A Pore Forming Toxin-like Protein Derived from Chinese Red Belly Toad Bombina maxima Triggers the Pyroptosis of Hippomal Neural Cells and Impairs the Cognitive Ability of Mice.

Toxin-like proteins and peptides of skin secretions from amphibians play important physiological and pathological roles in amphibians. ßγ-CAT is a Chinese red-belly toad-derived pore-forming toxin-like protein complex that consists of aerolysin domain, crystalline domain, and trefoil factor domain and induces various toxic effects via its membrane perforation process, including membrane binding, oligomerization, and endocytosis. Here, we observed the death of mouse hippocampal neuronal cells induced by ßγ-CAT at a concentration of 5 nM. Subsequent studies showed that the death of hippocampal neuronal cells was accompanied by the activation of Gasdermin E and caspase-1, suggesting that ßγ-CAT induces the pyroptosis of hippocampal neuronal cells. Further molecular mechanism studies revealed that the pyroptosis induced by ßγ-CAT is dependent on the oligomerization and endocytosis of ßγ-CAT. It is well known that the damage of hippocampal neuronal cells leads to the cognitive attenuation of animals. The impaired cognitive ability of mice was observed after intraperitoneal injection with 10 µg/kg ßγ-CAT in a water maze assay. Taken together, these findings reveal a previously unknown toxicological function of a vertebrate-derived pore-forming toxin-like protein in the nerve system, which triggers the pyroptosis of hippocampal neuronal cells, ultimately leading to hippocampal cognitive attenuation.

Cytotoxic peptides with insulin-releasing activities from skin secretions of the Italian stream frog Rana italica (Ranidae).

Peptidomic analysis of norepinephrine-stimulated skin secretions from Italian stream frog Rana italica led to the purification and characterization of two host-defense peptides differing by a single amino acid residue belonging to the brevinin-1 family (brevinin-1ITa and -1ITb), a peptide belonging to the temporin family (temporin-ITa) and a component identified as prokineticin Bv8. The secretions contained relatively high concentrations of the methionine-sulphoxide forms of brevinin-1ITa and -1ITb suggesting that these peptides may have a role as antioxidants in the skin of this montane frog. Brevinin-1ITa (IVPFLLGMVPKLVCLITKKC) displayed potent cytotoxicity against non-small cell lung adenocarcinoma A549 cells (LC50  = 18 µM), breast adenocarcinoma MDA-MB-231 cells (LC50  = 8 µM) and colorectal adenocarcinoma HT-29 cells (LC50  = 18 µM), but the peptide was also strongly hemolytic against mouse erythrocytes (LC50  = 7 µM). Temporin-ITa (VFLGAIAQALTSLLGKL.NH2 ) was between three and fivefold less potent against these cells. Brevinin-1ITa inhibited growth of both Gram-positive Staphylococcus epidermidis and Gram-negative Escherichia coli as well as a strain of the opportunist yeast pathogen Candida parapsilosis, whereas temporin-ITa was active only against S. epidermidis and C. parapsilosis. Both peptides stimulated the release of insulin from BRIN-BD11 clonal ß-cells at concentrations ≥1 nM, but brevinin-1ITa was cytotoxic to the cells at concentrations ≥3 µM. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Inhibitory and antimicrobial activities of OGTI and HV-BBI peptides, fragments and analogs derived from amphibian skin.

A series of linear and cyclic fragments and analogs of two peptides (OGTI and HV-BBI) isolated from skin secretions of frogs were synthesized by the solid-phase method. Their inhibitory activity against several serine proteinases: bovine ß-trypsin, bovine α-chymotypsin, human leukocyte elastase and cathepsin G from human neutrophils, was investigated together with evaluation of their antimicrobial activities against Gram-negative bacteria (Escherichia coli) and Gram-positive species isolated from patients (Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus sp., Streptococcus sp.). The cytotoxicity of the selected peptides toward an immortal human skin fibroblast cell line was also determined. Three peptides: HV-BBI, its truncated fragment HV-BBI(3-18) and its analog [Phe(8)]HV-BBI can be considered as bifunctional compounds with inhibitory as well as antibacterial properties. OGTI, although it did not display trypsin inhibitory activity as previously reported in the literature, exerted antimicrobial activity toward S. epidermidis. In addition, under our experimental conditions, this peptide did not show cytotoxicity.

Brevinin-2R(1) semi-selectively kills cancer cells by a distinct mechanism, which involves the lysosomal-mitochondrial death pathway.

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.

The antimicrobial peptide dermaseptin S4 inhibits HIV-1 infectivity in vitro.

Most of HIV-1 infections are acquired through sexual contact. In the absence of a preventive vaccine, the development of topical microbicides that can block infection at the mucosal tissues is needed. Dermaseptin S4 (DS4) is an antimicrobial peptide derived from amphibian skin, which displays a broad spectrum of activity against bacteria, yeast, filamentous fungi, and herpes simplex virus type 1. We show here that DS4 inhibits cell-free and cell-associated HIV-1 infection of P4-CCR5 indicator cells and human primary T lymphocytes. The peptide is effective against R5 and X4 primary isolates and laboratory-adapted strains of HIV-1. Its activity is directed against HIV-1 particles by disrupting the virion integrity. Increasing the number of DS4-positive charges reduced cytotoxicity without affecting the antiviral activity. The modified DS4 inhibited HIV-1 capture by dendritic cells and subsequent transmission to CD4(+) T cells, as well as HIV-1 binding on HEC-1 endometrial cells and transcytosis through a tight epithelial monolayer.

Synergism of Rana catesbeiana ribonuclease and IFN-gamma triggers distinct death machineries in different human cancer cells.

Rana catesbeiana ribonuclease (RC-RNase) possesses tumor-specific cytotoxicity, which can be synergized by IFN-gamma. However, it is unclear how RC-RNase and RC-RNase/IFN-gamma induce cell death. In this study, we use substrate cleavage assays to systematically investigate RC-RNase- and RC-RNase/IFN-gamma-induced caspase activation in HL-60, MCF-7, and SK-Hep-1 cells. We find that RC-RNase and RC-RNase/IFN-gamma induce mitochondria-mediated caspase activation in HL-60 and MCF-7 cells but not in SK-Hep-1 cells, although death of SK-Hep-1 cells is closely related to mitochondrial disruptions. Our findings provide evidence that RC-RNase and RC-RNase/IFN-gamma can kill different cancer cells by distinct mechanisms. Compared with onconase, RC-RNase seems to harbor a more specific anti-cancer activity.