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Tuning the drug efflux activity of an ABC transporter in vivo by in vitro selected DARPin binders.

Seeger MA, Mittal A, Velamakanni S, Hohl M, Schauer S, Salaa I, Grütter MG, van Veen HW - PLoS ONE (2012)

Bottom Line: Consistent with this, purified activator DARPins were found to stimulate the ATPase activity of LmrCD in vitro when reconstituted in proteoliposomes.This study suggests that membrane transporters are tunable in vivo by in vitro selected binding proteins.Our approach could be of biopharmaceutical importance and might facilitate studies on molecular mechanisms of ABC transporters.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Cambridge, Cambridge, United Kingdom. m.seeger@bioc.uzh.ch

ABSTRACT
ABC transporters use the energy from binding and hydrolysis of ATP to import or extrude substrates across the membrane. Using ribosome display, we raised designed ankyrin repeat proteins (DARPins) against detergent solubilized LmrCD, a heterodimeric multidrug ABC exporter from Lactococcus lactis. Several target-specific DARPin binders were identified that bind to at least three distinct, partially overlapping epitopes on LmrD in detergent solution as well as in native membranes. Remarkably, functional screening of the LmrCD-specific DARPin pools in L. lactis revealed three homologous DARPins which, when generated in LmrCD-expressing cells, strongly activated LmrCD-mediated drug transport. As LmrCD expression in the cell membrane was unaltered upon the co-expression of activator DARPins, the activation is suggested to occur at the level of LmrCD activity. Consistent with this, purified activator DARPins were found to stimulate the ATPase activity of LmrCD in vitro when reconstituted in proteoliposomes. This study suggests that membrane transporters are tunable in vivo by in vitro selected binding proteins. Our approach could be of biopharmaceutical importance and might facilitate studies on molecular mechanisms of ABC transporters.

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Related in: MedlinePlus

DARPin expression does not significantly alter expression of LmrCD proteins.(A, B) A V5-tag was introduced in frame at the 5′-end of genomic lmrD in L. lactis (denoted L. lactis NZ9000 lmrDV5). Plasmid-encoded DARPin activators or the control DARPin E3_5* were expressed in L. lactis NZ9000 lmrDV5 in the presence and absence of daunomycin (14 µM for DARPin_Act3 and E3_5* and 28 µM for DARPin_Act1 and DARPin_Act2, respectively). The expression levels of genomic LmrDV5 were then quantified by comparing the Western blot signal obtained using an anti-V5 antibody (A) with total protein detected by SYPRO ruby staining (B). (C) The relative amounts of LmrDV5 expression were quantified by densitometry. Each bar represents the average of three independent data points (n = 3) of which one data point is shown in (A) and (B).
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pone-0037845-g005: DARPin expression does not significantly alter expression of LmrCD proteins.(A, B) A V5-tag was introduced in frame at the 5′-end of genomic lmrD in L. lactis (denoted L. lactis NZ9000 lmrDV5). Plasmid-encoded DARPin activators or the control DARPin E3_5* were expressed in L. lactis NZ9000 lmrDV5 in the presence and absence of daunomycin (14 µM for DARPin_Act3 and E3_5* and 28 µM for DARPin_Act1 and DARPin_Act2, respectively). The expression levels of genomic LmrDV5 were then quantified by comparing the Western blot signal obtained using an anti-V5 antibody (A) with total protein detected by SYPRO ruby staining (B). (C) The relative amounts of LmrDV5 expression were quantified by densitometry. Each bar represents the average of three independent data points (n = 3) of which one data point is shown in (A) and (B).

Mentions: The observed gain of cellular drug resistance and enhanced rates of substrate efflux in DARPin producing cells could be explained if DARPin expression would upregulate the expression level of LmrCD. In order to compare the amounts of expressed LmrCD protein in the plasma membrane from DARPin-producing and control cells, we introduced a V5-tag downstream to the lmrD copy on the chromosome by homologous recombination. Cells producing the V5-tagged version of LmrD (LmrDV5) were as resistant to daunomycin as the wildtype cells. A specific band for LmrDV5 could be detected by Western blotting with an anti-V5 antibody (Figure 5A). L. lactis NZ9000 lmrDV5 expressing the activator DARPins and the control DARPin E3_5* were grown in the absence of drug and in the presence of the daunomycin concentration (see Materials and Methods). The amount of LmrDV5 was then analyzed by Western blotting whereas the total protein was quantified using SYPRO ruby staining. The LmrDV5 production level was consistently increased by a factor of around 1.5 upon the exposure to daunomycin irrespective of the DARPin expressed (Figure 5B). However, the activator DARPins did not lead to a significant increase in LmrDV5 production compared to the control cells, indicating that the DARPin activators directly stimulate the drug efflux activity of existing transporters.


