The silencing protein SIR2 and its homologs are NAD-dependent protein deacetylases

Abstract

Homologs of the chromatin-bound yeast silent information regulator 2 (SIR2) protein are found in organisms from all biological kingdoms. SIR2 itself was originally discovered to influence mating-type control in haploid cells by locus-specific transcriptional silencing. Since then, SIR2 and its homologs have been suggested to play additional roles in suppression of recombination, chromosomal stability, metabolic regulation, meiosis, and aging. Considering the far-ranging nature of these functions, a major experimental goal has been to understand the molecular mechanism(s) by which this family of proteins acts. We report here that members of the SIR2 family catalyze an NAD-nicotinamide exchange reaction that requires the presence of acetylated lysines such as those found in the N termini of histones. Significantly, these enzymes also catalyze histone deacetylation in a reaction that absolutely requires NAD, thereby distinguishing them from previously characterized deacetylases. The enzymes are active on histone substrates that have been acetylated by both chromatin assemblylinked and transcription-related acetyltransferases. Contrary to a recent report, we find no evidence that these proteins ADPribosylate histones. Discovery of an intrinsic deacetylation activity for the conserved SIR2 family provides a mechanism for modifying histones and other proteins to regulate transcription and diverse biological processes.Y east silent information regulator 2 (SIR2) protein functions in transcriptional silencing of the silent mating loci, telomeres, and rDNA (1-3). It is found in a chromatin-bound complex with SIR3 and SIR4 at the silent mating loci and telomeres, and in a different complex at rDNA (4-6). Four additional SIR2 homologs exist in yeast (HST1-4), and related proteins are found from archaeabacteria to eubacteria to mammals (7). Until recently, very little was known about the in vivo activity of this family of proteins. An important breakthrough came with the identification of the Salmonella typhimurium CobB protein as a SIR2 homolog (8). CobB can partially fulfill the requirement for CobT in vitamin B 12 synthesis. Because CobT protein was known to transfer ribose 5Ј-phosphate from nicotinic acid mononucleotide to a precursor of vitamin B 12 , it prompted tests of Sir2-like proteins for phosphoribosyltransferase activity. Indeed, Frye (9) found that Escherichia coli CobB had NAD-dependent ADP-ribosyltransferase activity. He also reported that both CobB and a human SIR2-like protein could transfer radioactivity from [ 32 P]NAD to albumin. Very recently, another group (10) reported that yeast SIR2 can ADP-ribosylate itself as well as histones and albumin.Here we show that members of the SIR2 family of enzymes catalyze an NAD-nicotinamide exchange reaction that requires the presence of acetylated lysines such as are found in the N termini of histones. Furthermore, these enzymes also catalyze histone deacetylation in a reaction that absolutely depends on NAD, thereby distinguishing them from previously known deacet...

Sirtuins: critical regulators at the crossroads between cancer and aging

“…The conserved catalytic domain shared by the sirtuins functions as a mono-ADP-ribosyltransferase and as a b-nicotinamide adenine dinucleotide (NAD þ )-dependent lysine deacetylase (Figure 2) (Frye, 1999;Imai et al, 2000;Landry et al, 2000;Smith et al, 2000). The deacetylase domain in the sirtuins is different from that of class I and II HDACs, which are Zn þ -dependent enzymes (North and Verdin, 2004;Longo and Kennedy, 2006).…”

“…The deacetylase domain in the sirtuins is different from that of class I and II HDACs, which are Zn þ -dependent enzymes (North and Verdin, 2004;Longo and Kennedy, 2006). Sirtuins use one NAD þ molecule and generate acetyl-ADP-ribose and nicotinamide during the deacetylation reaction (Landry et al, 2000). While SIRT1, 2, 3 and 5 have significant deacetylase activity towards a histone H4 peptide, SIRT4, 6 and 7 have low to undetectable deacetylase activity in vitro on tested substrates (Langley et al, 2002;North et al, 2003).…”

Sir2 Protein Deacetylases: Evidence for Chemical Intermediates and Functions of a Conserved Histidine

“…ADPr and OAADPr formation was measured using the previously described thin layer chromatography (TLC) assay (16,43,45) with the following modifications. Reactions were quenched by spotting 1 μL timepoints of the reactions directly onto the silica-gel TLC plates.…”

“…In the initial chemical step, nicotinamide is released upon formation of an enzyme:ADP-ribose:acetylated protein intermediate, which has been postulated to be an α-1'-O-alkylamidate (Scheme 1) (28)(29)(30). One line of evidence for an α-1'-O-alkylamidate is the ability of nicotinamide, a potent product inhibitor (15,16,(38)(39)(40), to rapidly react with the enzyme:ADP-ribose:acetylated protein intermediate, regenerating NAD + and acetylated protein by a process dubbed the nicotinamide exchange (or transglycosidation) reaction (39,40). In addition, the 2'-hydroxyl of NAD + does not play a significant role in the first chemical step since replacement with fluorine results in only a slight decrease in the nicotinamide exchange rate (39).…”

Do Sirtuins Promote Mammalian Longevity?: A Critical Review on Its Relevance to the Longevity Effect Induced by Calorie Restriction

“…SIRT1 activity is regulated by AMPKmediated increase of cellular NAD + levels and the [NAD + ]/ [NADH] ratio (Cantó et al, 2009;Imai et al, 2000;Landry et al, 2000). The observation that AMPK modulates the activity of SIRT1 targets, including PGC1a, FOXO1 and FOXO3a transcription factors, raises the possibility that AMPK and SIRT1 are implicated in beneficial functions of CR on metabolic adaptation (Cantó et al, 2009).…”