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Mar 14, 2014

Proteins under a Spotlight

A genetically encoded protein domain that is degraded upon exposure to nontoxic blue light is described in this literature review.

Proteins under a Spotlight

This system, the authors say, can work in vivo by injecting mRNA encoding a mCherry-BLID fusion into zebrafish. [© Jess Yu - Fotolia.com]

  • Click Image To Enlarge +
    Figure. Stability of the blue-light inducible degradation (B-LID) domain is regulated by light. (a) Schematic of the B-LID domain genetically fused to a protein of interest (POI). Expression of the protein and exposure to blue light results in degradation of the fusion construct. (b) NIH3T3 cells stably expressing the YFP-LOV24 fusion protein were treated with either vehicle or 10 µM MG132 and were kept in the dark or irradiated with blue light for 2 h and analyzed by flow cytometry and immunoblot using anti-YFP antibody. Tubulin is the loading control. The error bars represent the standard deviation of the mean on the basis of at least two experiments. (c) NIH3T3 cells expressing the YFP-LOV24 fusion protein were illuminated with blue light, and degradation of the fusion protein was monitored at various times using flow cytometry and anti-YFP immunoblotting. (d) Fluorescence micrograph of NIH3T3 cells stably expressing the YFP-LOV24 fusion protein. Cells were kept in the dark or illuminated with blue light for 2 h. The scale bar represents 100 µm.

    Methods to rapidly regulate the levels of proteins in cells are desirable to study the function of proteins of interest. Sequences of amino acids leading to protein degradation (so-called “degrons”) have been discovered, including a four amino acid peptide (RRRG) that leads to a protein's rapid degradation when fused to its C-terminus. Here the authors* describe the construction of a conditional degron where illumination with blue light leads to activation of the degron and degradation of the protein of interest.

    To achieve this, a light–oxygen–voltage (LOV) domain, a motif found in plant photoreceptor proteins that have a flavin cofactor absorbing blue light, was employed by engineering a degron into this domain. The flavin cofactor of the LOV domain forms a flavin–cysteine adduct upon exposure to blue light, leading to exposure and unfolding of a C-terminal helix.

    The authors reasoned that attaching a RRRG degron to the C-terminus of the LOV domain would lead to conditional degradation of the LOV domain because the C-terminus is buried in the protein core (shielding the degron) until the domain is activated by blue light. This “blue-light inducible degradation” (B-LID) domain can be fused to a protein of interest and protein levels controlled by exposure of cells to nontoxic blue light (465 nm). The RRRG sequence was found to be too potent, and so to achieve a conditional B-LID domain, the less potent RRRGN degron was used. Fusion of the B-LID domain to yellow fluorescent protein (YFP) showed degradation of YFP within in a few hours of exposure to blue light (see Figure).

    The authors also showed that this system can work in vivo by injecting mRNA encoding a mCherry-BLID fusion into zebrafish. This work provides a new tool for studying protein function in cells that does not require small molecules or RNAi transfection procedures.

  • *Abstract from ACS Chemical Biology 2014, Vol. 9: 111–115

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    Post-translational regulation of protein abundance in cells is a powerful tool for studying protein function. Here, we describe a novel genetically encoded protein domain that is degraded upon exposure to nontoxic blue light. We demonstrate that fusion proteins containing this domain are rapidly degraded in cultured cells and in zebrafish upon illumination.



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