Dataset Information

Unlocking Endosomal Entrapment with Supercharged Arginine-Rich Peptides.

ABSTRACT: Endosomal entrapment is a common bottleneck in various macromolecular delivery approaches. Recently, the polycationic peptide dfTAT was identified as a reagent that induces the efficient leakage of late endosomes and, thereby, enhances the penetration of macromolecules into the cytosol of live human cells. To gain further insights into the features that lead to this activity, the role of peptide sequence was investigated. We establish that the leakage activity of dfTAT can be recapitulated by polyarginine analogs but not by polylysine counterparts. Efficiencies of peptide endocytic uptake increase linearly with the number of arginine residues present. In contrast, peptide cytosolic penetration displays a threshold behavior, indicating that a minimum number of arginines is required to induce endosomal escape. Increasing arginine content above this threshold further augments delivery efficiencies. Yet, it also leads to increasing the toxicity of the delivery agents. Together, these data reveal a relatively narrow arginine-content window for the design of optimally active endosomolytic agents.

SUBMITTER: Najjar K

PROVIDER: S-EPMC5905407 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Unlocking Endosomal Entrapment with Supercharged Arginine-Rich Peptides.

Najjar Kristina K Erazo-Oliveras Alfredo A Mosior John W JW Whitlock Megan J MJ Rostane Ikram I Cinclair Joseph M JM Pellois Jean-Philippe JP Pellois Jean-Philippe JP

Bioconjugate chemistry 20171113 12

Endosomal entrapment is a common bottleneck in various macromolecular delivery approaches. Recently, the polycationic peptide dfTAT was identified as a reagent that induces the efficient leakage of late endosomes and, thereby, enhances the penetration of macromolecules into the cytosol of live human cells. To gain further insights into the features that lead to this activity, the role of peptide sequence was investigated. We establish that the leakage activity of dfTAT can be recapitulated by po ...[more]

PMID: 29065262

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Project description:The self-assembly behavior and antimicrobial activity of two designed amphiphilic peptides, R3F3 and R4F4, containing short hydrophobic phenylalanine (F) and cationic arginine (R) sequences, are investigated. The conformation of the peptides was examined using circular dichroism and FTIR spectroscopy, which show that they have a disordered secondary structure. Concentration-dependent fluorescence assays show the presence of a critical aggregation concentration (cac) for each peptide. Above the cac, small-angle X-ray scattering (SAXS) and transmission electron microscopy (TEM) reveal a population of twisted tapes for R3F3 and nanosheets for R4F4. The interaction of the peptides with model bacterial membranes comprising mixtures of the lipids DPPG [1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol] and DPPE [1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine], was studied using SAXS and cryogenic-TEM. Analysis of the SAXS structure factor indicates that R3F3 interacts with lipid bilayers by inducing correlation between bilayers, whereas R4F4 interacts with the bilayers causing an increase in polydispersity of the vesicle wall thickness. Both peptides break vesicles with a 1:3 DPPG:DPPE composition, which is close to the ratio of PG and PE lipids observed in the lipid membrane of Pseudomonas aeruginosa, a pathogen responsible for serious infections and which has developed antimicrobial resistant strains. Both peptides show activity against this bacterium in planktonic form. Peptide R4F4 shows particularly strong bioactivity against this microbe, with a minimum inhibitory concentration (MIC) value in the range of concentrations where the peptide is cytocompatible. It was further shown to have activity against other Pseudomonas species including the common plant pathogen Pseudomonas syringae. Finally, we show that R4F4 inhibits the development of P. aeruginosa biofilms. This was examined in detail and a proposed mechanism involving binding of the signaling molecule c-di-GMP is suggested, based on circular dichroism spectroscopy studies and Congo red assays of extracellular polysaccharides produced by the stressed bacteria. Thus, R4F4 is a promising candidate antimicrobial peptide with activity against Pseudomonas species.

Project description:Antimicrobial activity of many AMPs can be improved by lysine-to-arginine substitution due to a more favourable interaction of arginine guanidinium moiety with bacterial membranes. In a previous work, the structural and functional characterization of an amphipathic antimicrobial peptide named RiLK1, including lysine and arginine as the positively charged amino acids in its sequence, was reported. Specifically, RiLK1 retained its β-sheet structure under a wide range of environmental conditions (temperature, pH, and ionic strength), and exhibited bactericidal activity against Gram-positive and Gram-negative bacteria and fungal pathogens with no evidence of toxicity on mammalian cells. To further elucidate the influence of a lysine-to-arginine replacement on RiLK1 conformational properties, antimicrobial activity and peptide-liposome interaction, a new RiLK1-derivative, named RiLK3, in which the lysine is replaced with an arginine residue, was projected and characterised in comparison with its parental compound. The results evidenced that lysine-to-arginine mutation not only did not assure an improvement in the antimicrobial potency of RiLK1 in terms of bactericidal, virucidal and fungicidal activities, but rather it was completely abolished against the hepatitis A virus. Therefore, RiLK1 exhibited a wide range of antimicrobial activity like other cationic peptides, although the exact mechanisms of action are not completely understood. Moreover, tryptophan fluorescence measurements confirmed that RiLK3 bound to negatively charged lipid vesicles with an affinity lower than that of RiLK1, although no substantial differences from the structural and self-assembled point of view were evidenced. Therefore, our findings imply that antimicrobial efficacy and selectivity are affected by several complex and interrelated factors related to substitution of lysine with arginine, such as their relative proportion and position. In this context, this study could provide a better rationalisation for the optimization of antimicrobial peptide sequences, paving the way for the development of novel AMPs with broad applications.

Dataset Information

Unlocking Endosomal Entrapment with Supercharged Arginine-Rich Peptides.

Publications

Unlocking Endosomal Entrapment with Supercharged Arginine-Rich Peptides.

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