Peptide from sea anemone metridium senile affects transient receptor potential ankyrin-repeat 1 (TRPA1) function and produces analgesic effect

The Transient Receptor Potential Ankyrin-repeat 1 (TRPA1) is an important player in pain and inflammatory pathways. It is a promising target for novel drugs development for treatment of a number of pathological states. A novel peptide producing significant potentiating effect (up to ~90%) on AITC- and diclofenac-induced currents of TRPA1 was isolated from the venom of sea anemone Metridium senile. It is 35 amino acid peptide cross-linked by two disulfide bridges, named τ-AnmTX Ms 9a-1 (short name Ms 9a-1) according to structure similarity to other sea anemone peptides belonging to structural group 9a. The structure of two genes encoding different precursor proteins of Ms 9a-1 were determined. Peptide Ms 9a-1 acted as a positive modulator of TRPA1 in vitro but did not cause pain or thermal hyperalgesia when injected in mice hind paw. Intravenous injection of Ms 9a-1 (0.3 mg/kg) produced significant decrease of nociceptive and inflammatory response to AITC (agonist of TRPA1) and reversed CFA-induced inflammation and thermal hyperalgesia. Taken together, these data support the hypothesis that Ms 9a-1 potentiates response of TRPA1 to endogenous agonists followed by persistent functional loss of TRPA1-expressing neurons. We can conclude that TRPA1 potentiating may be useful as therapeutic approach since Ms 9a-1 produce significant analgesic and anti- inflammatory effect in mice models of pain

TRPV1 activation power can switch an action mode for its polypeptide ligands

<div><p>TRPV1 (vanilloid) receptors are activated by different types of stimuli including capsaicin, acidification and heat. Various ligands demonstrate stimulus-dependent action on TRPV1. In the present work we studied the action of polypeptides isolated from sea anemone <i>Heteractis crispa</i> (APHC1, APHC2 and APHC3) on rat TRPV1 receptors stably expressed in CHO cells using electrophysiological recordings, fluorescent Ca²⁺ measurements and molecular modeling. The APHCs potentiated TRPV1 responses to low (3–300 nM) concentrations of capsaicin but inhibited responses to high (>3.0 μM) concentrations. The activity-dependent action was also found for TRPV1 responses to 2APB and acidification. Thus the action mode of APHCs is bimodal and depended on the activation stimuli strength—potentiation of low-amplitude responses and no effect/inhibition of high-amplitude responses. The double-gate model of TRPV1 activation suggests that APHC-polypeptides may stabilize an intermediate state during the receptor activation. Molecular modeling revealed putative binding site at the outer loops of TRPV1. Binding to this site can directly affect activation by protons and can be allosterically coupled with capsaicin site. The results are important for further investigations of both TRPV1 and its ligands for potential therapeutic use.</p></div

Bimodal action of APHC3 on capsaicin-evoked response of TRPV1.

<p>[Ca²⁺] responses of rTRPV1-CHO cells to 3 nM capsaicin (<b>A</b>) and 3 μM capsaicin (<b>B</b>) alone (control) and in the presence of 500 nM APHC1, APHC2, APHC3. [Ca²⁺] responses were measured as changes in fluorescence intensity before (FI_base) and after agonist addition (FI). The data shown are representative average plots (n = 4) of the fluorescence signals against time during assays. <b>C</b>, Chart values of inhibiting/activating effects of APHC 1–3 (500 nM) relative to control in dependence on capsaicin concentration applied. In all graphs the values are given as mean ± SD of at least 5 independent experiments. Reliability (*) was checked by ANOVA followed by Tukey’s post hoc analysis, p = 0.05. <b>D</b>, “Dose-response” of TRPV1 activation by capsaicin alone (black squares) and in the presence of 500 nM APHC3 (red squares) determined in Fluo-4 based intracellular calcium assay tests. Relative responses were measured as (FI- FI_base)/FI_base, where FI is the measured peak fluorescence intensity after agonist addition, FI_base—fluorescence intensity of the cells before agonist addition. Data are expressed as mean ± SD (n = 4–8).</p

Action of APHCs on TRPV1 currents activated by 2APB or low pH stimuli.

<p><b>A-C</b>, effect of 100 nM APHC3 on TRPV1 currents activated by pH 6.2 (potentiation) (panel A) and pH 4.5 (no effect) (panel B), traces of cell responses in control and at peptide application are shown. Panel C summarize the effect of APHC3 (100 nM) for three different pH values, n = 9 in all series. <b>D-E</b>, Effect of 100 nM APHC3 on TRPV1 currents activated by 30 μM 2APB (potentiation) (panel D) and 300 μM 2APB (no effect) (panel E), traces of cell responses in control and at peptide application are shown. <b>F</b>, Chart values of APHC 1–3 (500 nM) inhibition effects relative to control in dependence on 2APB concentration applied in Fluo-4 based intracellular calcium assay tests. In all graphs the values are given as mean ± SD of at least 5 independent experiments. Reliability drug <i>vs</i> control (*) was checked by ANOVA followed by Tukey’s post hoc analysis, p = 0.05.</p

The model of APHC1 binding with TRPV1 outer region.

