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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

Codon optimization of FKBP** establishes a conditional fine-tuning protein regulation system.(A) The transcription of the destabilization cassette FKBP**-PTEN was subcloned downstream of LoxP-Stop-LoxP; transcription was driven by the ubiquitous CMV-enhanced chicken beta actin promoter (CAG). Presence of Cre induces FKBP**-PTEN gene transcription; however, the translated FKBP**-PTEN will be rapidly degraded. Addition of Shld1 confers FKBP**-PTEN stabilization in a tunable and reversible manner. (B) Cre-mediated cleavage of LoxP-Stop-LoxP (LSL) created a truncated FKBP**-PTEN fusion protein. Mouse forebrain neurons were nucleofected with LSL-FKBP**-PTEN and Cre-IresRFP (Cre (+)), or RFP (Cre (-)). Cells were treated with Shld1 (or control vehicle) overnight, before analyses of cell lysates using indicated antibodies. (C) Sequence alignment of FKBP** and LoxP. The third ATG codon region sequence (TATGCTA) of FKBP** (M3Lcta) is identical to the linker region of the LoxP stem-loop sequence (TATGCTA). Codon optimization of CTA with TTG (both code for amino acid Leu) will destroy the potential pseudo-cleavage site of Cre on FKBP**. (D) Codon optimization of M3Lcta to M3Lttg in FKBP** abolished the Cre-dependent FKBP**-PTEN truncation and produced a Shld1 dependent PTEN fusion protein. The M3Lttg modified LSL-FKBP**-PTEN construct was co-expressed with Cre-IresRFP (Cre (+)), or RFP (Cre (-)) in mouse forebrain neurons as before. (E) Combinatorial use of the Cre-LoxP system with the FKBP**-PTEN/Shld1 chemical-genetic protein control system in PTENflox/flox cells. Constructs were nucleofected into PTENloxp/loxp mouse primary neurons as before. Note that nucleofection of primary cells occurs with efficiencies at approx. 80%, and result in a residual endogenous PTEN signals detected by western blotting. (F) The generated system is able to couple PTEN-loss directly with the expression of tunable FKBP**-PTEN. Upon Cre-mediated recombination, PTENloxp/loxp cells will lose PTEN and, at the same time, activate expression of FKBP**-PTEN. In essence, endogenous PTEN is replaced by tuneable PTEN, which will enable to test whether PTEN-loss induced phenotypes can be rescued at different time-points (tShld1) and/or at different PTEN-concentrations (dShld1).
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pone.0145783.g005: Codon optimization of FKBP** establishes a conditional fine-tuning protein regulation system.(A) The transcription of the destabilization cassette FKBP**-PTEN was subcloned downstream of LoxP-Stop-LoxP; transcription was driven by the ubiquitous CMV-enhanced chicken beta actin promoter (CAG). Presence of Cre induces FKBP**-PTEN gene transcription; however, the translated FKBP**-PTEN will be rapidly degraded. Addition of Shld1 confers FKBP**-PTEN stabilization in a tunable and reversible manner. (B) Cre-mediated cleavage of LoxP-Stop-LoxP (LSL) created a truncated FKBP**-PTEN fusion protein. Mouse forebrain neurons were nucleofected with LSL-FKBP**-PTEN and Cre-IresRFP (Cre (+)), or RFP (Cre (-)). Cells were treated with Shld1 (or control vehicle) overnight, before analyses of cell lysates using indicated antibodies. (C) Sequence alignment of FKBP** and LoxP. The third ATG codon region sequence (TATGCTA) of FKBP** (M3Lcta) is identical to the linker region of the LoxP stem-loop sequence (TATGCTA). Codon optimization of CTA with TTG (both code for amino acid Leu) will destroy the potential pseudo-cleavage site of Cre on FKBP**. (D) Codon optimization of M3Lcta to M3Lttg in FKBP** abolished the Cre-dependent FKBP**-PTEN truncation and produced a Shld1 dependent PTEN fusion protein. The M3Lttg modified LSL-FKBP**-PTEN construct was co-expressed with Cre-IresRFP (Cre (+)), or RFP (Cre (-)) in mouse forebrain neurons as before. (E) Combinatorial use of the Cre-LoxP system with the FKBP**-PTEN/Shld1 chemical-genetic protein control system in PTENflox/flox cells. Constructs were nucleofected into PTENloxp/loxp mouse primary neurons as before. Note that nucleofection of primary cells occurs with efficiencies at approx. 80%, and result in a residual endogenous PTEN signals detected by western blotting. (F) The generated system is able to couple PTEN-loss directly with the expression of tunable FKBP**-PTEN. Upon Cre-mediated recombination, PTENloxp/loxp cells will lose PTEN and, at the same time, activate expression of FKBP**-PTEN. In essence, endogenous PTEN is replaced by tuneable PTEN, which will enable to test whether PTEN-loss induced phenotypes can be rescued at different time-points (tShld1) and/or at different PTEN-concentrations (dShld1).

