Blue light controls protein stability

Methods to reversibly regulate protein levels in cells are valuable for researchers in life science. A technology to induce degradation of a protein of interest by blue light has been developed and applied in cells and zebrafish embryos.

HFSP Cross-Disciplinary Fellow Kimberly Bonger and colleagues
authored on Thu, 13 March 2014

The ability to control protein activity in cells is effective to investigate the function of an unknown protein. Researchers use a wide range of strategies varying from gene depletions to small-molecule mediated inhibition of a specific protein. All methods may have benefits and drawbacks depending on the research question asked and the protein studied. Gene deletions, for example, are robust but not tunable and cannot be applied to study essential genes. Small-molecule inhibitors on the other hand may not be known or can be non-selective. Posttranslational regulation of protein levels may be beneficial in cases such as when the researcher can induce degradation of a protein of interest at a specific time in a tunable and reversible manner.

Figure: Top: General overview of the blue-light induced degradation (B-LID) system. Bottom: Zebrafish embryos were microinjected with mRNA encoding yellow fluorescent protein (YFP) fused to the light-sensitive B-LID domain. Embryos raised in dark were significantly more fluorescent than embryos raised in blue-light.

Over the years, our lab has developed several methods to regulate protein levels in cells that can be mediated by the addition of a small molecule.1,2. The protein of interest (POI) is fused on a genetic level to a regulatory domain that allows stabilization or degradation of the whole fusion protein, which allows the protein of interest to be present in a reversible and tunable manner. Besides the existing small-molecule control of protein degradation in cells, a method to control proteins by light may be beneficial, as light can be spatially and temporally applied. In this recent paper, we have engineered a known light sensitive protein domain (that is, a LOV2 domain from Avena sativa (AsLOV2)) to contain a peptide that can induce degradation mediated by the ubiquitin protein system (UPS). In the absence of light, the peptide is hidden and therefore it cannot be recognized by the UPS allowing the domain to be present in the cell. In the presence of non-toxic blue light, the protein domain undergoes a conformational change after which the peptide is exposed. This leads to recognition of the peptide by the UPS after which the domain, including a potential fusion protein, is degraded by the proteasome.

We have successfully applied the method in cells and in zebrafish embryos. The major benefit of the system is that the protein tunability depends on fusion of a single protein regulatory domain. With the current gene editing methods, this method has great potential to be applied to other higher organisms to allow local activation of a specific protein of interest, for example in the field of neurobiology.

Reference

General method for regulating protein stability with light. Bonger, K.M., Rakhit R, Payumo A.Y., Chen J.K., Wandless T.J.. ACS Chem Biol. 9, 111-115 (2014).

Other References

 1.   A rapid, reversible, and tunable method to regulate protein function in living cells using synthetic small molecules. Banaszynski, L. A., Chen, L. C., Maynard-Smith, L. A., Ooi, A. G. L. & Wandless, T. J.  Cell 126, 995-1004 (2006).

2.    Small-molecule displacement of a cryptic degron causes conditional protein degradation. Bonger, K.M., Chen, L.C., Liu, C.W., Wandless, T.J.  Nat. Chem. Biol. 7, 531–537. (2011).

Pubmed link