Kunitz-Type Peptides from Sea Anemones Protect Neuronal Cells against Parkinson's Disease Inductors via Inhibition of ROS Production and ATP-Induced P2X7 Receptor Activation.

Parkinson's disease (PD) is a socially significant disease, during the development of which oxidative stress and inflammation play a significant role. Here, we studied the neuroprotective effects of four Kunitz-type peptides from Heteractis crispa and Heteractis magnifica sea anemones against PD inductors. The peptide HCIQ1c9, which was obtained for the first time, inhibited trypsin less than other peptides due to unfavorable interactions of Arg17 with Lys43 in the enzyme. Its activity was reduced by up to 70% over the temperature range of 60-100 °C, while HCIQ2c1, HCIQ4c7, and HMIQ3c1 retained their conformation and stayed active up to 90-100 °C. All studied peptides inhibited paraquat- and rotenone-induced intracellular ROS formation, in particular NO, and scavenged free radicals outside the cells. The peptides did not modulate the TRPV1 channels but they affected the P2X7R, both of which are considered therapeutic targets in Parkinson's disease. HMIQ3c1 and HCIQ4c7 almost completely inhibited the ATP-induced uptake of YO-PRO-1 dye in Neuro-2a cells through P2X7 ion channels and significantly reduced the stable calcium response in these cells. The complex formation of the peptides with the P2X7R extracellular domain was determined via SPR analysis. Thus, these peptides may be considered promising compounds to protect neuronal cells against PD inductors, which act as ROS production inhibitors and partially act as ATP-induced P2X7R activation inhibitors.

Porin from Marine Bacterium Marinomonas primoryensis KMM 3633T: Isolation, Physico-Chemical Properties, and Functional Activity.

Marinomonas primoryensis KMM 3633T, extreme living marine bacterium was isolated from a sample of coastal sea ice in the Amursky Bay near Vladivostok, Russia. The goal of our investigation is to study outer membrane channels determining cell permeability. Porin from M. primoryensis KMM 3633T (MpOmp) has been isolated and characterized. Amino acid analysis and whole genome sequencing were the sources of amino acid data of porin, identified as Porin_4 according to the conservative domain searching. The amino acid composition of MpOmp distinguished by high content of acidic amino acids and low content of sulfur-containing amino acids, but there are no tryptophan residues in its molecule. The native MpOmp existed as a trimer. The reconstitution of MpOmp into black lipid membranes demonstrated its ability to form ion channels whose conductivity depends on the electrolyte concentration. The spatial structure of MpOmp had features typical for the classical gram-negative porins. However, the oligomeric structure of isolated MpOmp was distinguished by very low stability: heat-modified monomer was already observed at 30 °C. The data obtained suggest the stabilizing role of lipids in the natural membrane of marine bacteria in the formation of the oligomeric structure of porin.

Magnificamide, a ß-Defensin-Like Peptide from the Mucus of the Sea Anemone Heteractis magnifica, Is a Strong Inhibitor of Mammalian α-Amylases.

Sea anemones' venom is rich in peptides acting on different biological targets, mainly on cytoplasmic membranes and ion channels. These animals are also a source of pancreatic α-amylase inhibitors, which have the ability to control the glucose level in the blood and can be used for the treatment of prediabetes and type 2 diabetes mellitus. Recently we have isolated and characterized magnificamide (44 aa, 4770 Da), the major α-amylase inhibitor of the sea anemone Heteractis magnifica mucus, which shares 84% sequence identity with helianthamide from Stichodactyla helianthus. Herein, we report some features in the action of a recombinant analog of magnificamide. The recombinant peptide inhibits porcine pancreatic and human saliva α-amylases with Ki's equal to 0.17 ± 0.06 nM and 7.7 ± 1.5 nM, respectively, and does not show antimicrobial or channel modulating activities. We have concluded that the main function of magnificamide is the inhibition of α-amylases; therefore, its functionally active recombinant analog is a promising agent for further studies as a potential drug candidate for the treatment of the type 2 diabetes mellitus.

Production of recombinant porin from Y. pseudotuberculosis in a water-soluble form for pseudotuberculosis diagnostics.

OmpF porin from the outer membrane of Yersinia pseudotuberculosis was cloned into pET-40b(+) plasmid. Using E. coli Rosetta (DE3) strain, MX medium, IPTG concentration of 0.2 mm and post-induction cultivation at 14°C overnight allowed us to obtain a water-soluble form of the recombinant protein (rs-OmpF). Rs-OmpF was shown to have the ordered spatial structure at the levels of secondary and tertiary structure. Rs-OmpF was found to be effective as diagnostic antigen in ELISA for pseudotuberculosis diagnostics.

