Esculentin-1B

Long-standing Type 2 diabetes is associated with loss of both β-cell function and β-cell mass. Peptides derived from the frog-skin host-defense peptide esculentin-1 have been shown to exhibit potent, broad-spectrum antimicrobial activity. The aim of the present study is to determine whether such peptides also show insulinotropic and β-cell protective activities. Esculentin-1a(1-21).NH2, esculentin-1b(1-18).NH2, and esculentin-1a(1-14).NH2 produced concentration-dependent stimulations of insulin release from BRIN-BD11 rat clonal β-cells, 1.1B4 human-derived pancreatic β-cells, and isolated mouse islets with no cytotoxicity at concentrations of up to 3 μM. The mechanism of insulinotropic action involved membrane depolarization and an increase in intracellular Ca2+ concentrations. The analogue [D-Lys14, D-Ser17]esculentin-1a(1-21).NH2 (Esc(1-21)-1c) was less potent in vitro than the all L-amino acid containing peptides and esculentin-1a(9-21) was inactive indicating that helicity is an important determinant of insulinotropic activity. However, intraperitoneal injection of Esc(1-21)-1c (75 nmol/kg body weight) together with a glucose load (18 mmol/kg body weight) in C57BL6 mice improved glucose tolerance with a concomitant increase in insulin secretion, whereas administration of esculentin-1a(1-21).NH2, esculentin-1b(1-18).NH2, and esculentin-1a(1-14) was without significant effect on plasma glucose levels. Esc(1-21)-1c (1 µM) protected BRIN-BD11 cells against cytokine-induced apoptosis (P < 0.01) and augmented proliferation of the cells (P < 0.01) to a similar extent as glucagon-like peptide-1. The data demonstrate that the multifunctional peptide Esc(1-21)-1c, as well as showing therapeutic potential as an anti-infective and wound-healing agent, may constitute a template for development of compounds for treatment of patients with Type 2 diabetes.

Antimicrobial peptides constitute one of the main classes of molecular weapons deployed by the innate immune system of all multicellular organisms to resist microbial invasion. A good proportion of all antimicrobial peptides currently known, numbering hundreds of molecules, have been isolated from frog skin. Nevertheless, very little is known about the effect(s) and the mode(s) of action of amphibian antimicrobial peptides on intact bacteria, especially when they are used at subinhibitory concentrations and under conditions closer to those encountered in vivo. Here we show that esculentin-1b(1-18) [Esc(1-18)] (GIFSKLAGKKLKNLLISG-NH(2)), a linear peptide encompassing the first 18 residues of the full-length esculentin-1b, rapidly kills Escherichia coli at the minimal inhibitory concentration. The lethal event is concomitant with the permeation of the outer and inner bacterial membranes. This is in contrast to what is found for many host defense peptides, which do not destabilize membranes at their minimal inhibitory concentrations. Importantly, proteomic analysis revealed that Esc(1-18) has a limited ability to modify the bacterium's protein expression profile, at either bactericidal or sublethal concentrations. To the best of our knowledge, this is the first report on the effects of an antimicrobial peptide from frog skin on the proteome of its bacterial target, and underscores the fact that the bacterial membrane is the major target for the killing mechanism of Esc(1-18), rather than intracellular processes.

Antimicrobial peptides (AMPs) are found in various classes of organisms as part of the innate immune system. Despite high sequence variability, they share common features such as net positive charge and an amphipathic fold when interacting with biologic membranes. Esculentin-1b is a 46-mer frog-skin peptide, which shows an outstanding antimicrobial activity. Experimental studies revealed that the N-terminal fragment encompassing the first 18 residues, Esc(1-18), is responsible for the antimicrobial activity of the whole peptide, with a negligible toxicity toward eukaryotic cells, thus representing an excellent candidate for future pharmaceutical applications. Similarly to most of the known AMPs, Esc(1-18) is expected to act by destroying/permeating the bacterial plasma-membrane but, to date, its 3D structure and the detailed mode of action remains unexplored. Before an in-depth investigation on peptide/membranes interactions could be undertaken, it is necessary to characterize peptide's folding propensity in solution, to understand what is intrinsically due to the peptide sequence, and what is actually driven by the membrane interaction. Circular dichroism and nuclear magnetic resonance spectroscopy were used to determine the structure adopted by the peptide, moving from water to increasing amounts of trifluoroethanol. The results showed that Esc(1-18) has a clear tendency to fold in a helical conformation as hydrophobicity of the environment increases, revealing an intriguing amphipathic structure. The helical folding is adopted only by the N-terminal portion of the peptide, while the rest is unstructured. The presence of a hydrophobic cluster of residues in the C-terminal portion suggests its possible membrane-anchoring role.