<p><b>A</b>, Aligned sequences of APHC1 and trypsin inhibitor SHPI-1. <b>B</b>, Different views on the minimal-energy complex calculated for APHC1 and close stated TRPV1. Backbone of APHC1 is shown magenta, backbone of the channel is colored green. The <i>C</i>-terminal helix of the APHC1 intercalates between diverging P-loops. <b>C</b>, The details of polypeptide-channel interaction. Key residues involved in the interaction are shown as sticks. <b>D</b>, Superimposition of two APHC1 binding models—APHC1—closed TRPV1 (green and magenta) and APHC1—opened TRPV1 (cyan and orange). In the open state the slot between P-loops is narrower and APHC1 is shifted in the external direction.</p

Kinetics of 100 nM APHC3 action in the continuous presence of activation stimuli.

<p><b>A</b>, Reversible inhibition of the current evoked by 3 μM capsaicin. <b>B</b>, Reversible potentiation of the current evoked by 0.3 μM capsaicin. <b>C</b>, Reversible potentiation of the current evoked by pH drop to 6.2. Single-exponential fitting in panels A-C are shown as red curves. <b>D</b>, Averaged values of time constants of APHC3 effects development and recovery (n = 5 in all series). The kinetics of action is significantly slower in the case of TRPV1 activation by pH drop.</p

New Disulfide-Stabilized Fold Provides Sea Anemone Peptide to Exhibit Both Antimicrobial and TRPA1 Potentiating Properties

A novel bioactive peptide named τ-AnmTx Ueq 12-1 (short name Ueq 12-1) was isolated and characterized from the sea anemone Urticina eques. Ueq 12-1 is unique among the variety of known sea anemone peptides in terms of its primary and spatial structure. It consists of 45 amino acids including 10 cysteine residues with an unusual distribution and represents a new group of sea anemone peptides. The 3D structure of Ueq 12-1, determined by NMR spectroscopy, represents a new disulfide-stabilized fold partly similar to the defensin-like fold. Ueq 12-1 showed the dual activity of both a moderate antibacterial activity against Gram-positive bacteria and a potentiating activity on the transient receptor potential ankyrin 1 (TRPA1). Ueq 12-1 is a unique peptide potentiator of the TRPA1 receptor that produces analgesic and anti-inflammatory effects in vivo. The antinociceptive properties allow us to consider Ueq 12-1 as a potential analgesic drug lead with antibacterial properties

Physical and Biological Features of the Waters in the Outer Patagonian Shelf and the Malvinas Current

The aim of this study is to trace how the fine-thermohaline and kinematic structure, formed over a section along 45.8° S in the interaction zone of the outer Patagonian Shelf (PS) and Malvinas (Falkland) Current (MC) System waters, affect the spatial distribution of bio-optical characteristics, phyto/zooplankton, birds, and marine mammals. For the first time, simultaneous multidisciplinary observations at high spatial resolution (~2.5 km) were performed in this region during the cruise of the R/V “Akademic Mstislav Keldysh” in February 2022. A fine structure of alternating upwelling and downwelling zones over the PS and slope was identified, which resulted from the interaction between the MC inshore branch (MCi), bottom topography, and wind. This interaction significantly affects all the physical, and optical characteristics analyzed in the work, as well as the biota of the region. It was found that the euphotic zone is larger in the downwelling zones than in the upwelling zones, and all spatially local maxima of phytoplankton photosynthetic efficiency are observed in the zones between upwelling and downwelling. Phytoplankton along the section were represented by 43 species. A total of 30 zooplankton species/taxa were identified. Three species of marine mammals and 11 species of birds were recorded in the study site. Most of the phytoplankton species list were formed by dinoflagellates, and picoplankton Prasinoderma colonial quantitatively dominated everywhere. Two floristic and three assemblage groups were distinguished among the analyzed phytoplankton communities. High phytoplankton biodiversity was observed above the PS and low above the PS edge and in the MCi core. Copepods mostly dominated in zooplankton. Subantarctic species/taxa of zooplankton concentrated in the nearshore waters of the PS, while Antarctic species/taxa were most abundant in the zone between the MCi and the MC offshore branch (MCo). The relative abundance of birds in the PS was several times higher than in the MCo. The minimum abundance of birds was in the MCi in the zone of the strongest upwelling identified above the PS edge