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Role of Membrane Potential on Entry of Cell-Penetrating Peptide Transportan 10 into Single Vesicles.

ABSTRACT: Cell-penetrating peptides (CPPs) can translocate across plasma membranes to enter the cytosol of eukaryotic cells without decreasing cell viability. We revealed the mechanism underlying this translocation by examining the effect of membrane potential, ?m, on the entry of a CPP, transportan 10 (TP10), into the lumen of single giant unilamellar vesicles (GUVs). For this purpose, we used the single GUV method to detect the entry of carboxyfluorescein (CF)-labeled TP10 (CF-TP10) into the lumen of single GUVs. First, we used various K+ concentration differences to apply different negative membrane potentials on single GUVs containing gramicidin A in their membrane and confirmed these potentials using the ?m-sensitive fluorescent probe 3,3'-dihexyloxacarbocyanine iodine. The fluorescence intensity of the GUV membranes (i.e., the rim intensity) due to 3,3'-dihexyloxacarbocyanine iodine increased with |?m| up to 118 mV, and its dependence on |?m| less than 28 mV agreed with a theoretical estimation (i.e., the dye concentration in the inner leaflet of a GUV is larger than that in the outer leaflet according to the Boltzmann distribution). We then examined the effect of ?m on the entry of CF-TP10 into GUVs using single GUVs containing small GUVs or large unilamellar vesicles inside the mother GUV lumen. We found that CF-TP10 entered the GUV lumen without pore formation and the rate of entry of CF-TP10 into the GUV lumen, Ventry, increased with an increase in |?m|. The rim intensity due to CF-TP10 increased with an increase in |?m|, indicating that the CF-TP10 concentration in the inner leaflet of the GUV increased with |?m|. These results indicate that the ?m-induced elevation in Ventry can be explained by the increase in CF-TP10 concentration in the inner leaflet with |?m|. We discuss the mechanism underlying this effect of membrane potential based on the pre-pore model of the translocation of CF-TP10 across a GUV membrane.

SUBMITTER: Moghal MMR

PROVIDER: S-EPMC6950768 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Role of Membrane Potential on Entry of Cell-Penetrating Peptide Transportan 10 into Single Vesicles.

Moghal Md Mizanur Rahman MMR Islam Md Zahidul MZ Hossain Farzana F Saha Samiron Kumar SK Yamazaki Masahito M

Biophysical journal 20191120 1

Cell-penetrating peptides (CPPs) can translocate across plasma membranes to enter the cytosol of eukaryotic cells without decreasing cell viability. We revealed the mechanism underlying this translocation by examining the effect of membrane potential, φ<sub>m</sub>, on the entry of a CPP, transportan 10 (TP10), into the lumen of single giant unilamellar vesicles (GUVs). For this purpose, we used the single GUV method to detect the entry of carboxyfluorescein (CF)-labeled TP10 (CF-TP10) into the ...[more]

PMID: 31810658

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Project description:Structure analysis of the cell-penetrating peptide transportan 10 (TP10) revealed an exemplary range of different conformations in the membrane-bound state. The bipartite peptide (derived N-terminally from galanin and C-terminally from mastoparan) was found to exhibit prominent characteristics of (i) amphiphilic ?-helices, (ii) intrinsically disordered peptides, as well as (iii) ?-pleated amyloid fibrils, and these conformational states become interconverted as a function of concentration. We used a complementary approach of solid-state (19)F-NMR and circular dichroism in oriented membrane samples to characterize the structural and dynamical behaviour of TP10 in its monomeric and aggregated forms. Nine different positions in the peptide were selectively substituted with either the L- or D-enantiomer of 3-(trifluoromethyl)-bicyclopent-[1.1.1]-1-ylglycine (CF3-Bpg) as a reporter group for (19)F-NMR. Using the L-epimeric analogs, a comprehensive three-dimensional structure analysis was carried out in lipid bilayers at low peptide concentration, where TP10 is monomeric. While the N-terminal region is flexible and intrinsically unstructured within the plane of the lipid bilayer, the C-terminal ?-helix is embedded in the membrane with an oblique tilt angle of ? 55° and in accordance with its amphiphilic profile. Incorporation of the sterically obstructive D-CF3-Bpg reporter group into the helical region leads to a local unfolding of the membrane-bound peptide. At high concentration, these helix-destabilizing C-terminal substitutions promote aggregation into immobile ?-sheets, which resemble amyloid fibrils. On the other hand, the obstructive D-CF3-Bpg substitutions can be accommodated in the flexible N-terminus of TP10 where they do not promote aggregation at high concentration. The cross-talk between the two regions of TP10 thus exerts a delicate balance on its conformational switch, as the presence of the ?-helix counteracts the tendency of the unfolded N-terminus to self-assemble into ?-pleated fibrils.

Project description:The antimicrobial peptide (AMP) derived from lactoferricin B, LfcinB (4-9) (RRWQWR), and lissamine rhodamine B red-labeled peptide (Rh-LfcinB (4-9)) exhibit strong antimicrobial activities, and they can enter Escherichia coli cells without damaging the cell membranes. Thus, these peptides are cell-penetrating peptide (CPP) -type AMPs. In this study, to elucidate the effect of the membrane potential (Δφ) on the action of the CPP-type AMP, Rh-LfcinB (4-9), we investigated the interactions of Rh-LfcinB (4-9) with single E. coli cells and spheroplasts containing calcein in the cytosol using confocal laser scanning microscopy. At low peptide concentrations, Rh-LfcinB (4-9) entered the cytosol of single E. coli cells and spheroplasts without damaging the cell membranes, and the H+-ionophore carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) suppressed its entry. The studies using the time-kill method indicate that these low concentrations of peptide exhibit antimicrobial activity but CCCP inhibits this activity. Next, we investigated the effect of Δφ on the interaction of Rh-LfcinB (4-9) with single giant unilamellar vesicles (GUVs) comprising E. coli polar lipid extracts and containing a fluorescent probe, Alexa Fluor 647 hydrazide. At low concentrations (0.2-0.5 μM), Rh-LfcinB (4-9) showed significant entry to the single GUV lumen without pore formation in the presence of Δφ. The fraction of entry of peptide increased with increasing negative membrane potential, indicating that the rate of peptide entry into the GUV lumen increased with increasing negative membrane potential. These results indicate that Δφ enhances the entry of Rh-LfcinB (4-9) into single E. coli cells, spheroplasts, and GUVs and its antimicrobial activity.IMPORTANCE: Bacterial cells have a membrane potential (Δφ), but the effect of Δφ on action of cell-penetrating peptide-type antimicrobial peptides (AMPs) is not clear. Here, we investigated the effect of Δφ on the action of fluorescent probe-labeled AMP derived from lactoferricin B, Rh-LfcinB (4-9). At low peptide concentrations, Rh-LfcinB (4-9) enters the cytosol of Escherichia coli cells and spheroplasts without damaging their cell membrane, but a protonophore suppresses this entry and its antimicrobial activity. The rate of entry of Rh-LfcinB (4-9) into the giant unilamellar vesicles (GUVs) comprising E. coli lipids without pore formation increases with increasing Δφ. These results indicate that Δφ enhances the antimicrobial activity of Rh-LfcinB (4-9) and hence LfcinB (4-9) by increasing the rate of their entry into the cytosol.

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Role of Membrane Potential on Entry of Cell-Penetrating Peptide Transportan 10 into Single Vesicles.

Publications

Role of Membrane Potential on Entry of Cell-Penetrating Peptide Transportan 10 into Single Vesicles.

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