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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.

Show MeSH
Identification of LmrCD-activating DARPins.(A) Overexpression of DARPin_Act1 (•), DARPin_Act2 (○), DARPin_Act3 (▾) in wildtype L. lactis increases the resistance towards daunomycin compared to cells expressing control DARPin E3_5* (not interacting with LmrCD) (Δ). (B) No differences were observed when experiments in (A) were performed with cells lacking the chromosomal copy of lmrCD. (C) BCECF-AM transport measurements in pre-energized wildtype L. lactis cells demonstrate activation of LmrCD-mediated extrusion upon expression of DARPin_Act2 (trace 1) but not of control DARPin E3_5* (trace 2). No activation of LmrCD activity was observed upon expression of DARPin_Act2 (trace 3) or control DARPin E3_5* (trace 4) in L. lactis ΔlmrCD cells. Shown are representative data from at least three independent measurements (n≥3).
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pone-0037845-g004: Identification of LmrCD-activating DARPins.(A) Overexpression of DARPin_Act1 (•), DARPin_Act2 (○), DARPin_Act3 (▾) in wildtype L. lactis increases the resistance towards daunomycin compared to cells expressing control DARPin E3_5* (not interacting with LmrCD) (Δ). (B) No differences were observed when experiments in (A) were performed with cells lacking the chromosomal copy of lmrCD. (C) BCECF-AM transport measurements in pre-energized wildtype L. lactis cells demonstrate activation of LmrCD-mediated extrusion upon expression of DARPin_Act2 (trace 1) but not of control DARPin E3_5* (trace 2). No activation of LmrCD activity was observed upon expression of DARPin_Act2 (trace 3) or control DARPin E3_5* (trace 4) in L. lactis ΔlmrCD cells. Shown are representative data from at least three independent measurements (n≥3).

Mentions: LmrCD-mediated daunomycin resistance in L. lactis[53] was used for screening of DARPins that affect LmrCD activity. Individual DARPins of the pool obtained after four selection rounds (Figure 2; note: these are not the DARPin binders identified by ELISA from the previous section) were expressed at high levels in the cytoplasm of L. lactis using the nisin-inducible lactococcal vector pNZ8048 (estimated to 2–5% of total soluble protein, not shown) [54]. We first attempted to find DARPins whose expression lead to a decrease of LmrCD-dependent daunomycin resistance (inhibitors). Around 20 apparent inhibitors were found by screening 400 DARPin clones expressed in L. lactis. A closer inspection of these initial hits however, revealed that they were false positives; lactococcal cells expressing these DARPins grew considerably slower than cells expressing the control DARPin E3_5*. When these DARPin inhibitors were expressed in the L. lactis strain lacking the chromosomal lmrCD genes (L. lactis NZ9000 ΔlmrA ΔlmrCD[55]), the apparent inhibition was also observed. Hence, the increased drug susceptibility of L. lactis expressing these DARPins was independent of LmrCD. Surprisingly, we also found DARPins the expression of which increased daunomycin resistance in L. lactis, suggesting enhancement of LmrCD activity. Three strong activators (DARPin_Act1-3) were found in a screen including 1128 clones (Figure 2). In cell growth experiments, the daunomycin resistance of L. lactis NZ9000 expressing the activator DARPins was compared to the control DARPin E3_5* in the wildtype and the ΔlmrCD background (Figure 4A and B). In wildtype cells, the IC50 for daunomycin was increased by a factor of 3.3, 2.6 and 1.7 upon the production of DARPin_Act1, DARPin_Act2, and DARPin_Act3, respectively. Importantly, the expression of the activator DARPins in the L.lactis NZ9000 ΔlmrA ΔlmrCD background did not affect the daunomycin resistance of the cells, indicating an LmrCD-specific functional stimulation. The knock-out of lmrCD in L. lactis results in an 8.3-fold decrease of the IC50 for daunomycin (Figure 4A and B). Therefore, the DARPin-induced stimulation of LmrCD-mediated drug transport by a factor up to 3.2 is substantial. The DARPins α-LmrCD#1-5 that were identified in the ELISA screen to bind to LmrCD (see previous section) were also assayed regarding the potential modulation of the LmrCD-mediated drug resistance in L. lactis. Although DARPins α-LmrCD#1-5, the DARPin activators and DARPin E3_5* were overproduced equally well in L. lactis, expression of DARPins α-LmrCD#1-5 did not alter the drug resistance of lactococcal cells towards daunomycin (not shown).


