Increasing lifespan by modulation of WWP-1 and UBC-18
View Patent ↗Ubiquitin ligase wwp-1 and ubiquitin conjugating enzyme ubc-18 are identified in nematodes as mediators of dietary restriction induced longevity and therefore as targets for modulation of lifespan in animals. Methods of screening for compounds that modulate longevity by assaying wwp-1 ubiquitination pathway parameters are provided, as are related systems. In addition, methods of using wwp-1 and/or ubc-18 to modulate longevity or delay onset of age-related diseases are described.
1. A method of identifying a modulator of longevity, the method comprising:
providing a C. elegans animal;
administering a test compound to the C. elegans animal; and
monitoring expression or activity of a C. elegans wwp-1 E3 ubiquitin ligase or a homolog thereof, expression or activity of a C. elegans ubc-18 E2 ubiquitin conjugation enzyme or a homolog thereof, and/or post-translational modification state of a C. elegans wwp-1 E3 ubiquitin ligase or a homolog thereof or a substrate thereof in the animal, wherein an increase in expression or activity of the C. elegans wwp-1 or the homolog thereof, an increase in expression or activity of the C. elegans ubc-18 or the homolog thereof, increased phosphorylation of the C. elegans wwp-1 or the homolog thereof, and/or increased ubiquitination of the substrate indicates that the test compound modulates longevity.
2. The method of claim 1 , wherein the C. elegans animal expresses a fusion protein comprising GFP or a homolog thereof and either a C. elegans wwp-1 or a homolog thereof or a C. elegans ubc-18 or a homolog thereof, wherein monitoring expression of the C. elegans wwp-1 or ubc-18 or the homolog thereof comprises monitoring an optical signal from the GFP.
3. A method of identifying a modulator of longevity, the method comprising:
providing an isolated cell;
contacting the cell with a test compound; and
monitoring expression or activity of a C. elegans wwp-1 E3 ubiquitin ligase or a homolog thereof, expression or activity of a C. elegans ubc-18 E2 ubiquitin conjugation enzyme or a homolog thereof, and/or post-translational modification state of a C. elegans wwp-1 E3 ubiquitin ligase or a homolog thereof or a substrate thereof in the cell, wherein an increase in expression or activity of the C. elegans wwp-1 or the homolog thereof, an increase in expression or activity of the C. elegans ubc-18 or the homolog thereof, increased phosphorylation of the C. elegans wwp-1 or the homolog thereof, and/or increased ubiquitination of the substrate indicates that the test compound modulates longevity.
4. The method of claim 3 , wherein the cell expresses a fusion protein comprising GFP or a homolog thereof and either a C. elegans wwp-1 or a homolog thereof or a C. elegans ubc-18 or a homolog thereof, wherein monitoring expression of the C. elegans wwp-1 or ubc-18 or the homolog thereof comprises monitoring an optical signal from the GFP.
5. The method of claim 1 , wherein the C elegans animal is dietary-restricted.
6. The method of claim 1 , wherein the C. elegans wwp-1 homolog is selected from Drosophila and mammalian homologs.
7. The method of claim 1 , wherein the C. elegans wwp-1 homolog comprises an amino acid sequence at least 90% identical to the amino acid sequence depicted in SEQ ID NO: 9.
8. The method of claim 1 , wherein the C. elegans wwp-1 comprises the amino acid sequence depicted in SEQ ID NO: 9.
9. The method of claim 1 , wherein the C. elegans ubc-18 homolog is selected from Drosophila and mammalian homologs.
10. The method of claim 1 , wherein the C. elegans ubc-18 homolog comprises an amino acid sequence at least 90% identical to the amino acid sequence depicted in SEQ ID NO: 11.
11. The method of claim 1 , wherein the C. elegans ubc-18 comprises the amino acid sequence depicted in SEQ ID NO: 11.
12. The method of claim 1 , wherein the C. elegans animal exhibits increased phosphorylation of the C. elegans wwp-1 or the homolog thereof, and/or increased ubiquitination of the substrate.
13. The method of claim 5 , wherein the dietary-restricted C. elegans animal exhibits reduced expression or activity of the C. elegans wwp-1 or the homolog thereof and/or reduced expression or activity of the C. elegans ubc-18 or the homolog thereof.
14. The method of claim 5 , wherein the dietary-restricted C. elegans animal expresses a dominant negative of the C. elegans wwp-1 or the homolog thereof or a dominant negative form of the C. elegans ubc-18 or the homolog thereof.
15. The method of claim 1 , wherein the C. elegans animal overexpresses C. elegans wwp-1 or the homolog thereof.
16. The method of claim 1 , wherein the test compound is selected from the group consisting of: a small molecule, a polypeptide, an antibody, a nucleic acid, an antisense molecule, and a double-stranded RNA.
17. The method of claim 1 , wherein the C. elegans wwp-1 homolog is encoded by a nucleic acid sequence at least 90% identical to the nucleic acid sequence depicted in SEQ ID NO: 10.
18. The method of claim 1 , wherein the C. elegans ubc-18 homolog is encoded by a nucleic acid sequence at least 90% identical to the nucleic acid sequence depicted in SEQ ID NO: 12.