Brevinin-2R

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.

Brevinin-2R is a novel non-hemolytic defensin that was isolated from the skin of the frog Rana ridibunda. It exhibits preferential cytotoxicity towards malignant cells, including Jurkat (T-cell leukemia), BJAB (B-cell lymphoma), HT29/219, SW742 (colon carcinomas), L929 (fibrosarcoma), MCF-7 (breast adenocarcinoma), A549 (lung carcinoma), as compared to primary cells including peripheral blood mononuclear cells (PBMC), T cells and human lung fibroblasts. Jurkat and MCF-7 cells overexpressing Bcl2, and L929 and MCF-7 over-expressing a dominant-negative mutant of a pro-apoptotic BNIP3 (DeltaTM-BNIP3) were largely resistant towards Brevinin-2R treatment. The decrease in mitochondrial membrane potential (DeltaPsim), or total cellular ATP levels, and increased reactive oxygen species (ROS) production, but not caspase activation or the release of apoptosis-inducing factor (AIF) or endonuclease G (Endo G), were early indicators of Brevinin-2R-triggered death. Brevinin-2R interacts with both early and late endosomes. Lysosomal membrane permeabilization inhibitors and inhibitors of cathepsin-B and cathepsin-L prevented Brevinin-2R-induced cell death. Autophagosomes have been detected upon Brevinin-2R treatment. Our results show that Brevinin-2R activates the lysosomalmitochondrial death pathway, and involves autophagy-like cell death.

Objectives: Polyethylenimine (PEI) is one of the most widely used polymers in gene delivery. The aim of this study was to modify PEI by replacing some of its primary amines with Brevinin 2R (BR-2R) peptide in order to increase the efficiency of gene delivery. Materials and methods: Polyethylenimine was modified by BR-2R peptide by two different approaches; A) conjugation methods including (І) using succinimidyl 3-(2-pyridyldithio) propionate (SPDP), (П) EDC/NHS protocol and (ПІ) EDC/NHS+6-bromohexanoic acid protocol, and B) physical interaction method. The modified polymers were characterized for their ability of plasmid condensation, number of primary amines, size and zeta potential. The transfection efficiency and cytotoxicity were evaluated on HEK293, L929, WEHI164 and Neuro2A cell lines by green fluorescent protein (GFP)-based plasmid (pGFP) reporter gene and viability assays, respectively. Apoptosis induction ability was also evaluated via PI/Annexin V assay. Results: Polyplex had size and zeta potential between 200-270 nm and +21.5- +28.4 mV, respectively. All vectors were able to condense plasmid DNA in C/P=4 (carrier-plasmid ratio). Transfection results on the Neuro2A cell line showed that the vector containing the BR-2R peptide, which was synthesized using EDC-NHS protocol had the best transfection efficiency. Conclusion: Our results showed that conjugation of Brevinin 2R as cell penetrating peptide to polyethyleneimine could enhance the transfection ability of the polymer.