrogenase (GDH), but does not deacetylate GDH. This demonstrates that SIRT4 ADP-ribosylation is not dependent on the deacetylation reaction. This is significant as the enzyme responsible for GDH ADP-ribosylation in the mitochondria was not previously identified. Unlike SIRT4, the nuclear sirtuin, TbSIR2RP1, has detectable deacetylase and ADP-ribosyltransferase activity. Biochemical data demonstrated that unacetylated and acetylated substrates were both amenable to TbSIR2 ADP-ribosylation, thus indicating that ADP-ribosylation is not a side reaction of deacetylation. In addition, ADP-ribosylation by TbSIR2RP1 showed arginine specificity in unacetylated substrates. Interestingly, arginine is not the major ADP-ribose acceptor in reactions with acetylated histone H1.1. These results suggest that TbSIR2RP1 ADP-ribosylation can occur using two distinct mechanisms, a direct ADP-ribosyltransfer reaction and a deacetylation-dependent reaction.