Roles of individual domains in the function of DHX29, an essential factor required for translation of structured mammalian mRNAs.

On most eukaryotic mRNAs, initiation codon selection involves base-by-base inspection of 5' UTRs by scanning ribosomal complexes. Although the eukaryotic initiation factors 4A/4B/4G can mediate scanning through medium-stability hairpins, scanning through more stable structures additionally requires DHX29, a member of the superfamily 2 DEAH/RNA helicase A (RHA) helicase family that binds to 40S subunits and possesses 40S-stimulated nucleoside triphosphatase (NTPase) activity. Here, sequence alignment and structural modeling indicated that DHX29 comprises a unique 534-aa-long N-terminal region (NTR), central catalytic RecA1/RecA2 domains containing a large insert in the RecA2 domain, and the C-terminal part, which includes winged-helix, ratchet, and oligonucleotide/oligosaccharide-binding (OB) domains that are characteristic of DEAH/RHA helicases. Functional characterization revealed that specific ribosomal targeting is required for DHX29's activity in initiation and is determined by elements that map to the NTR and to the N-terminal half of the winged-helix domain. The ribosome-binding determinant located in the NTR was identified as a putative double-stranded RNA-binding domain. Mutational analyses of RecA1/RecA2 domains confirmed the essential role of NTP hydrolysis for DHX29's function in initiation and validated the significance of a ß-hairpin protruding from RecA2. The large RecA2 insert played an autoinhibitory role in suppressing DHX29's intrinsic NTPase activity but was not essential for its 40S-stimulated NTPase activity and function in initiation. Deletion of the OB domain also increased DHX29's basal NTPase activity, but more importantly, abrogated the responsiveness of the NTPase activity to stimulation, which abolished DHX29's function in initiation. This finding suggests that the OB domain, which is specific for DEAH/RHA helicases, plays an important role in their NTPase cycle.

Control of postprandial hyperglycemia by oral administration of the sea anemone mucus-derived α-amylase inhibitor (magnificamide).

Diabetes mellitus is a serious threat to human health in both developed and developing countries. Optimal disease control requires the use of a diet and a combination of several medications, including oral hypoglycemic agents such as α-glucosidase inhibitors. Currently, the arsenal of available drugs is insufficient, which determines the relevance of studying new potent α-amylase inhibitors. We implemented the recombinant production of sea anemone derived α-amylase inhibitor magnificamide in Escherichia coli. Peptide was isolated by a combination of liquid chromatography techniques. Its folding and molecular weight was proved by 1H NMR and mass spectrometry. The Ki value of magnificamide against human pancreatic α-amylase is 3.1 nM according to Morrison equation for tight binding inhibitors. Our study of the thermodynamic characteristics of binding of magnificamide to human salivary and pancreatic α-amylases by isothermal titration calorimetry showed the presence of different binding mechanisms with Kd equal to 0.11 µM and 0.1 nM, respectively. Experiments in mice with streptozotocin-induced diabetes mimicking diabetes mellitus type 1 were used to study the efficiency of magnificamide against postprandial hyperglycemia. It was found that at a dose of 0.005 mg kg-1, magnificamide effectively blocks starch breakdown and prevents the development of postprandial hyperglycemia in T1D mice. Our results demonstrated the therapeutic potential of magnificamide for the control of postprandial hyperglycemia.

Synaptic transmission block by presynaptic injection of oligomeric amyloid beta.

Early Alzheimer's disease (AD) pathophysiology is characterized by synaptic changes induced by degradation products of amyloid precursor protein (APP). The exact mechanisms of such modulation are unknown. Here, we report that nanomolar concentrations of intraaxonal oligomeric (o)Abeta42, but not oAbeta40 or extracellular oAbeta42, acutely inhibited synaptic transmission at the squid giant synapse. Further characterization of this phenotype demonstrated that presynaptic calcium currents were unaffected. However, electron microscopy experiments revealed diminished docked synaptic vesicles in oAbeta42-microinjected terminals, without affecting clathrin-coated vesicles. The molecular events of this modulation involved casein kinase 2 and the synaptic vesicle rapid endocytosis pathway. These findings open the possibility of a new therapeutic target aimed at ameliorating synaptic dysfunction in AD.

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

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.

