Dataset Information

COPA and SLC4A4 are required for cellular entry of arginine-rich peptides.

ABSTRACT: Cell-penetrating peptides (CPPs) have gained attention as promising tools to enable the delivery of various molecules in a non-invasive manner. Among the CPPs, TAT and poly-arginine have been extensively utilized in numerous studies for the delivery of functional proteins, peptides, and macromolecules to analyze cellular signaling. However, the molecular mechanisms of cellular entry remain largely unknown. Here, we applied siRNA library screening to identify the regulatory genes for the cellular entry of poly-arginine peptide based on microscopic observation of the entry of fluorescent peptides in siRNA-treated cells. In this screening, we identified the cell membrane gene SLC4A4 and the trafficking regulator gene COPA, which also plays an important role in early endosome maturation. These results demonstrated that cellular entry of poly-arginine requires at least two different steps, probably binding on the cell surface and endosomal entry. The identification of genes for cellular entry of poly-arginine provides insights into its mechanisms and should further aid in the development of highly efficient cell-penetrating peptides.

SUBMITTER: Tsumuraya T

PROVIDER: S-EPMC3904941 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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COPA and SLC4A4 are required for cellular entry of arginine-rich peptides.

Tsumuraya Tomoyuki T Matsushita Masayuki M

PloS one 20140128 1

Cell-penetrating peptides (CPPs) have gained attention as promising tools to enable the delivery of various molecules in a non-invasive manner. Among the CPPs, TAT and poly-arginine have been extensively utilized in numerous studies for the delivery of functional proteins, peptides, and macromolecules to analyze cellular signaling. However, the molecular mechanisms of cellular entry remain largely unknown. Here, we applied siRNA library screening to identify the regulatory genes for the cellular ...[more]

PMID: 24489756

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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.

Dataset Information

COPA and SLC4A4 are required for cellular entry of arginine-rich peptides.

Publications

COPA and SLC4A4 are required for cellular entry of arginine-rich peptides.

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