Methods of identifying longevity modulators and therapeutic methods of use thereof
The present invention is based at least in part on the discovery of a role for the JNK signaling pathway in longevity. In particular, the present inventors have shown that overexpression of c-jun N-terminal kinase 1 (jnk-1) extends lifespan and that said extended lifespan is associated with DAF-16 phosphorylation by JNK-1 and the consequent DAF-16 localization to the nucleus. Accordingly, the present invention features methods of identifying modulators of longevity in assays featuring organisms and/or cells having a JNK signaling pathway and, optionally, an IR signaling pathway. Also featured is an in vitro method of identifying an agent capable of enhancing longevity featuring an assay composition having a JNK signaling pathway molecule and insulin signaling pathway molecule. Further featured are therapeutic methods for the use of JNK signaling pathway modulators to enhance longevity, to prevent or reduce obesity and to prevent or treat type II diabetes.
1. A method for identifying an agent capable of modulating the physical interaction between c-jun N-terminal kinase 1 (JNK-1) or a mammalian ortholog thereof and DAF-16 or a mammalian ortholog thereof, comprising:
contacting a cell with a test agent, said cell expressing JNK-1 or a mammalian ortholog thereof and DAF-16 or a mammalian ortholog thereof; and
detecting physical interaction between said JNK-1 or mammalian ortholog thereof and said DAF-16 or mammalian ortholog thereof by examining the N-terminal phosphorylation and nuclear localization of said DAF-16 or mammalian ortholog thereof in the presence of said test agent as compared to a control cell to which no agent has been added;
wherein an increase in the N-terminal phosphorylation and nuclear localization of said DAF-16 or mammalian ortholog thereof in the presence of said test agent identifies said agent as increasing the physical interaction between said DAF-16 or mammalian ortholog thereof and said JNK-1 or mammalian ortholog thereof, and wherein a decrease in the N-terminal phosphorylation and nuclear localization of said DAF-16 or mammalian ortholog thereof in the presence of said test agent identifies said agent as decreasing the physical interaction between said DAF-16 or mammalian ortholog thereof and said JNK-1 or mammalian ortholog thereof.
2. The method of claim 1 , wherein the cell is a mammalian cell.
3. The method of claim 1 , wherein the cell is a human cell.
4. The method of claim 1 , wherein the cell is a bacterial cell or a yeast cell.
5. The method claim 1 , wherein the cell is a cell derived from a nematode.
6. A method for identifying an agent capable of modulating the physical interaction between JNK-1 or a mammalian ortholog thereof and DAF-16 or a mammalian ortholog thereof, comprising:
contacting an assay composition with a test compound, wherein said assay composition comprises JNK-1 or a mammalian ortholog thereof and DAF-16 or a mammalian ortholog thereof; and
detecting the phosphorylation of the N-terminal region of said DAF-16 or mammalian ortholog thereof as compared to a control assay composition to which no agent has been added;
wherein an increase in the phosphorylation of the N-terminal region of said DAF-16 or mammalian ortholog thereof in the presence of said agent identifies said agent as increasing the physical interaction between said JNK-1 or mammalian ortholog thereof and said DAF-16 or mammalian ortholog thereof, and wherein a decrease in the phosphorylation of the N-terminal region of said DAF-16 or mammalian ortholog thereof in the presence of said agent identifies said agent as decreasing the physical interaction between said JNK-1 or mammalian ortholog thereof and said DAF-16 or mammalian ortholog thereof.
7. The method of claim 6 , wherein said assay composition is a cell-free extract.
8. The method of claim 6 , wherein said assay composition comprises purified proteins.
9. The method of claim 1 or 6 , wherein said agent is identified based on its ability to decrease said physical interaction.
10. The method of claim 1 or 6 , wherein said agent is identified based on its ability to increase said physical interaction.
11. The method of claim 1 or 6 , wherein said agent is identified based on its ability to increase the N-terminal phosphorylation of said DAF-16 or mammalian orthologue thereof.
12. A method of identifying an agent that modulates the phosphorylation of DAF-16 or a mammalian ortholog thereof by c-jun N-terminal kinase 1 (JNK-1) or a mammalian ortholog thereof comprising:
contacting a cell with a test agent, said cell expressing JNK-1 or a mammalian ortholog thereof and DAF-16 or a mammalian ortholog thereof;
detecting phosphorylation of the N-terminal region of said DAF-16 or mammalian ortholog thereof in the presence of said test agent as compared to a control cell to which no agent has been added;
wherein an increase in said phosphorylation of said N-terminal region of said DAF-16 or mammalian ortholog thereof in the presence of said test agent identifies said agent as increasing the phosphorylation of said DAF-16 or mammalian ortholog thereof by said JNK-1 or mammalian ortholog thereof, and wherein a decrease in said phosphorylation of said N-terminal region of said DAF-16 or mammalian ortholog thereof in the presence of said test agent identifies said agent as decreasing the phosphorylation of DAF-16 or mammalian ortholog thereof by said JNK-1 or mammalian ortholog thereof.
13. The method of claim 12 , wherein the cell is a mammalian cell.
14. The method of claim 12 , wherein the cell is a human cell.
15. The method of claim 12 , wherein the cell is a bacterial cell or a yeast cell.
16. The method of claim 12 , wherein the cell is a cell derived from a nematode.
17. A method for identifying an agent capable of modulating the physical interaction between c-jun N-terminal kinase 1 (JNK-1) or a mammalian ortholog thereof and DAF-16 or a mammalian ortholog thereof, comprising:
contacting an assay composition with a test agent, wherein said assay composition comprises JNK-1 or a mammalian ortholog thereof and DAF-16 or a mammalian ortholog thereof; and
examining the co-immunoprecipitation of said JNK-1 or mammalian ortholog thereof and said DAF-16 or mammalian ortholog thereof in the presence of said test agent as compared to a control assay composition to which no agent has been added;
wherein an increase of said co-immunoprecipitation in the presence of said test agent identifies said agent as increasing the physical interaction between said DAF-16 or mammalian ortholog thereof and said JNK-1 or mammalian ortholog thereof, and wherein a decrease of said co-immunoprecipitation in the presence of said test agent identifies said agent as decreasing the physical interaction between said DAF-16 or mammalian ortholog thereof and said JNK-1 or mammalian ortholog thereof.