Возможный механизм лиганд-рецепторного связывания синтетического трипептида Ac-RRR-NH2 с мембраной ноцицептивного нейрона

Several arginine-containing short peptides have been shown by the patch-clamp method to effectively modulate the NaV1.8 channel activation gating system, which makes them promising candidates for the role of a novel analgesic medicinal substance. As demonstrated by the organotypic tissue culture method, all active and inactive peptides studied do not trigger the downstream signaling cascades controlling neurite outgrowth and should not be expected to evoke adverse side effects on the tissue level upon their medicinal administration. The conformational analysis of Ac-RAR-NH2, Ac-RER-NH2, Ac-RAAR-NH2, Ac-REAR-NH2, Ac-RERR-NH2, Ac-REAAR-NH2, Ac-PRERRA-NH2, and Ac-PRARRA-NH2 has made it possible to find the structural parameter, the value of which is correlated with the target physiological effect of arginine-containing short peptides. The distances between the positively charged guanidinium groups of the arginine side chains involved in intermolecular ligand–receptor ion–ion bonds between the attacking peptide molecules and the NaV1.8 channel molecule should fall within a certain range, the lower threshold of which is estimated to be around 9 Å. The distance values have been calculated to be below 9 Å in the inactive peptide molecules, except for Ac-RER-NH2, and in the range of 9–12 Å in the active peptide molecules.

MULTIPLE SITES OF ACTION IN THE INHIBITORY EFFECT OF NOCICEPTIN ON THE MICTURITION REFLEX

Interactions between positively charged side-chain residues and C-terminal carboxylate groups are of great importance in stabilizing secondary structures of many biologically active peptide sequences. The conformational properties of the hexapeptide sequence L-Arg-L-Val-Gly-L-Arg-L-Val-Gly with an Arg residue located at the third position from the carboxy terminus and a second Arg moiety at the amino terminus of the molecule, have been investigated in DMSO solution at pH 6.5, by means of 1D and 2D 1H NMR spectroscopy. The considerable downfield shift of the Arg4NIµH resonance, when the pH of the solution is raised from 2 to 6.5, and the magnetically non-equivalent CβH and Cδ H methylene protons of the same residue at pH 6.5 provide evidence for a non-covalent hydrogen-bonding interaction between Arg4 guanidinium and C-terminal carboxylate groups. The intense backbone–backbone NOE connectivities observed for the L-Arg4-L-Val5-Gly6 C-terminal sequence, the NOE connectivities of Arg4 methylene protons and the retained ABX system of Gly6 C-terminal residue at high temperature are evidence for some mobility restrictions around this part of the molecule, and provide additional confirmation for a particular propensity of the arginine side chain, when occupying a position near the carboxy terminus, to interact with the C-terminal carboxylate group at neutral pH. Absence of ROE connectivities between protons of the Arg1 side chain, and the fact that almost all methylene protons of the same residue appear magnetically equivalent, point out that this Arg moiety does not participate in any intramolecular interaction. Thus, differences in the behaviour of guanidinium side chains of the reported peptide sequence can be used as a general probe for the study of Arg side-chain mobility and for its ability to generate hydrogen-bonding interactions in other peptides and proteins.

Nociceptin activation of the human ORL1 receptor expressed in Chinese hamster ovary cells: Functional homology with opioid receptors

In the primary sensory neuron, ouabain activates the dual mechanism that modulates the functional activity of NaV1.8 channels. Ouabain at endogenous concentrations (EO) triggers two different signaling cascades, in which the Na,K-ATPase/Src complex is the EO target and the signal transducer. The fast EO effect is based on modulation of the NaV1.8 channel activation gating device. EO triggers the tangential signaling cascade along the neuron membrane from Na,K-ATPase to the NaV1.8 channel. It evokes a decrease in effective charge transfer of the NaV1.8 channel activation gating device. Intracellular application of PP2, an inhibitor of Src kinase, completely eliminated the effect of EO, thus indicating the absence of direct EO binding to the NaV1.8 channel. The delayed EO effect probably controls the density of NaV1.8 channels in the neuron membrane. EO triggers the downstream signaling cascade to the neuron genome, which should result in a delayed decrease in the NaV1.8 channels' density. PKC and p38 MAPK are involved in this pathway. Identification of the dual mechanism of the strong EO effect on NaV1.8 channels makes it possible to suggest that application of EO to the primary sensory neuron membrane should result in a potent antinociceptive effect at the organismal level.