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.

Structures of eremophilane-type sesquiterpene glucosides, alticolosides A-G, from the Far Eastern endemic Ligularia alticola Worosch.

Seven eremophilane-type sesquiterpene glucosides, alticolosides A-G, have been isolated from aerial parts of the endemic Far Eastern species Ligularia alticola Worosch. (Family Asteraceae) along with two known compounds, monoterpenoid glycoside (4S)-α-terpineol 8-O-ß-D-glucopyranoside and norditerpenoid glycoside 7(8)-dihydro-ß-ionone 3-O-ß-D-glucopyranoside. Alticoloside D was identified with the earlier known 8-O-(ß-D-glucopyranosyl)-2-oxo-eremophila-1(10),8,11-triene, but the stereostructure of the latter was revised on the basis of ROESY and CD data. All the glycosides are derivatives of new eremophilane-type aglycones, differing from known eremophilanes in details of planar and/or stereo structures except for the aglycone of alticoloside E.

On the possibility of lipid-induced regulation of conformation and immunogenicity of influenza a virus H1/N1 hemagglutinin as antigen of TI-complexes.

The tubular immunostimulating complex (TI-complex) consisting of cucumarioside A2-2, cholesterol and monogalactosyldiacylglycerol (MGDG) from marine macrophytes is the perspective antigen delivery system for subunit vaccines. MGDG is a lipid matrix for the protein antigen incorporated in the TI-complex. The aim of the present work was to study the influence of MGDGs from different macrophytes on conformation and immunogenicity of the secreted recombinant uncleaved hemagglutinin monomer (HA0S) of influenza A virus H1/N1. Differential scanning calorimetry, fluorescence spectroscopy and circular dichroism showed a dependence of the conformational changes of HA0S on the microviscosity of MGDG. The most viscous MGDG from Zostera marina induced the strongest rearrangements in protein conformation. Immunization of mice with HA0S within TI-complexes comprising different MGDGs resulted in an approximately 2-fold increase of the levels of anti-HA0S antibodies and granulocyte-macrophage colony-stimulating factor (GM-CSF) compared with those induced by HA0S alone. TI-complexes based on MGDG from Z. marina stimulated the maximal production of GM-CSF. However, humoral immune response (anti-HA0S antibodies), unlike cell-mediated immune response (GM-CSF), did not depend on the physicochemical properties of MGDGs. It is assumed that this is due to the different localization and conformational lipid sensitivity of the HA0S regions, which are responsible for these types of immune responses.

An endo-(1-->3)-beta-D-glucanase from the scallop Chlamys albidus: catalytic properties, cDNA cloning and secondary-structure characterization.

An endo-(1-->3)-beta-d-glucanase (L(0)) with molecular mass of 37 kDa was purified to homogeneity from the crystalline style of the scallop Chlamys albidus. The endo-(1-->3)-beta-d-glucanase was extremely thermolabile with a half-life of 10 min at 37 degrees C. L(0) hydrolyzed laminaran with K(m) approximately 0.75 mg/mL, and catalyzed effectively transglycosylation reactions with laminaran as donor and p-nitrophenyl betad-glucoside as acceptor (K(m) approximately 2mg/mL for laminaran) and laminaran as donor and as acceptor (K(m) approximately 5mg/mL) yielding p-nitrophenyl betad-glucooligosaccharides (n=2-6) and high-molecular branching (1-->3),(1-->6)-beta-d-glucans, respectively. Efficiency of hydrolysis and transglycosylation processes depended on the substrate structure and decreased appreciably with the increase of the percentage of beta-(1-->6)-glycosidic bonds, and laminaran with 10% of beta-(1-->6)-glycosidic bonds was the optimal substrate for both reactions. The CD spectrum of L(0) was characteristic for a protein with prevailing beta secondary-structural elements. Binding L(0) with d-glucose as the best acceptor for transglycosylation was investigated by the methods of intrinsic tryptophan fluorescence and CD. Glucose in concentration sufficient to saturate the enzyme binding sites resulted in a red shift in the maximum of fluorescence emission of 1-1.5 nm and quenching the Trp fluorescence up to 50%. An apparent association constant of L(0) with glucose (K(a)=7.4 x 10(5)+/-1.1 x 10(5)M(-1)) and stoichiometry (n=13.3+/-0.7) was calculated. The cDNA encoding L(0) was sequenced, and the enzyme was classified in glycoside hydrolases family 16 on the basis of the amino acid sequence similarity.