Entry of Cell-Penetrating Peptide Transportan 10 into a Single Vesicle by Translocating Across Lipid Membrane and Its Induced Pores

Islam, Md. Zahidul; Ariyama, Hirotaka; Alam, Jahangir Md.; Yamazaki, Masahito

doi:10.1021/bi401406p

Cited by 75 publications

(233 citation statements)

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“…wall may have a "sieving" effect with some larger peptides that would be lost in spheroplasts, requiring additional controls comparing spheroplasts and "normal" cells in other assays (18). However, even with these caveats we believe that spheroplasts provide an excellent model system compared to other alternatives to overcome size and shape limitations, such as giant unilamellar vesicles (19)(20)(21), as spheroplasts preserve a physiological bacterial membrane composition and are viable if returned to growth conditions (13,22). Moreover, although spheroplasts have generally been produced from E. coli, protocols can be adjusted to make them from strains of other species (23).…”

mentioning

confidence: 99%

Bacterial Spheroplasts as a Model for Visualizing Membrane Translocation of Antimicrobial Peptides

Lei

LaBouyer

Darling

et al. 2016

Antimicrob Agents Chemother

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Studies attempting to characterize the membrane translocation of antimicrobial and cell-penetrating peptides are frequently limited by the resolution of conventional light microscopy. This study shows that spheroplasts provide a valuable approach to overcome these limits. Spheroplasts produce less ambiguous images and allow for more systematic analyses of localization. Data collected with spheroplasts are consistent with studies using normal bacterial cells and imply that a particular peptide may not always follow the same mechanism of action.A ntimicrobial peptides (AMPs) represent a promising alternative to conventional therapeutics in the face of concerns about the rise of antibiotic-resistant bacteria in clinical settings (1). Traditionally, AMPs were believed to kill bacteria through membrane disruption. While many AMPs do induce membrane permeabilization, researchers have identified increasing numbers of peptides that function by translocating into bacterial cells and targeting intracellular components (2). Thus, it has become increasingly important for researchers to reliably determine whether AMPs are able to effectively translocate into bacterial cells (3). Many researchers have turned to confocal microscopy in order to assess cell entry (4-11). However, bacterial cells are so small that effective imaging is limited by the resolution of conventional light microscopes. For example, in order to distinguish whether any observed signal from peptides arises from inside the cell versus on the cell membrane, researchers ideally should examine individual focal plane images throughout cells. However, if signal on the membrane is sufficiently strong it can "contaminate" slices ostensibly taken "inside" the cell, as we have observed in measurements of the membrane-localized dye di-8-ANEPPS (Fig. 1).In order to overcome these resolution limits, we have employed bacterial spheroplasts (12)(13)(14). Spheroplasts are produced by culturing bacteria in the presence of an antibiotic, such as cephalexin, that prevents division while still allowing cells to grow. The resulting elongated bacterial "snakes" are then exposed to lysozyme, which digests the outer cell wall and produces spherical spheroplasts that are typically 2 to 5 m in diameter (see Fig. S1 in the supplemental material). Perhaps even more important than larger size, the spherical shape allows one to obtain consistent slices regardless of how a spheroplast is oriented during imaging.In order to test the validity of using spheroplasts to assess peptide translocation, we have measured the cellular localization of four previously characterized peptides (Table 1). To this end, we exposed Escherichia coli spheroplasts to peptides with an N-terminally conjugated fluorescein isothiocyanate (FITC) label for imaging; detailed methods for spheroplast preparation and peptide incubation are provided in the supplemental material. As one set of positive and negative controls, we chose buforin II (BF2), arguably the best-studied membrane-translocating AMP (15), and BF2 with...

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mentioning

confidence: 99%

Bacterial Spheroplasts as a Model for Visualizing Membrane Translocation of Antimicrobial Peptides

Lei

LaBouyer

Darling

et al. 2016

Antimicrob Agents Chemother

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“…Interaction of the amphipathic peptide, specifically tryptophan residues in the hydrophobic domain, with peptidoglycans are critical in the eventual internalization process [81]. Among the oldest hydrophobic peptides is transportan 10 (TP10), derived from transportan which is a chimeric peptide of the neuropeptide galanin and wasp venom mastoparan linked by a lysine chain [32,82] It was reported to sink deeply into the bilayer and cross it carrying its cargo such as green fluorescent protein (GFP) [83].…”