Imaging the Activity and Localization of Single Voltage-Gated Ca 2+ Channels by Total Internal Reflection Fluorescence Microscopy

A growing body of evidence suggests that the aggregation and toxic potentials of amyloidogenic proteins, including amyloid β-protein (Aβ), α-synuclein, and prion protein, emerge through the interaction of these proteins with neuronal and/or glial membranes. The aggregation and deposition of Aβ are the initial events of Alzheimer's disease (AD), and the toxicity of aggregated Aβ is the basis for the neuronal loss in AD brains. Thus, the Aβ behavior on neuronal membranes should be one of the critical issues to be clarified for our further understanding of the pathogenesis of AD and to develop therapeutic strategies. To accelerate studies in this field, we have invited original research articles as well as review articles that will provide novel information for our special issue. The first three papers of this special issue describe the crucial involvement of lipid rafts, which are specific membrane microdomains on the cell surface that are rich in sphingolipids and cholesterol, in the production, aggregation, and toxicities of Aβ. The subsequent three papers focus on the gangliosides, which are the major constituent of lipid rafts, particularly in terms of their role in the induction of conformational changes of Aβ, leading to their aggregation and emerging toxicities. The next two articles address how Aβ causes neuronal injury by showing the possibility of formation of amyloid channels in the neuronal membranes, resulting in the disruption of calcium homeostasis that is critical for the function and survival of neurons, and the possibility of generation of radicals. In regard to the Aβ toxicities, much attention has been paid to the argument that the accumulation of Aβ inside neurons may be the critical step. In this context, the next two papers propose a mechanism by which Aβ enters the neurons, which are followed by another two papers showing how the internalized Aβ acts pathologically inside neurons, emphasizing the possibility that the mitochondria may be a target of intraneuronal Aβ. A further argument for the possible interaction between Aβ and neuronal membranes is presented in the next four papers. In these papers, it is presented how Aβ affects the properties of neuronal membranes or, conversely, how the alteration of membrane properties affects the processing of amyloid precursor protein (APP) leading to Aβ generation. Note that the metabolism of neuronal lipids, particularly sphingolipids and ceramide, can be regulated in association with APP processing. The final paper of this special issue describes a foresighted aspect of science and technology of nanochemistry with respect to the pathological protein aggregation, which is likely based on the catalysts of membrane lipids, suggesting an opportunity for developing novel nanomedicines and nanodiagnostics for various amyloidoses. We all look forward to seeing further expansion of studies in this field in the near future. Katsuhiko Yanagisawa Jacques Fantini Avijit Chakrabartty Anne Eckert

The present work continues our recent series of articles that aim to elucidate the ligand–receptor binding mechanism of short cationic peptides to the NaV1.8 channel in the nociceptive neuron. The applied methodological approach has involved several methods: the patch-clamp experimental evaluation of the effective charge of the NaV1.8 channel activation gating system, the organotypic tissue culture method, the formalin test, and theoretical conformational analysis. The lysine-containing short peptide Ac-KEKK-NH2 has been shown to effectively modulate the NaV1.8 channel activation gating system. As demonstrated by the organotypic tissue culture method, the studied short peptide does not trigger the downstream signaling cascades controlling neurite outgrowth and should not be expected to evoke adverse side effects. Conformational analysis of the Ac-KEKK-NH2 molecule has revealed that the distances between the positively charged amino groups of the lysine side chains are equal to 11–12 Å. According to the previously suggested mechanism of ligand–receptor binding of short peptides to the NaV1.8 channel molecule, Ac-KEKK-NH2 should exhibit an analgesic effect, which has been confirmed by the formalin test. The data obtained unequivocally indicate that the studied lysine-containing short peptide is a promising candidate for the role of a novel analgesic medicinal substance.

