Sir2 activity
Methods for screening a compound, by providing a test mixture comprising a transcription factor, Sir2, and a Sir2 cofactor with the compound, and evaluating an activity of a component of the test mixture in the presence of the compound are described.
1. A method of screening a compound, comprising the steps of:
(a) contacting a compound that modulates Sir2-mediated deacetylation of a transcription factor, with a cell or a system that includes a Sir2 protein or an active portion thereof and the transcription factor or an active portion thereof; and
(b) measuring modulation of transcription of a gene whose expression is regulated by the transcription factor in the cell or the system, thereby screening for the compound.
2. The method of claim 1 , wherein the transcription factor is a p53 protein or an active portion thereof.
3. The method of claim 1 , wherein the transcription factor binds a specific DNA site.
4. The method of claim 1 , wherein measuring modulation of transcription of the gene includes comparing the transcription of the gene at a first concentration of the compound and at a second concentration of the compound.
5. The method of claim 4 , wherein the first or second concentration of the compound is zero.
6. The method of claim 1 , wherein the cell or system further includes a Sir2 cofactor.
7. The method of claim 6 , wherein the Sir2 cofactor is NAD or an NAD analog.
8. The method of claim 1 , wherein the Sir2 protein is a Sir2 variant that has reduced deacetylase activity.
9. The method of claim 1 , wherein the Sir2 protein is a human Sir2 protein.
10. The method of claim 9 , wherein the human Sir2 protein is human SIRT1.
11. The method of claim 1 , wherein the Sir2 protein is a murine Sir2 protein.
12. The method of claim 11 , wherein the murine Sir2 protein is murine Sir2α.
13. The method of claim 1 , wherein the Sir2 protein is exogenous and expressed from a heterologous nucleic acid.
14. The method of claim 1 , wherein the transcription factor is exogenous and expressed from a heterologous nucleic acid.
15. The method of claim 1 , further including the steps of:
(c) administering the compound to a cell or an animal, wherein modulation of transcription of the gene is measured by evaluating the effect of the compound on the cell or animal.
16. The method of claim 1 , wherein the active portion includes a core domain of the Sir2 protein.
17. The method of claim 16 , wherein the core domain includes amino acid residues 258-451 of SEQ ID No: 12.
18. The method of claim 10 , wherein the human SIRT1 comprises the amino acid sequence of SEQ ID NO: 12.
19. The method of claim 9 , wherein the human Sir2 protein is selected from the group consisting of SIRT3, SIRT4, and SIRT5.
20. The method of claim 19 , wherein the human Sir2 protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 18.
21. The method of claim 2 , wherein the p53 protein or active portion thereof comprises K382 of p53.