Section: Cell Penetrating Peptidesmentioning

confidence: 99%

“…The uptake of the peptide across the membrane can be followed using GUVs or liposomes [71,83,222]. These membrane mimics are often composed of mixture of predefined proportions of certain lipids while live cell membrane is composed of many different components in varying proportions with respect to time and space.…”

Section: Live Imagingmentioning

confidence: 99%

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Membrane Active Peptides and Their Biophysical Characterization

Avcı

Akbulut

Özkırımlı

2018

Preprint

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In the last 20 years, an increasing number of studies have been reported on membrane active peptides, which exert their biological activity by interacting with the cell membrane either to disrupt it and lead to cell lysis or to translocate through it to deliver cargos into the cell and reach their target. These peptides are attractive alternatives to currently used pharmaceuticals. Antimicrobial peptides (AMPs) and peptides designed for drug and gene delivery currently in the drug pipeline suggest that these membrane active peptides will soon constitute a significant percentage of the drug market. Here, we focus on two most prominent classes of membrane active peptides; AMPs and cell-penetrating peptides (CPPs). AMPs are a group of membrane active peptides that disrupt the membrane integrity or inhibit the cellular functions of bacteria, virus and fungi. CPPs are another group of membrane active peptides that mainly function as cargo-carriers even though they may also show antimicrobial activity to some extent. Biophysical techniques to understand how they interact with the membrane have shed light on the peptide-membrane interaction at various levels of detail. Structural investigation of membrane active peptides in the presence of the membrane provides important clues on the effect of the membrane environment on peptide conformations. Advances in live imaging techniques have allowed examination of peptide action at a single cell or single molecule level. In addition to these experimental biophysical techniques, molecular dynamics simulations provided clues on the peptide-lipid interactions and dynamics of the cell entry process at atomic detail. In this review, we summarize the recent advances in experimental and computational investigation of membrane active peptides with particular emphasis on two amphipathic membrane active peptides, the AMP melittin and the CPP pVEC.

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“…Yamazaki and coworkers proposed a different model, based on experiments with tp10 and a single GUV composed of DOPC (80 mol%) and DOPG (20 mol%) lipids [72]. They observed that tp10 translocation and membrane pore formation appear to be two independent processes.…”

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confidence: 99%

Current Understanding of the Mechanisms by which Membrane-Active Peptides Permeate and Disrupt Model Lipid Membranes

Sun

Forsman²,

Woodward

2015

CTMC

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Link to publicationCitation for published version (APA): Sun, D., Forsman, J., & Woodward, C. E. (2016). Current understanding of the mechanisms by which membrane-active peptides permeate and disrupt model lipid membranes. Current Topics in Medicinal Chemistry, 16(2), 170-186. DOI: 10.2174/1568026615666150812121241 General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.• Users may download and print one copy of any publication from the public portal for the purpose of private study or research.• You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal translocation, but associated with this, is membrane rupture to form large pores, which are subsequently stabilized by peptide adsorption to the pore edges. This disruption to the membrane is presumably responsible for cell death. Amyloidal peptides also show evidence of stable large pore formation, however, the mechanism for pore stabilization appears linked with their ability to form fibrils and prefibrillar aggregates and oligomers. There is some evidence that pores and membrane defects in fact act as nucleation sites for these structures. Where possible we have related the experimental and theoretical work to our own simulation findings in an effort to produce a comprehensive, albeit speculative picture for the mechanisms of action for this important group of peptides. Keywords:Membrane active peptides; anti-microbial peptides; cell-penetrating peptides; amyloid peptides; lipid membrane; pore-formation 3 Graphical AbstractWe review the modes of activity of three classes of membrane active peptides: cell penetrating; antimicrobial, and amyloid peptides.4

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Entry of Cell-Penetrating Peptide Transportan 10 into a Single Vesicle by Translocating Across Lipid Membrane and Its Induced Pores

Cited by 75 publications

References 36 publications

Bacterial Spheroplasts as a Model for Visualizing Membrane Translocation of Antimicrobial Peptides

Bacterial Spheroplasts as a Model for Visualizing Membrane Translocation of Antimicrobial Peptides

Membrane Active Peptides and Their Biophysical Characterization

Current Understanding of the Mechanisms by which Membrane-Active Peptides Permeate and Disrupt Model Lipid Membranes

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