L-arginine deiminase (ADI) catalyzes the irreversible hydrolysis of L-arginine to citrulline and ammonia. In a previous report of the structure of apoADI from Pseudomonas aeruginosa, the four residues that form the catalytic motif were identified as Cys406, His278, Asp280, and Asp166. The function of Cys406 in nucleophilic catalysis has been demonstrated by transient kinetic studies. In this study, the structure of the C406A mutant in complex with L-arginine is reported to provide a snapshot of the enzyme.substrate complex. Through the comparison of the structures of apoenzyme and substrate-bound enzyme, a substrate-induced conformational transition, which might play an important role in activity regulation, was discovered. Furthermore, the position of the guanidinium group of the bound substrate relative to the side chains of His278, Asp280, and Asp166 indicated that these residues mediate multiple proton transfers. His278 and Asp280, which are positioned to activate the water nucleophile in the hydrolysis of the S-alkylthiouronium intermediate, were replaced with alanine to stabilize the intermediate for structure determination. The structures determined for the H278A and D280A mutants co-crystallized with L-arginine provide a snapshot of the S-alkylthiouronium adduct formed by attack of Cys406 on the guanidinium carbon of L-arginine followed by the elimination of ammonia. Asp280 and Asp166 engage in ionic interactions with the guanidinium group in the C406A ADI. L-arginine structure and might orient the reaction center and participate in proton transfer. Structure determination of D166A revealed the apoD166A ADI. The collection of structures is interpreted in the context of recent biochemical data to propose a model for ADI substrate recognition and catalysis.

Arginine amide radicals are generated by femtosecond electron transfer to protonated arginine amide cations in the gas phase. A fraction of the arginine radicals formed (2-amino-5-dihydroguanid-1'-yl-pentanamide, 1H) is stable on the 6.7 micros time scale and is detected after collisional reionization. The main dissociation of 1H is loss of a guanidine molecule from the side chain followed by consecutive dissociations of the 2-aminopentanamid-5-yl radical intermediate. Intramolecular hydrogen atom transfer from the guanidinium group onto the amide group is not observed. These results are explained by ab initio and density functional theory calculations of dissociation and transition state energies. Loss of guanidine from 1H is calculated to require a transition state energy of 68 kJ mol(-)(1), which is substantially lower than that for hydrogen atom migration from the guanidine group. The loss of guanidine competes with the reverse migration of the arginine alpha-hydrogen atom onto the guanidyl radical. RRKM calculations of dissociation kinetics predict the loss of guanidine to account for >95% of 1H dissociations. The anomalous behavior of protonated arginine amide upon electron transfer provides an insight into electron capture and transfer dissociations of peptide cations containing arginine residues as charge carriers. The absence of efficient hydrogen atom transfer from charge-reduced arginine onto sterically proximate amide group blocks one of the current mechanisms for electron capture dissociation. Conversely, charge-reduced guanidine groups in arginine residues may function as radical traps and induce side-chain dissociations. In light of the current findings, backbone dissociations in arginine-containing peptides are predicted to involve excited electronic states and proceed by the amide superbase mechanism that involves electron capture in an amide pi* orbital, which is stabilized by through-space coulomb interaction with the remote charge carriers.

A general labelling method is presented which allows the determination of the number of guanidine groups (related to arginine and homoarginine in peptides and proteins) by means of mass spectrometry. It implies a guanidine-selective derivatization step with 2,3-butanedione and an arylboronic acid under aqueous, alkaline conditions (pH 8-10). The reaction mixture is then directly analysed by electrospray ionization mass spectrometry without further sample pretreatment. Other amino acids are not affected by this reaction although it is demonstrated that lysine side-chains may be unambiguously identified when they are converted to homoarginine prior to derivatization. Guanidine functionalities, as e.g. in the amino acid arginine, are easily identified by the characteristic mass shift between underivatized and derivatized analyte. The tagging procedure is straightforward and selective for guanidine groups. The influence of several experimental parameters, especially the pH of the solution and the choice of reagents, is examined and the method is applied to various arginine-containing peptides and to lysozyme as a representative protein. Possible applications of this technique and its limitations are discussed.

The inhibitory effects of the pyrazine derivative, amiloride, on sodium transport in an amphibian epithelium has been studied as a function of pH. It is concluded that the charged (guanidinium) group interacts with a negatively charged acid grouping in the membrane. Similarities between sodium channels in excitable membranes and epithelia are highlighted.