Tuning the drug efflux activity of an ABC transporter in vivo by in vitro selected DARPin binders.

Seeger MA, Mittal A, Velamakanni S, Hohl M, Schauer S, Salaa I, Grütter MG, van Veen HW - PLoS ONE (2012)

DARPin expression does not significantly alter expression of LmrCD proteins.(A, B) A V5-tag was introduced in frame at the 5′-end of genomic lmrD in L. lactis (denoted L. lactis NZ9000 lmrDV5). Plasmid-encoded DARPin activators or the control DARPin E3_5* were expressed in L. lactis NZ9000 lmrDV5 in the presence and absence of daunomycin (14 µM for DARPin_Act3 and E3_5* and 28 µM for DARPin_Act1 and DARPin_Act2, respectively). The expression levels of genomic LmrDV5 were then quantified by comparing the Western blot signal obtained using an anti-V5 antibody (A) with total protein detected by SYPRO ruby staining (B). (C) The relative amounts of LmrDV5 expression were quantified by densitometry. Each bar represents the average of three independent data points (n = 3) of which one data point is shown in (A) and (B).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3366976&req=5

pone-0037845-g005: DARPin expression does not significantly alter expression of LmrCD proteins.(A, B) A V5-tag was introduced in frame at the 5′-end of genomic lmrD in L. lactis (denoted L. lactis NZ9000 lmrDV5). Plasmid-encoded DARPin activators or the control DARPin E3_5* were expressed in L. lactis NZ9000 lmrDV5 in the presence and absence of daunomycin (14 µM for DARPin_Act3 and E3_5* and 28 µM for DARPin_Act1 and DARPin_Act2, respectively). The expression levels of genomic LmrDV5 were then quantified by comparing the Western blot signal obtained using an anti-V5 antibody (A) with total protein detected by SYPRO ruby staining (B). (C) The relative amounts of LmrDV5 expression were quantified by densitometry. Each bar represents the average of three independent data points (n = 3) of which one data point is shown in (A) and (B).
Mentions: The observed gain of cellular drug resistance and enhanced rates of substrate efflux in DARPin producing cells could be explained if DARPin expression would upregulate the expression level of LmrCD. In order to compare the amounts of expressed LmrCD protein in the plasma membrane from DARPin-producing and control cells, we introduced a V5-tag downstream to the lmrD copy on the chromosome by homologous recombination. Cells producing the V5-tagged version of LmrD (LmrDV5) were as resistant to daunomycin as the wildtype cells. A specific band for LmrDV5 could be detected by Western blotting with an anti-V5 antibody (Figure 5A). L. lactis NZ9000 lmrDV5 expressing the activator DARPins and the control DARPin E3_5* were grown in the absence of drug and in the presence of the daunomycin concentration (see Materials and Methods). The amount of LmrDV5 was then analyzed by Western blotting whereas the total protein was quantified using SYPRO ruby staining. The LmrDV5 production level was consistently increased by a factor of around 1.5 upon the exposure to daunomycin irrespective of the DARPin expressed (Figure 5B). However, the activator DARPins did not lead to a significant increase in LmrDV5 production compared to the control cells, indicating that the DARPin activators directly stimulate the drug efflux activity of existing transporters.

Bottom Line: Consistent with this, purified activator DARPins were found to stimulate the ATPase activity of LmrCD in vitro when reconstituted in proteoliposomes.This study suggests that membrane transporters are tunable in vivo by in vitro selected binding proteins.Our approach could be of biopharmaceutical importance and might facilitate studies on molecular mechanisms of ABC transporters.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Cambridge, Cambridge, United Kingdom. m.seeger@bioc.uzh.ch

ABSTRACT
ABC transporters use the energy from binding and hydrolysis of ATP to import or extrude substrates across the membrane. Using ribosome display, we raised designed ankyrin repeat proteins (DARPins) against detergent solubilized LmrCD, a heterodimeric multidrug ABC exporter from Lactococcus lactis. Several target-specific DARPin binders were identified that bind to at least three distinct, partially overlapping epitopes on LmrD in detergent solution as well as in native membranes. Remarkably, functional screening of the LmrCD-specific DARPin pools in L. lactis revealed three homologous DARPins which, when generated in LmrCD-expressing cells, strongly activated LmrCD-mediated drug transport. As LmrCD expression in the cell membrane was unaltered upon the co-expression of activator DARPins, the activation is suggested to occur at the level of LmrCD activity. Consistent with this, purified activator DARPins were found to stimulate the ATPase activity of LmrCD in vitro when reconstituted in proteoliposomes. This study suggests that membrane transporters are tunable in vivo by in vitro selected binding proteins. Our approach could be of biopharmaceutical importance and might facilitate studies on molecular mechanisms of ABC transporters.

Show MeSH
Related in: MedlinePlus