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Engineering FKBP-Based Destabilizing Domains to Build Sophisticated Protein Regulation Systems.

An W, Jackson RE, Hunter P, Gögel S, van Diepen M, Liu K, Meyer MP, Eickholt BJ - PLoS ONE (2015)

Bottom Line: Targeting protein stability with small molecules has emerged as an effective tool to control protein abundance in a fast, scalable and reversible manner.Another drawback of this approach is the remaining endogenous protein.This new system will consolidate and extend the use of DD-technology to control protein function precisely in living cells and animal models.

View Article: PubMed Central - PubMed

Affiliation: MRC Centre for Developmental Neurobiology, King's College London, London, SE1 1UL, United Kingdom.

ABSTRACT
Targeting protein stability with small molecules has emerged as an effective tool to control protein abundance in a fast, scalable and reversible manner. The technique involves tagging a protein of interest (POI) with a destabilizing domain (DD) specifically controlled by a small molecule. The successful construction of such fusion proteins may, however, be limited by functional interference of the DD epitope with electrostatic interactions required for full biological function of proteins. Another drawback of this approach is the remaining endogenous protein. Here, we combined the Cre-LoxP system with an advanced DD and generated a protein regulation system in which the loss of an endogenous protein, in our case the tumor suppressor PTEN, can be coupled directly with a conditionally fine-tunable DD-PTEN. This new system will consolidate and extend the use of DD-technology to control protein function precisely in living cells and animal models.

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Related in: MedlinePlus

FKBP surface engineering restores PTEN activity of FKBP**-PTEN fusion protein.(A) Electrostatic potential map of PTEN (PDB ID: 1d5r) and FKBP (PDB ID: 1BL4). Positively charged binding pocket of the PTEN PIP3 substrate is circled with a white dashed line; the negatively charged lump formed by E31/D32 residues on the FKBP surface is circled by a black dashed line. (B) Schematic representation of amino acid residue substitutions of FKBP* protein surface at E31/D32. (C) Different FKBP*-PTEN mutants were transiently co-tansfected with AKT-GFP into U87MG cells, the expression of fusion protein and pAKT were examined by Western blotting. (D) E31S/D32S amino acid substitutions on FKBP* (FKBP**) restores PTEN activity of the FKBP**-PTEN fusion protein. FKBP**-PTEN (or GFP/GFP-PTEN) was transiently co-expressed with AKT-GFP in U87MG cells. Cells were treated with Shld1 and analyzed as before.
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pone.0145783.g002: FKBP surface engineering restores PTEN activity of FKBP**-PTEN fusion protein.(A) Electrostatic potential map of PTEN (PDB ID: 1d5r) and FKBP (PDB ID: 1BL4). Positively charged binding pocket of the PTEN PIP3 substrate is circled with a white dashed line; the negatively charged lump formed by E31/D32 residues on the FKBP surface is circled by a black dashed line. (B) Schematic representation of amino acid residue substitutions of FKBP* protein surface at E31/D32. (C) Different FKBP*-PTEN mutants were transiently co-tansfected with AKT-GFP into U87MG cells, the expression of fusion protein and pAKT were examined by Western blotting. (D) E31S/D32S amino acid substitutions on FKBP* (FKBP**) restores PTEN activity of the FKBP**-PTEN fusion protein. FKBP**-PTEN (or GFP/GFP-PTEN) was transiently co-expressed with AKT-GFP in U87MG cells. Cells were treated with Shld1 and analyzed as before.

Mentions: Transfection of plasmid DNA into U87MG cells was carried out using GeneJuice (Novagen). Nucleofection of mouse primary cortical neurons was performed by using Amaxa® Mouse Neuron Nucleofector® Kit (Lonza, UK). In order to monitor PTEN activity in transiently transfected U87MG cells, the co-transfection of AKT-GFP with FKBP*-PTEN or GFP-PTEN constructs was exploited as a fast and effective experimental protocol that allowed the analysis of PTEN activity towards PIP3-dependent signaling in transfected cell, only. This strategy was pursued in transient transfections (Figs 1 and 2), but not in stable FKBP**-PTEN expressing U87MG cells lines (Fig 3).


