RESUMO
We report the molecular mechanism of action of gausemycins and the isolation of new members of the family, gausemycins C (1c), D (1d), E (1e), and F (1f), the minor components of the mixture. To elucidate the mechanism of action of gausemycins, we investigated the antimicrobial activity of the most active compounds, gausemycins A and B, in the presence of Ca2+, other metal ions, and phosphate. Gausemycins require a significantly higher Ca2+ concentration for maximum activity than daptomycin but lower than that required for malacidine and cadasides. Species-specific antimicrobial activity was found upon testing against a wide panel of Gram-positive bacteria. Membranoactivity of gausemycins was demonstrated upon their interactions with model lipid bilayers and micelles. The pore-forming ability was found to be dramatically dependent on the Ca2+ concentration and the membrane lipid composition. An NMR study of gausemycin B in zwitterionic and anionic micelles suggested the putative structure of the gausemycin/membrane complex and revealed the binding of Ca2+ by the macrocyclic domain of the antibiotic.
Assuntos
Antibacterianos , Cálcio , Bactérias Gram-Positivas , Testes de Sensibilidade Microbiana , Antibacterianos/farmacologia , Antibacterianos/química , Cálcio/metabolismo , Estrutura Molecular , Bactérias Gram-Positivas/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Daptomicina/farmacologia , Daptomicina/química , Bicamadas Lipídicas/química , MicelasRESUMO
Alzheimer disease (AD) is a widespread neurodegenerative disease characterized by the accumulation of oligomeric toxic forms of ß-amyloid (Aß1-42) and dysfunction of the cholinergic system in the different brain regions. However, the exact mechanisms of AD pathogenesis and the role of the nicotinic acetylcholine receptors (nAChRs) in the disease progression remain unclear. Here, we revealed a decreased expression of a number of the Ly6/uPAR proteins targeting nAChRs in the cerebellum of 2xTg-AD mice (model of early AD) in comparison with non-transgenic mice both at mRNA and protein levels. We showed that co-localization of one of them, - neuromodulator Lynx1, with α7-nAChR was diminished in the vicinity of cerebellar astrocytes of 2xTg-AD mice, while Aß1-42 co-localization with this receptor present was increased. Moreover, the expression of anti-inflammatory transcription factor KLF4 regulating transcription of the Ly6/uPAR genes was decreased in the cerebellum of 2xTg-AD mice, while expression of inflammatory cytokine TNF-α was increased. Based on these data together with observed astrocyte degeneration in the cerebellum of 2xTg-AD mice, we suggest the mechanism by which expression of the Ly6/uPAR proteins upon Aß pathology results in dysregulation of the cholinergic system and particularly of α7-nAChR function in the cerebellum. This leads to enhanced neuroinflammation and cerebellar astrocyte degeneration.
Assuntos
Doença de Alzheimer , Doenças Neurodegenerativas , Receptores Nicotínicos , Camundongos , Animais , Doença de Alzheimer/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/genética , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Astrócitos/metabolismo , Doenças Neurodegenerativas/metabolismo , Peptídeos beta-Amiloides/metabolismo , Receptores Nicotínicos/metabolismo , Cerebelo/metabolismo , Colinérgicos/metabolismoRESUMO
To date, a number of lantibiotics have been shown to use lipid II-a highly conserved peptidoglycan precursor in the cytoplasmic membrane of bacteria-as their molecular target. The α-component (Lchα) of the two-component lantibiotic lichenicidin, previously isolated from the Bacillus licheniformis VK21 strain, seems to contain two putative lipid II binding sites in its N-terminal and C-terminal domains. Using NMR spectroscopy in DPC micelles, we obtained convincing evidence that the C-terminal mersacidin-like site is involved in the interaction with lipid II. These data were confirmed by the MD simulations. The contact area of lipid II includes pyrophosphate and disaccharide residues along with the first isoprene units of bactoprenol. MD also showed the potential for the formation of a stable N-terminal nisin-like complex; however, the conditions necessary for its implementation in vitro remain unknown. Overall, our results clarify the picture of two component lantibiotics mechanism of antimicrobial action.