Insertion of short peptides into the cell membrane is energetically unfavorable and challenges the commonly accepted hydrophobic matching principle. Yet there has been evidence that many short peptides can penetrate into the cells to perform the biological functions in salt solution. On the basis of the previous study (J. Phys. Chem. C 2013, 117, 11095−11103), here we further performed a systematic study on the interaction of mastoparan with various neutral lipid bilayers with different lipid chain lengths in situ to examine the hydrophobic matching principle in different aqueous salt environments using sum frequency generation vibrational spectroscopy. It is found that the hydrophobic matching is the dominant driving force for the association of MP with a lipid bilayer in a pure water environment. However, in a kosmotropic ion environment, the hydration of ions can overcome the hydrophobic mismatching effects, leading to the insertion of MP into lipid bilayers with much longer hydrophobic lengths. When the hydrophobic thickness of the bilayer is much longer than MP’s hydrophobic length, MP diffuses on a single monolayer, rather than spanning the bilayer to prevent the exposure of the hydrophilic part of MP to the lipid hydrophobic moiety. Findings from the present study suggest that the interaction between the positively charged choline group of a lipid and kosmotropic ions could be an important step for effective peptide insertion into a cell membrane. Results from our studies will provide an insight into how the short peptides form the ion channel in a thick membrane and offer some ideas for cellular delivery.

1. The effect of nociceptin (NC) on 5-hydroxytryptamine (5-HT) release was studied in rat cerebral cortex slices preincubated with [3H]-5-HT and electrically stimulated (3 Hz, for 2 min) at the 45th (St1) and the 75th (St2) min of superfusion. 2. NC (0.1 - 3 microM), present in the medium from the 70th min onward, concentration-dependently reduced electrically evoked [3H]-5-HT efflux (pEC50=6.54, Emax -54%). The inhibition was not antagonized by naloxone (1 microM) ruling out the involvement of opioid receptors. 3. Phe1psi(CH2-NH2)Gly2]NC(1-13)NH2, which acts as an opioid-like receptor (ORL1) antagonist at the peripheral level, behaved as a partial agonist in cerebral cortex slices i.e. it inhibited [3H]-5-HT efflux when added before St2, however, when present in the medium throughout the whole experiment, [Phe1psi(CH2-NH2)Gly2]NC(1-13)NH2 prevented the action of NC added at the 70th min. 4. The non-selective ORL1 receptor antagonist, naloxone benzoylhydrazone (3 microM), in the presence of 10 microM naloxone, did not modify the St2/St1 ratio but completely abolished the NC effect. 5. These findings demonstrate that NC inhibits 5-HT release from rat cerebral cortex slices via ORL1 receptors, suggesting its involvement in central processes mediated by 5-HT.

Functional characterization of new short glyproline peptides, which are able to provide a regulatory effect on the functional state of the hemostasis system, as well as lipid and carbohydrate metabolism in the body, is an actual task of physiology and medicine. In the present study, we used a model of experimental metabolic syndrome developed in animals due to continuous feeding with high-calorie food. This leads to increased clotting, glucose concentration, low-density lipoprotein cholesterol, triglycerides, and the level of total cholesterol in the blood, which is accompanied by an increase in the body weight of rats. Arginine-containing peptides (Arg-Glu-Arg-Pro-Gly-Pro, Arg-Glu-Arg-Val-Gly-Pro, Arg-Glu-Arg-Gly-Pro) were intranasally administered every 24 h to rats seven times 6 weeks after the development of metabolic syndrome. These peptides provided a unique combined effect on the body, restoring parameters of lipid metabolism, the hemostasis system, and the concentration of blood glucose to normal values. The corrective effect of the studied peptides was detected 20 h after the last administration and was maintained for 168 h even under further feeding of rats with high-calorie diet. The studied glyproline peptides belong to therapeutic normoglycemic and lipid-lowering drugs. They block the accumulation of new fat deposits in the body, and also have anticoagulant and antithrombotic effects in disorders of lipid metabolism. The Arg-Glu-Arg-Pro-Gly-Pro peptide possessed the most pronounced and stable positive effect on the body.