Engineering FKBP-Based Destabilizing Domains to Build Sophisticated Protein Regulation Systems.

An W, Jackson RE, Hunter P, Gögel S, van Diepen M, Liu K, Meyer MP, Eickholt BJ - PLoS ONE (2015)

FKBP surface engineering restores PTEN activity of FKBP**-PTEN fusion protein.(A) Electrostatic potential map of PTEN (PDB ID: 1d5r) and FKBP (PDB ID: 1BL4). Positively charged binding pocket of the PTEN PIP3 substrate is circled with a white dashed line; the negatively charged lump formed by E31/D32 residues on the FKBP surface is circled by a black dashed line. (B) Schematic representation of amino acid residue substitutions of FKBP* protein surface at E31/D32. (C) Different FKBP*-PTEN mutants were transiently co-tansfected with AKT-GFP into U87MG cells, the expression of fusion protein and pAKT were examined by Western blotting. (D) E31S/D32S amino acid substitutions on FKBP* (FKBP**) restores PTEN activity of the FKBP**-PTEN fusion protein. FKBP**-PTEN (or GFP/GFP-PTEN) was transiently co-expressed with AKT-GFP in U87MG cells. Cells were treated with Shld1 and analyzed as before.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4696822&req=5

pone.0145783.g002: FKBP surface engineering restores PTEN activity of FKBP**-PTEN fusion protein.(A) Electrostatic potential map of PTEN (PDB ID: 1d5r) and FKBP (PDB ID: 1BL4). Positively charged binding pocket of the PTEN PIP3 substrate is circled with a white dashed line; the negatively charged lump formed by E31/D32 residues on the FKBP surface is circled by a black dashed line. (B) Schematic representation of amino acid residue substitutions of FKBP* protein surface at E31/D32. (C) Different FKBP*-PTEN mutants were transiently co-tansfected with AKT-GFP into U87MG cells, the expression of fusion protein and pAKT were examined by Western blotting. (D) E31S/D32S amino acid substitutions on FKBP* (FKBP**) restores PTEN activity of the FKBP**-PTEN fusion protein. FKBP**-PTEN (or GFP/GFP-PTEN) was transiently co-expressed with AKT-GFP in U87MG cells. Cells were treated with Shld1 and analyzed as before.
Mentions: Transfection of plasmid DNA into U87MG cells was carried out using GeneJuice (Novagen). Nucleofection of mouse primary cortical neurons was performed by using Amaxa® Mouse Neuron Nucleofector® Kit (Lonza, UK). In order to monitor PTEN activity in transiently transfected U87MG cells, the co-transfection of AKT-GFP with FKBP*-PTEN or GFP-PTEN constructs was exploited as a fast and effective experimental protocol that allowed the analysis of PTEN activity towards PIP3-dependent signaling in transfected cell, only. This strategy was pursued in transient transfections (Figs 1 and 2), but not in stable FKBP**-PTEN expressing U87MG cells lines (Fig 3).

Bottom Line: Targeting protein stability with small molecules has emerged as an effective tool to control protein abundance in a fast, scalable and reversible manner.Another drawback of this approach is the remaining endogenous protein.This new system will consolidate and extend the use of DD-technology to control protein function precisely in living cells and animal models.

View Article: PubMed Central - PubMed

Affiliation: MRC Centre for Developmental Neurobiology, King's College London, London, SE1 1UL, United Kingdom.

ABSTRACT
Targeting protein stability with small molecules has emerged as an effective tool to control protein abundance in a fast, scalable and reversible manner. The technique involves tagging a protein of interest (POI) with a destabilizing domain (DD) specifically controlled by a small molecule. The successful construction of such fusion proteins may, however, be limited by functional interference of the DD epitope with electrostatic interactions required for full biological function of proteins. Another drawback of this approach is the remaining endogenous protein. Here, we combined the Cre-LoxP system with an advanced DD and generated a protein regulation system in which the loss of an endogenous protein, in our case the tumor suppressor PTEN, can be coupled directly with a conditionally fine-tunable DD-PTEN. This new system will consolidate and extend the use of DD-technology to control protein function precisely in living cells and animal models.

Show MeSH
Related in: MedlinePlus