Recombinant Analogs of Sea Anemone Kunitz-Type Peptides Influence P2X7 Receptor Activity in Neuro-2a Cells.

Purinergic P2X7 receptors (P2X7) have now been proven to play an important role and represent an important therapeutic target in many pathological conditions including neurodegeneration. Here, we investigated the impact of peptides on purinergic signaling in Neuro-2a cells through the P2X7 subtype in in vitro models. We have found that a number of recombinant peptides, analogs of sea anemone Kunitz-type peptides, are able to influence the action of high concentrations of ATP and thereby reduce the toxic effects of ATP. The influx of calcium, as well as the fluorescent dye YO-PRO-1, was significantly suppressed by the studied peptides. Immunofluorescence experiments confirmed that the peptides reduce the P2X7 expression level in neuronal Neuro-2a cells. Two selected active peptides, HCRG1 and HCGS1.10, were found to specifically interact with the extracellular domain of P2X7 and formed stable complexes with the receptor in surface plasmon resonance experiments. The molecular docking approach allowed us to establish the putative binding sites of the most active HCRG1 peptide on the extracellular domain of the P2X7 homotrimer and propose a mechanism for regulating its function. Thus, our work demonstrates the ability of the Kunitz-type peptides to prevent neuronal death by affecting signaling through the P2X7 receptor.

First Anti-Inflammatory Peptide AnmTX Sco 9a-1 from the Swimming Sea Anemone Stomphia coccinea.

A novel peptide AnmTX Sco 9a-1 with the ß-hairpin fold was isolated from the swimming sea anemone Stomphia coccinea (Actinostolidae family). The peptide consists of 28 amino acid residues, including modified hydroxyproline residue, and its measured molecular mass is 2960 Da. The peptide was not toxic on mice; however, it stimulated their exploratory motivation and active search behavior, and demonstrated an anti-anxiety effect. AnmTX Sco 9a-1 at doses of 0.1 and 1 mg/kg reduced the volume of edema during 24 h better than the nonsteroidal anti-inflammatory drug, Diclofenac, at dose of 1 mg/kg in a model of acute local λ-carrageenan-induced inflammation. ELISA analysis of the animal's blood showed that peptide at a dose of 1 mg/kg reduced the content of tumor necrosis factor-α (TNF-α), a pro-inflammatory mediator responsible in the edema development, up to the level of TNF-α in the intact group. Besides, AnmTX Sco 9a-1 demonstrated a significant analgesic effect on acute pain sensitivity in the carrageenan-induced thermal hyperalgesia model at doses of 0.1 and 1 mg/kg. Activity of AnmTX Sco 9a-1 was shown not to be associated with modulation of nociceptive ASIC channels.

New Insights into the Type II Toxins from the Sea Anemone Heteractis crispa.

Toxins modulating NaV channels are the most abundant and studied peptide components of sea anemone venom. Three type-II toxins, δ-SHTX-Hcr1f (= RpII), RTX-III, and RTX-VI, were isolated from the sea anemone Heteractis crispa. RTX-VI has been found to be an unusual analog of RTX-III. The electrophysiological effects of Heteractis toxins on nine NaV subtypes were investigated for the first time. Heteractis toxins mainly affect the inactivation of the mammalian NaV channels expressed in the central nervous system (NaV1.1-NaV1.3, NaV1.6) as well as insect and arachnid channels (BgNaV1, VdNaV1). The absence of Arg13 in the RTX-VI structure does not prevent toxin binding with the channel but it has changed its pharmacological profile and potency. According to computer modeling data, the δ-SHTX-Hcr1f binds within the extracellular region of the rNaV1.2 voltage-sensing domain IV and pore-forming domain I through a network of strong interactions, and an additional fixation of the toxin at the channel binding site is carried out through the phospholipid environment. Our data suggest that Heteractis toxins could be used as molecular tools for NaV channel studies or insecticides rather than as pharmacological agents.