Giardia duodenalis GlSir2.2, homolog of SIRT1, is a nuclear-located and NAD+-dependent deacethylase

Abstract

Sir2 family proteins are highly conserved and catalyze Nicotinamide Adenine Dinucleotide (NAD+)-dependent protein deacetylation reaction that regulates multiple cellular processes. Little is known about Sir2 family proteins in Giardia. In this research, Sir2 homologs of Giardia were Phylogenetically analyzed. GL50803_10707 (GlSIR2.2) showed strong homology to SIRT1 and was the only parasite SIRT1 homolog being reported to date. Recombinant GlSIR2.2 (rGlSIR2.2) was expressed and purified. The renaturied recombinant protein showed a typical NAD-dependent protein deacetylase activity that could be inhibited by nicotinamide, with IC50 of 4.47 mM rGlSIR2.2 displayed deacetylase activity under varied NAD+, with Km, kcat and kcat/Km values of 31.71 μM, 1.4 × 10−3 s−1, and 4.42 × 10−5 μM−1 s−1. Similarly, the steady-state kinetic parameters with varied ZMAL, yielded Km, kcat and kcat/Km values of 96.89 μM, 4.7 × 10−3 s−1, and 4.85 × 10−5 μM−1 s−1. Anti-rGlSIR2.2 serum was used to probe subcellular localization of GlSIR2.2 and strong staining was found predominantly in the nucleus. So we demonstrated that GlSIR2.2 was a SIRT1-like, nuclear-located, NAD+-dependent deacetylase. This is the first report of deacetylase activity of Sir2 family protein in Giardia.