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)

Identification of LmrCD-activating DARPins.(A) Overexpression of DARPin_Act1 (•), DARPin_Act2 (○), DARPin_Act3 (▾) in wildtype L. lactis increases the resistance towards daunomycin compared to cells expressing control DARPin E3_5* (not interacting with LmrCD) (Δ). (B) No differences were observed when experiments in (A) were performed with cells lacking the chromosomal copy of lmrCD. (C) BCECF-AM transport measurements in pre-energized wildtype L. lactis cells demonstrate activation of LmrCD-mediated extrusion upon expression of DARPin_Act2 (trace 1) but not of control DARPin E3_5* (trace 2). No activation of LmrCD activity was observed upon expression of DARPin_Act2 (trace 3) or control DARPin E3_5* (trace 4) in L. lactis ΔlmrCD cells. Shown are representative data from at least three independent measurements (n≥3).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3366976&req=5

pone-0037845-g004: Identification of LmrCD-activating DARPins.(A) Overexpression of DARPin_Act1 (•), DARPin_Act2 (○), DARPin_Act3 (▾) in wildtype L. lactis increases the resistance towards daunomycin compared to cells expressing control DARPin E3_5* (not interacting with LmrCD) (Δ). (B) No differences were observed when experiments in (A) were performed with cells lacking the chromosomal copy of lmrCD. (C) BCECF-AM transport measurements in pre-energized wildtype L. lactis cells demonstrate activation of LmrCD-mediated extrusion upon expression of DARPin_Act2 (trace 1) but not of control DARPin E3_5* (trace 2). No activation of LmrCD activity was observed upon expression of DARPin_Act2 (trace 3) or control DARPin E3_5* (trace 4) in L. lactis ΔlmrCD cells. Shown are representative data from at least three independent measurements (n≥3).
Mentions: LmrCD-mediated daunomycin resistance in L. lactis[53] was used for screening of DARPins that affect LmrCD activity. Individual DARPins of the pool obtained after four selection rounds (Figure 2; note: these are not the DARPin binders identified by ELISA from the previous section) were expressed at high levels in the cytoplasm of L. lactis using the nisin-inducible lactococcal vector pNZ8048 (estimated to 2–5% of total soluble protein, not shown) [54]. We first attempted to find DARPins whose expression lead to a decrease of LmrCD-dependent daunomycin resistance (inhibitors). Around 20 apparent inhibitors were found by screening 400 DARPin clones expressed in L. lactis. A closer inspection of these initial hits however, revealed that they were false positives; lactococcal cells expressing these DARPins grew considerably slower than cells expressing the control DARPin E3_5*. When these DARPin inhibitors were expressed in the L. lactis strain lacking the chromosomal lmrCD genes (L. lactis NZ9000 ΔlmrA ΔlmrCD[55]), the apparent inhibition was also observed. Hence, the increased drug susceptibility of L. lactis expressing these DARPins was independent of LmrCD. Surprisingly, we also found DARPins the expression of which increased daunomycin resistance in L. lactis, suggesting enhancement of LmrCD activity. Three strong activators (DARPin_Act1-3) were found in a screen including 1128 clones (Figure 2). In cell growth experiments, the daunomycin resistance of L. lactis NZ9000 expressing the activator DARPins was compared to the control DARPin E3_5* in the wildtype and the ΔlmrCD background (Figure 4A and B). In wildtype cells, the IC50 for daunomycin was increased by a factor of 3.3, 2.6 and 1.7 upon the production of DARPin_Act1, DARPin_Act2, and DARPin_Act3, respectively. Importantly, the expression of the activator DARPins in the L.lactis NZ9000 ΔlmrA ΔlmrCD background did not affect the daunomycin resistance of the cells, indicating an LmrCD-specific functional stimulation. The knock-out of lmrCD in L. lactis results in an 8.3-fold decrease of the IC50 for daunomycin (Figure 4A and B). Therefore, the DARPin-induced stimulation of LmrCD-mediated drug transport by a factor up to 3.2 is substantial. The DARPins α-LmrCD#1-5 that were identified in the ELISA screen to bind to LmrCD (see previous section) were also assayed regarding the potential modulation of the LmrCD-mediated drug resistance in L. lactis. Although DARPins α-LmrCD#1-5, the DARPin activators and DARPin E3_5* were overproduced equally well in L. lactis, expression of DARPins α-LmrCD#1-5 did not alter the drug resistance of lactococcal cells towards daunomycin (not shown).

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