Mentions: In order to achieve advanced regulation of protein function, it would be desirable to combine tunable FKBP**-PTEN with established systems enabling precise control of gene expression, such as the Cre/LoxP system (Fig 5a). For this purpose, FKBP**-PTEN was subcloned immediately downstream of the second LoxP site of the LoxP-Stop-LoxP (LSL) cassette. We tested the pCAG-LSL-FKBP**-PTEN construct in mouse cortical neuronal primary cultures by transient co-transfection with Cre-IresRFP (or, in control experiments, RFP). The FKBP**-PTEN fusion protein was only detectable by western blotting in the presence of Cre, and following application of the synthetic ligand Shld1 (Fig 5b). Surprisingly, when Cre and FKBP**-PTEN were co-expressed, the anti-PTEN antibody also identified a distinct protein band with lower molecular weight than full-length FKBP**-PTEN, which was stable even in the absence of Shld1. We reasoned that this protein could represent a form of FKBP**-PTEN with a truncation in the FKBP** domain which allows it to escape Shld1 regulation. A mechanism involving Cre-dependent truncation of FKBP**-PTEN suggests that these two regulation systems could be incompatible. Due to the sequence similarity of FKBP* and the engineered FKBP** domain it is likely that this might also be true of the parental DD system. We therefore investigated the potential sites of Cre action within the DD domain.


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)

Codon optimization of FKBP** establishes a conditional fine-tuning protein regulation system.(A) The transcription of the destabilization cassette FKBP**-PTEN was subcloned downstream of LoxP-Stop-LoxP; transcription was driven by the ubiquitous CMV-enhanced chicken beta actin promoter (CAG). Presence of Cre induces FKBP**-PTEN gene transcription; however, the translated FKBP**-PTEN will be rapidly degraded. Addition of Shld1 confers FKBP**-PTEN stabilization in a tunable and reversible manner. (B) Cre-mediated cleavage of LoxP-Stop-LoxP (LSL) created a truncated FKBP**-PTEN fusion protein. Mouse forebrain neurons were nucleofected with LSL-FKBP**-PTEN and Cre-IresRFP (Cre (+)), or RFP (Cre (-)). Cells were treated with Shld1 (or control vehicle) overnight, before analyses of cell lysates using indicated antibodies. (C) Sequence alignment of FKBP** and LoxP. The third ATG codon region sequence (TATGCTA) of FKBP** (M3Lcta) is identical to the linker region of the LoxP stem-loop sequence (TATGCTA). Codon optimization of CTA with TTG (both code for amino acid Leu) will destroy the potential pseudo-cleavage site of Cre on FKBP**. (D) Codon optimization of M3Lcta to M3Lttg in FKBP** abolished the Cre-dependent FKBP**-PTEN truncation and produced a Shld1 dependent PTEN fusion protein. The M3Lttg modified LSL-FKBP**-PTEN construct was co-expressed with Cre-IresRFP (Cre (+)), or RFP (Cre (-)) in mouse forebrain neurons as before. (E) Combinatorial use of the Cre-LoxP system with the FKBP**-PTEN/Shld1 chemical-genetic protein control system in PTENflox/flox cells. Constructs were nucleofected into PTENloxp/loxp mouse primary neurons as before. Note that nucleofection of primary cells occurs with efficiencies at approx. 80%, and result in a residual endogenous PTEN signals detected by western blotting. (F) The generated system is able to couple PTEN-loss directly with the expression of tunable FKBP**-PTEN. Upon Cre-mediated recombination, PTENloxp/loxp cells will lose PTEN and, at the same time, activate expression of FKBP**-PTEN. In essence, endogenous PTEN is replaced by tuneable PTEN, which will enable to test whether PTEN-loss induced phenotypes can be rescued at different time-points (tShld1) and/or at different PTEN-concentrations (dShld1).
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Related In: Results  -  Collection

