Uptake Kinetics of Cell-Penetrating Peptides

Florén, Anders; Mäger, Imre; Langel, Ülo

doi:10.1007/978-1-60761-919-2_9

Cited by 14 publications

(8 citation statements)

References 18 publications

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“…The penetration abilities of the YopM-derived CPPs were investigated in different eukaryotic cell lines and compared to the performance of the Tat peptide. The fluorescein isothiocyanate (FITC)-labeled peptides were incubated in HeLa cells and human brain microvascular endothelial cells (HBMEC) at 37°C and analyzed by using flow cytometry in a quenched time-lapse uptake assay (46). In this assay, 0.2% trypan blue was added to completely quench the extracellular fluorescence and to measure only fluorescence arising from the intracellular FITC-labeled peptides.…”

Section: Resultsmentioning

confidence: 99%

“…To determine whether direct penetration was also involved in the translocation of these peptides, a membranolysis assay was performed (46). This assay is based on the measurement of the fluorescence emission of the cell-impermeable compound propidium iodide (PI) once it intercalates into DNA during an ongoing incubation of cells with FITC-labeled CPPs.…”

Section: Resultsmentioning

confidence: 99%

“…The kinetics of CPP-FITC peptide uptake were measured by performing quenched time-lapse uptake assays (46). To measure peptide uptake rates, trypan blue was added to quench extracellular fluorescence.…”

Section: Methodsmentioning

confidence: 99%

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Bacterium-Derived Cell-Penetrating Peptides Deliver Gentamicin To Kill Intracellular Pathogens

Gomarasca

Martins

Greune

et al. 2017

Antimicrob Agents Chemother

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Commonly used antimicrobials show poor cellular uptake and often have limited access to intracellular targets, resulting in low antimicrobial activity against intracellular pathogens. An efficient delivery system to transport these drugs to the intracellular site of action is needed. Cell-penetrating peptides (CPPs) mediate the internalization of biologically active molecules into the cytoplasm. Here, we characterized two CPPs, ␣1H and ␣2H, derived from the Yersinia enterocolitica YopM effector protein. These CPPs, as well as Tat (trans-activator of transcription) from HIV-1, were used to deliver the antibiotic gentamicin to target intracellular bacteria. The YopM-derived CPPs penetrated different endothelial and epithelial cells to the same extent as Tat. CPPs were covalently conjugated to gentamicin, and CPP-gentamicin conjugates were used to target infected cells to kill multiple intracellular Gram-negative pathogenic bacteria, including Escherichia coli K1, Salmonella enterica serovar Typhimurium, and Shigella flexneri. Taken together, CPPs show great potential as delivery vehicles for antimicrobial agents and may contribute to the generation of new therapeutic tools to treat infectious diseases caused by intracellular pathogens.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Bacterium-Derived Cell-Penetrating Peptides Deliver Gentamicin To Kill Intracellular Pathogens

Gomarasca

Martins

Greune

et al. 2017

Antimicrob Agents Chemother

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“…The mechanism of cell entry of these peptides has been widely studied (13,(17)(18)(19)(21)(22)(23). Although there is still some disagreement on the contribution of spontaneous membrane translocation, in recent years, a consensus has begun to emerge that the highly cationic CPPs (such as Tat and polyarginine) are actively taken up into cells mostly by one or more type of endocytosis (10, 12, 13, 15, 19, 21, 24 -26).…”

mentioning

confidence: 99%

Direct Cytosolic Delivery of Polar Cargo to Cells by Spontaneous Membrane-translocating Peptides

He¹,

Kauffman²,

Fuselier³

et al. 2013

Journal of Biological Chemistry

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Background: Spontaneous membrane-translocating peptides were discovered by screening in synthetic lipid vesicles. Results: The translocating peptides carry membrane-impermeant cargos directly across cell membranes and drive systemic biodistribution in small animals. Conclusion: These peptides constitute a new class of delivery vehicle for membrane-impermeant cargos. Significance: Spontaneous membrane-translocating peptides could expand the universe of useful drugs.

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“…FACS analysis was employed to monitor uptake kinetics of the recombinant proteins Ssph1, YopM, and Tat-GFP [16] . CTT TAA GAA GGA GAT ATA CAT ATG GCT AGC TTT AAT ATC CGC AAT ACA CAA CCT TCT GTA R-SspHI-3xFLAG…”

Section: Facs Analysesmentioning

confidence: 99%

Analysis of T3SS-independent Autonomous Internalization of the Bacterial Effector Protein SspH1 from Salmonella typhimurium

Lubos

Norkowski

Stolle

et al. 2014

Inflamm Cell Signal

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Previous studies identified the effector protein YopM of Yersinia enterocolitica as a novel bacterial cell-penetrating protein. YopM's ability to translocate across the host cell plasma membrane independently of Yersinia's type III secretion system (T3SS) is mediated by its two N-terminal α-helices. The SspH1 effector protein of Salmonella typhimurium shares significant homology in sequence and structure with YopM, which prompted us to investigate potential cell-penetrating abilities of this effector protein. For this, we recombinantly expressed SspH1 in Escherichia coli and analyzed a potential T3SS-independent translocation of the protein by cell fractionation of HeLa cells, immunofluorescence microscopy and FACS analyses. The functionality of the recombinant protein as an E3 ubiquitin-ligase was determined using in vitro-ubiquitination assays. Additionally, an effect of the recombinant protein on the expression of pro-inflammatory cytokines was analyzed by quantitative real time PCR. In this study, we could show that the SspH1 effector protein of Salmonella typhimurium is able to translocate autonomously into eukaryotic cells without requiring additional factors. Furthermore, we could show that recombinant SspH1 is a functional E3 ubiquitin ligase that is able to undergo auto-ubiquitination following T3SS-independent translocation and to reduce the expression of Interleukin-8 in IL-1β stimulated cells.

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Uptake Kinetics of Cell-Penetrating Peptides

Cited by 14 publications

References 18 publications

Bacterium-Derived Cell-Penetrating Peptides Deliver Gentamicin To Kill Intracellular Pathogens

Bacterium-Derived Cell-Penetrating Peptides Deliver Gentamicin To Kill Intracellular Pathogens

Direct Cytosolic Delivery of Polar Cargo to Cells by Spontaneous Membrane-translocating Peptides

Analysis of T3SS-independent Autonomous Internalization of the Bacterial Effector Protein SspH1 from Salmonella typhimurium

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