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Characterization and Evaluation of Cell-Penetrating Activity of Brevinin-2R: An Amphibian Skin Antimicrobial Peptide

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Abstract

Natural peptides have been the source of some important tools to address challenges in protein therapy of diseases. Bypassing cell plasma membrane has been a bottleneck in the intracellular delivery of biomolecules. Among others, cell-penetrating peptides (CPPs) provide an efficient strategy for intracellular delivery of various cargos. Brevinin-2R peptide is an antimicrobial peptide isolated from the skin secretions of marsh frog, Rana ridibunda with semi-selective anticancer properties. Here, we investigated cell-penetrating properties of Brevinin-2R peptide and its ability to deliver functional protein cargos. Bioinformatics studies showed that Brevinin-2R is a cationic peptide with a net charge of + 5 with an alpha-helix structure and a heptameric ring at the carboxylic terminal due to disulfide bond between C19 and C25 amino acids and a hinge region at A10. To evaluate the ability of this peptide as a CPP, β-galactosidase protein and GFP were transfected into HeLa cells. The entry pathway of the peptide/protein complex into the cell was investigated by inhibiting endocytic pathways at 4 °C. It was observed that Brevinin-2R can efficiently transfer β-galactosidase and GFP with 21% and 90% efficacy, respectively. Brevinin-2R opts for endocytosis pathways to enter cells. The cytotoxicity of this peptide against HeLa cells was studied using MTT assay. The results showed that at the concentration of 131.5 μg/ml of Brevinin-2R peptide, the proliferation of 50% of HeLa cells was inhibited. The results of this study suggest that Brevinin-2R peptide can act as a CPP of natural origin and low cytotoxicity.

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Acknowledgements

This study was supported by research Grant No. 951791, provided by Mashhad University of Medical Sciences, Mashhad, Iran. This study was part of the first author MSc thesis.

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Authors and Affiliations

  1. Department of Medical Biotechnology and Nanotechnology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

    Samin Nooranian & Amin Jalili

  2. Applied Biomedical Research Center, Mashhad University of Medical Sciences, Mashhad, Iran

    Reza Kazemi Oskuee

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  1. Samin Nooranian
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  2. Reza Kazemi Oskuee
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  3. Amin Jalili
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Correspondence to Amin Jalili.

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Nooranian, S., Kazemi Oskuee, R. & Jalili, A. Characterization and Evaluation of Cell-Penetrating Activity of Brevinin-2R: An Amphibian Skin Antimicrobial Peptide. Mol Biotechnol 64, 546–559 (2022). https://doi.org/10.1007/s12033-021-00433-5

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