Sir2 products and activities
View Patent ↗A novel compound, 2′/3′-O-acetyl-ADP-ribose, is provided. The compound is a mixture of the 2′ and 3′ regioisomers of O-acetyl-ADP ribose, and is formed nonenzymatically from 2′-O-acetyl-ADP-ribose, which is the newly discovered product of the reaction of Sir2 enzymes with acetylated peptides and NAD + . Analogs of 2′/3′-O-acetyl-ADP-ribose are also provided. Additionally, methods of preparing 2′/3′-O-acetyl-ADP-ribose, methods of determining whether a test compound is an inhibitor of a Sir2 enzyme, methods of detecting Sir2 activity in a composition, methods of deacetylating an acetylated peptide, and methods of inhibiting the deacetylation of an acetylated peptide are provided. Prodrugs of 2′/3′-O-acetyl-ADP-ribose are also provided.
1. A method of detecting activity of a Sir2 in a composition, the method comprising
combining the composition with NAD + and an acetylated peptide substrate of a Sir2 in a reaction mixture under conditions and for a time sufficient to deacetylate the peptide in the presence of Sir2 activity, wherein the acetylated peptide substrate is not from a histone; then
identifying 2′/3′-O-acetyl-ADP-ribose in the mixture to determine the presence of Sir2 activity in the composition.
2. The method of claim 1 , wherein the 2′/3′-O-acetyl-ADP-ribose is identified after subjecting the reaction mixture to chromatography.
3. The method of claim 1 , wherein the chromatography is HPLC.
4. The method of claim 1 , wherein the chromatography is on a Sephadex-DEAE column.
5. The method of claim 1 , wherein the NAD + is radiolabeled such that the 2′/3′-O-acetyl-ADP-ribose is radiolabeled after the peptide is deacetylated.
6. The method of claim 1 , wherein the acetylated peptide substrate is homologous to an acetylated region of a regulatory domain of a p53.
7. The method of claim 6 , wherein the acetylated peptide comprises JB11 or JB12.
8. The method of claim 1 , wherein the composition is an extract of a prokaryote.
9. The method of claim 1 , wherein the composition is an extract from a eukaryote.
10. The method of claim 1 , wherein the Sir2 activity is quantified.
11. A method of detecting activity of a Sir2 in a composition, the method comprising
combining the composition with radiolabeled NAD + and an acetylated peptide substrate of a Sir2 in a reaction mixture under conditions and for a time sufficient to deacetylate the peptide in the presence of Sir2 activity; then
identifying radiolabeled 2′/3′-O-acetyl-ADP-ribose in the mixture to determine the presence of Sir2 activity in the composition.
12. The method of claim 11 , wherein the 2′/3′-O-acetyl-ADP-ribose is identified after subjecting the reaction mixture to chromatography.
13. The method of claim 12 , wherein the chromatography is on a Sephadex-DEAE column.
14. The method of claim 11 , wherein the NAD + is radiolabeled at the 5′ position with 14 C.
15. The method of claim 11 , wherein the acetylated peptide substrate comprises at least two amino acids, wherein at least one of the amino acids comprise a lysine residue that is acetylated at the ε-amino moiety.
16. The method of claim 11 , wherein the acetylated peptide substrate is homologous to an acetylated region of a regulatory domain of a p53.
17. The method of claim 11 , wherein the acetylated peptide substrate is homologous to an acetylated region of a histone.
18. The method of claim 11 , wherein the composition is an extract of a prokaryote.
19. The method of claim 11 , wherein the composition is an extract from a eukaryote.
20. The method of claim 11 , wherein the Sir2 activity is quantified.