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Small-molecule displacement of a cryptic degron causes conditional protein degradation.

Bonger KM, Chen LC, Liu CW, Wandless TJ - Nat. Chem. Biol. (2011)

Bottom Line: The LID domain is comprised of the FK506- and rapamycin-binding protein (FKBP) and a 19-amino-acid degron fused to the C terminus of FKBP.When present, Shield-1 binds tightly to FKBP, displacing the degron and inducing rapid and processive degradation of the LID domain and any fused partner protein.Structure-function studies of the 19-residue peptide showed that a 4-amino-acid sequence within the peptide is responsible for degradation.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical & Systems Biology, Stanford University, Stanford, California, USA.

ABSTRACT
The ability to rapidly regulate the functions of specific proteins in living cells is a valuable tool for biological research. Here we describe a new technique by which the degradation of a specific protein is induced by a small molecule. A protein of interest is fused to a ligand-induced degradation (LID) domain, resulting in the expression of a stable and functional fusion protein. The LID domain is comprised of the FK506- and rapamycin-binding protein (FKBP) and a 19-amino-acid degron fused to the C terminus of FKBP. In the absence of the small molecule Shield-1, the degron is bound to the FKBP fusion protein and the protein is stable. When present, Shield-1 binds tightly to FKBP, displacing the degron and inducing rapid and processive degradation of the LID domain and any fused partner protein. Structure-function studies of the 19-residue peptide showed that a 4-amino-acid sequence within the peptide is responsible for degradation.

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Shield-1 can simultaneously stabilize and destabilize specific targets. NIH3T3 cells stably expressing both YFP-LID and DD-mCherry were treated with various concentrations of Shield-1 for 24 h, and the fluorescent signals in the appropriate channels were monitored by flow cytometry (a) and fluorescence microscopy (b). Insert scalebars represent 10 μm.
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Figure 6: Shield-1 can simultaneously stabilize and destabilize specific targets. NIH3T3 cells stably expressing both YFP-LID and DD-mCherry were treated with various concentrations of Shield-1 for 24 h, and the fluorescent signals in the appropriate channels were monitored by flow cytometry (a) and fluorescence microscopy (b). Insert scalebars represent 10 μm.

Mentions: In some experimental designs, investigators may desire the rapid depletion of one protein and the simultaneous induction of another protein to acutely perturb their system of interest. Therefore, we set out to test whether the LID domain and our previously reported destabilizing domain (FKBP(L106P) mutant) could be used simultaneously in cells.10 Addition of Shield-1 would be expected to induce the degradation of a protein that is fused to the LID domain and simultaneously stabilize a second protein that is fused to the DD. We doubly transduced NIH3T3 cells with YFP-LID and DD-mCherry and these cells were treated with various concentrations of Shield-1. After 24 hours, the cells were analyzed by flow cytometry and epifluorescence microscopy revealing the dose-dependent depletion of YFP-LID and simultaneous stabilization of mCherry (Fig. 6).


Small-molecule displacement of a cryptic degron causes conditional protein degradation.

Bonger KM, Chen LC, Liu CW, Wandless TJ - Nat. Chem. Biol. (2011)

Shield-1 can simultaneously stabilize and destabilize specific targets. NIH3T3 cells stably expressing both YFP-LID and DD-mCherry were treated with various concentrations of Shield-1 for 24 h, and the fluorescent signals in the appropriate channels were monitored by flow cytometry (a) and fluorescence microscopy (b). Insert scalebars represent 10 μm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3139708&req=5

Figure 6: Shield-1 can simultaneously stabilize and destabilize specific targets. NIH3T3 cells stably expressing both YFP-LID and DD-mCherry were treated with various concentrations of Shield-1 for 24 h, and the fluorescent signals in the appropriate channels were monitored by flow cytometry (a) and fluorescence microscopy (b). Insert scalebars represent 10 μm.
Mentions: In some experimental designs, investigators may desire the rapid depletion of one protein and the simultaneous induction of another protein to acutely perturb their system of interest. Therefore, we set out to test whether the LID domain and our previously reported destabilizing domain (FKBP(L106P) mutant) could be used simultaneously in cells.10 Addition of Shield-1 would be expected to induce the degradation of a protein that is fused to the LID domain and simultaneously stabilize a second protein that is fused to the DD. We doubly transduced NIH3T3 cells with YFP-LID and DD-mCherry and these cells were treated with various concentrations of Shield-1. After 24 hours, the cells were analyzed by flow cytometry and epifluorescence microscopy revealing the dose-dependent depletion of YFP-LID and simultaneous stabilization of mCherry (Fig. 6).

Bottom Line: The LID domain is comprised of the FK506- and rapamycin-binding protein (FKBP) and a 19-amino-acid degron fused to the C terminus of FKBP.When present, Shield-1 binds tightly to FKBP, displacing the degron and inducing rapid and processive degradation of the LID domain and any fused partner protein.Structure-function studies of the 19-residue peptide showed that a 4-amino-acid sequence within the peptide is responsible for degradation.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical & Systems Biology, Stanford University, Stanford, California, USA.

ABSTRACT
The ability to rapidly regulate the functions of specific proteins in living cells is a valuable tool for biological research. Here we describe a new technique by which the degradation of a specific protein is induced by a small molecule. A protein of interest is fused to a ligand-induced degradation (LID) domain, resulting in the expression of a stable and functional fusion protein. The LID domain is comprised of the FK506- and rapamycin-binding protein (FKBP) and a 19-amino-acid degron fused to the C terminus of FKBP. In the absence of the small molecule Shield-1, the degron is bound to the FKBP fusion protein and the protein is stable. When present, Shield-1 binds tightly to FKBP, displacing the degron and inducing rapid and processive degradation of the LID domain and any fused partner protein. Structure-function studies of the 19-residue peptide showed that a 4-amino-acid sequence within the peptide is responsible for degradation.

Show MeSH
Related in: MedlinePlus