Kalina RS et al. (2020), APETx-Like Peptides from the Sea Anemone Hetera...

XB-ART-57868

Toxins (Basel) 2020 Apr 20;124:. doi: 10.3390/toxins12040266.

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APETx-Like Peptides from the Sea Anemone Heteractis crispa, Diverse in Their Effect on ASIC1a and ASIC3 Ion Channels.

Kalina RS , Koshelev SG , Zelepuga EA , Kim NY , Kozlov SA , Kozlovskaya EP , Monastyrnaya MM , Gladkikh IN .

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Currently, five peptide modulators of acid-sensing ion channels (ASICs) attributed to structural class 1b of sea anemone toxins have been described. The APETx2 toxin is the first and most potent ASIC3 inhibitor, so its homologs from sea anemones are known as the APETx-like peptides. We have discovered that two APETx-like peptides from the sea anemone Heteractis crispa, Hcr 1b-3 and Hcr 1b-4, demonstrate different effects on rASIC1a and rASIC3 currents. While Hcr 1b-3 inhibits both investigated ASIC subtypes with IC50 4.95 ± 0.19 μM for rASIC1a and 17 ± 5.8 μM for rASIC3, Hcr 1b-4 has been found to be the first potentiator of ASIC3, simultaneously inhibiting rASIC1a at similar concentrations: EC50 1.53 ± 0.07 μM and IC50 1.25 ± 0.04 μM. The closest homologs, APETx2, Hcr 1b-1, and Hcr 1b-2, previously demonstrated the ability to inhibit hASIC3 with IC50 63 nM, 5.5, and 15.9 μM, respectively, while Hcr 1b-2 also inhibited rASIC1a with IC50 4.8 ± 0.3 μM. Computer modeling allowed us to describe the peculiarities of Hcr 1b-2 and Hcr 1b-4 interfaces with the rASIC1a channel and the stabilization of the expanded acidic pocket resulting from peptides binding which traps the rASIC1a channel in the closed state.

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Species referenced: Xenopus laevis
Genes referenced: asic1 asic3

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	Figure 1. Multiple sequence alignment of the toxins: Hcr 1b-1 (P0DL87), Hcr 1b-2 (C0HL52), Hcr 1b-3 (C0HL53), Hcr 1b-4 (C0HL54) from H. crispa, and APETx2 (P61542) from A. elegantissima. Identical and conserved amino acid residues are shown on a dark and light gray background, respectively. Vector NTI software (Invitrogen, USA) [37] was used for multiple sequence alignment.
	Figure 2. Circular dichroism (CD) spectra of H. crispa peptides Hcr 1b-2, -3, and -4.
	Figure 3. Homology models of Hcr 1b-2, -3, -4 and spatial structures of APETx2 and PcTx1. (a) Ribbon representation of peptide molecules and hydrophobic, basic, and acidic residues are colored green, blue, and red, respectively. Dipole moments are shown as blue arrows; magnitude of dipole moments is indicated as Debye. Visualization is performed with Discovery studio 4.0 Visualizer software [41]. (b) The spherical projection maps of surface electrostatic and hydrophobic properties for Hcr 1b-2, -3, -4, and APETx2 peptides performed with Patch analysis suite in MOE 2019.0102 CCG® software [42]; the molecules on panel (b) are presented in one orientation which shows the functionally important residues of APETx2. The residue projections are labeled in a one letter code. The hydrophobic, basic, and acidic areas are presented as green, blue, and red, respectively.
	Figure 4. Modulatory activity of peptides Hcr 1b-3 and Hcr 1b-4 toward rASIC1a and rASIC3 channels. Acid-induced currents through rASIC1a (a,b) and rASIC3 (c,d) expressed in X. laevis oocytes were evoked by pH drop from 7.4 to 5.5 and 4.0; the effect of Hcr 1b-3 (a,c) and Hcr 1b-4 (b,d) at a concentration of 10 μM on the transient currents. Concentration-response curves for rASIC1a and rASIC3 indicating the inhibitory effect of peptides (e) and potentiating effect of Hcr 1b-4 on rASIC3 channels (f). Each point is means ± SD (obtained from 5 cells for each rASIC1a and rASIC3). Data were fitted by a logistic equation. The resulting values of the fitting parameters for rASIC1a: IC50 4.95 ± 0.19 μM; nH of 1.20 ± 0.05; A 30.3 ± 0.7% (Hcr 1b-3) and IC50 1.25 ± 0.04 μM; nH 1.08 ± 0.03; A 13.7 ± 0.8% (Hcr 1b-4). The resulting values of the fitting parameters for rASIC3: IC50 17 ± 5.8 μM; nH of 0.9 ± 0.2; A 26 ± 7%; (Hcr 1b-3) and EC50 1.53 ± 0.07 μM; nH 1.30 ± 0.06.; A 207.78 ± 1.56% (Hcr 1b-4).
	Figure 5. Amino acid sequences of ASIC1a and/or ASIC3 inhibitors from spider, snake, and sea anemone. Active sites are indicated by asterisks above the residues: PcTx1, mambalgin-1, and APETx2 residues that, when mutated to alanine, have a major impact on the peptides inhibitory activity toward ASICs [13,31,45,46], and Hcr 1b-2 and Hcr 1b-4 residues forming hot spot interactions. Homologous sea anemone peptides are grouped and identical amino acid residues are shown on a dark gray background.
	Figure 6. Interaction of Hcr 1b-2 and Hcr 1b-4 peptides with rASIC1a. Ribbon diagrams of direct intermolecular interactions of rASIC1a with Hcr 1b-2 (a) and Hcr 1b-4 (b,c) are shown. (c) The abnormally expanded acidic pocket of rASIC1a channel in complex with HcrTxs is shown. The distances separated Cα atoms of Asp237, Asp345, and Asp349 (proton sensors) in Hcr 1b-4–rASIC1 complex at pH 5.5 are shown. The non-covalent intermolecular interactions of Hcr 1b-4 C-terminal residues are also shown. (d) Network of intra- and inter-subunit ionic and hydrogen bonds between Glu277‒Arg279, Gln278‒Arg369‒Glu79, and Glu416‒Gln278 are shown. It forms a ring in the region of the central vestibule and prevents the increase in channel pore size in the complex with HcrTxs and stabilizes the closed pore state. Figure style: side chains of peptides and channel residues involved in binding are represented as sticks; hydrogen bonds as blue dotted line; ionic interactions are represented as blue colored contours as well as the hot spot interactions as magenta-colored contours; color intensity is proportional to the energy contribution.

References [+] :

Andreev, Analgesic Activity of Acid-Sensing Ion Channel 3 (ASIС3) Inhibitors: Sea Anemones Peptides Ugr9-1 and APETx2 versus Low Molecular Weight Compounds. 2018, Pubmed, Xenbase