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pone.0145783.g005: Codon optimization of FKBP** establishes a conditional fine-tuning protein regulation system.(A) The transcription of the destabilization cassette FKBP**-PTEN was subcloned downstream of LoxP-Stop-LoxP; transcription was driven by the ubiquitous CMV-enhanced chicken beta actin promoter (CAG). Presence of Cre induces FKBP**-PTEN gene transcription; however, the translated FKBP**-PTEN will be rapidly degraded. Addition of Shld1 confers FKBP**-PTEN stabilization in a tunable and reversible manner. (B) Cre-mediated cleavage of LoxP-Stop-LoxP (LSL) created a truncated FKBP**-PTEN fusion protein. Mouse forebrain neurons were nucleofected with LSL-FKBP**-PTEN and Cre-IresRFP (Cre (+)), or RFP (Cre (-)). Cells were treated with Shld1 (or control vehicle) overnight, before analyses of cell lysates using indicated antibodies. (C) Sequence alignment of FKBP** and LoxP. The third ATG codon region sequence (TATGCTA) of FKBP** (M3Lcta) is identical to the linker region of the LoxP stem-loop sequence (TATGCTA). Codon optimization of CTA with TTG (both code for amino acid Leu) will destroy the potential pseudo-cleavage site of Cre on FKBP**. (D) Codon optimization of M3Lcta to M3Lttg in FKBP** abolished the Cre-dependent FKBP**-PTEN truncation and produced a Shld1 dependent PTEN fusion protein. The M3Lttg modified LSL-FKBP**-PTEN construct was co-expressed with Cre-IresRFP (Cre (+)), or RFP (Cre (-)) in mouse forebrain neurons as before. (E) Combinatorial use of the Cre-LoxP system with the FKBP**-PTEN/Shld1 chemical-genetic protein control system in PTENflox/flox cells. Constructs were nucleofected into PTENloxp/loxp mouse primary neurons as before. Note that nucleofection of primary cells occurs with efficiencies at approx. 80%, and result in a residual endogenous PTEN signals detected by western blotting. (F) The generated system is able to couple PTEN-loss directly with the expression of tunable FKBP**-PTEN. Upon Cre-mediated recombination, PTENloxp/loxp cells will lose PTEN and, at the same time, activate expression of FKBP**-PTEN. In essence, endogenous PTEN is replaced by tuneable PTEN, which will enable to test whether PTEN-loss induced phenotypes can be rescued at different time-points (tShld1) and/or at different PTEN-concentrations (dShld1).
Mentions: In order to achieve advanced regulation of protein function, it would be desirable to combine tunable FKBP**-PTEN with established systems enabling precise control of gene expression, such as the Cre/LoxP system (Fig 5a). For this purpose, FKBP**-PTEN was subcloned immediately downstream of the second LoxP site of the LoxP-Stop-LoxP (LSL) cassette. We tested the pCAG-LSL-FKBP**-PTEN construct in mouse cortical neuronal primary cultures by transient co-transfection with Cre-IresRFP (or, in control experiments, RFP). The FKBP**-PTEN fusion protein was only detectable by western blotting in the presence of Cre, and following application of the synthetic ligand Shld1 (Fig 5b). Surprisingly, when Cre and FKBP**-PTEN were co-expressed, the anti-PTEN antibody also identified a distinct protein band with lower molecular weight than full-length FKBP**-PTEN, which was stable even in the absence of Shld1. We reasoned that this protein could represent a form of FKBP**-PTEN with a truncation in the FKBP** domain which allows it to escape Shld1 regulation. A mechanism involving Cre-dependent truncation of FKBP**-PTEN suggests that these two regulation systems could be incompatible. Due to the sequence similarity of FKBP* and the engineered FKBP** domain it is likely that this might also be true of the parental DD system. We therefore investigated the potential sites of Cre action within the DD domain.

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