Assuntos
Antibacterianos , Bacteriocinas , Antibacterianos/química , Peptidoglicano/metabolismo , Bacteriocinas/química , Uridina Difosfato Ácido N-Acetilmurâmico/química , Uridina Difosfato Ácido N-Acetilmurâmico/metabolismoRESUMO
Two forms were found in the NMR spectra of N6-substituted 2-chloroadenosines. The proportion of the mini-form was 11-32% of the main form. It was characterized by a separate set of signals in COSY, 15N-HMBC and other NMR spectra. We assumed that the mini-form arises due to the formation of an intramolecular hydrogen bond between the N7 atom of purine and the N6-CH proton of the substituent. The 1H,15N-HMBC spectrum confirmed the presence of a hydrogen bond in the mini-form of the nucleoside and its absence in the main form. Compounds incapable of forming such a hydrogen bond were synthesized. In these compounds, either the N7 atom of the purine or the N6-CH proton of the substituent was absent. The mini-form was not found in the NMR spectra of these nucleosides, confirming the importance of the intramolecular hydrogen bond in its formation.
Assuntos
Prótons , Ligação de Hidrogênio , 2-Cloroadenosina , Espectroscopia de Ressonância MagnéticaRESUMO
A number of purine arabinosides containing chiral amino acid amides at the C6 position of the purine were synthesized using a transglycosylation reaction with recombinant E. coli nucleoside phosphorylases. Arsenolysis of 2-chloropurine ribosides with chiral amino acid amides at C6 was used for the enzymatic synthesis, and the reaction equilibrium shifted towards the synthesis of arabinonucleosides. The synthesized nucleosides were shown to be resistant to the action of E. coli adenosine deaminase. The antiproliferative activity of the synthesized nucleosides was studied on human acute myeloid leukemia cell line U937. Among all the compounds, the serine derivative exhibited an activity level (IC50 = 16 µM) close to that of Nelarabine (IC50 = 3 µM) and was evaluated as active.
Assuntos
Escherichia coli , Nucleosídeos de Purina , Humanos , Nucleosídeos de Purina/farmacologia , Escherichia coli/metabolismo , Aminoácidos , Nucleosídeos/química , ArabinonucleosídeosRESUMO
Selectively 15N-labeled tetrazolo[1,5-b][1,2,4]triazines and tetrazolo[1,5-a]pyrimidines bearing one, two, or three 15N labels were synthesized. The synthesized compounds were studied by 1H, 13C, and 15N NMR spectroscopy in DMSO and TFA solutions, where the azide-tetrazole equilibrium can lead to the formation of two tetrazole (T, T') isomers and one azide (A) isomer for each compound. Incorporation of the 15N-label(s) leads to the appearance of 15N-15N coupling constants (JNN), which can be easily measured via simple 1D 15N NMR spectra, even at natural abundance between labeled and unlabeled 15N atoms. The chemical shifts for the 15N nuclei in the azole moiety are very sensitive to the ring opening and azide formation, thus providing information about the azido-tetrazole equilibrium. At the same time, the 1-2JNN couplings between 15N-labeled atoms in the azole and azine fragments unambiguously determine the fusion type between tetrazole and azine rings in the cyclic isomers T and T'. Thus, combined analysis of 15N chemical shifts and JNN values in selectively isotope-enriched compounds provides an effective diagnostic tool for direct structural determination of tetrazole isomers and azide form in solution. This method was found to be the most simple and efficient way to study the azido-tetrazole equilibrium.
Assuntos
Azidas , Tetrazóis , Isomerismo , Espectroscopia de Ressonância Magnética , TriazinasRESUMO
A series of purine ribonucleosides bearing chiral amino acid amides at the C6 position of 2-chloropurine was synthesized. Molecular docking of the synthesized analogs of 2-chloroadenosine by their affinity for A1 adenosine receptors (A1ARs) was conducted. The investigation of A1AR stimulating activity of synthesized nucleosides was carried out in a model of an isolated mouse atrium. We have shown that derivatives with tyrosine, valine, and serine residues exhibit the properties of A1AR partial agonists. Animal experiments in the open field test have shown that these compounds have different profiles of psychoactive action. These nucleosides have an ophthalmic hypotensive effect and reduce intraocular pressure in a manner slightly inferior to that of timolol and brimonidine. The synthesized nucleosides can be the basis for further design and synthesis of new A1AR agonists.
Assuntos
Aminoácidos , Agonistas do Receptor Purinérgico P1 , Amidas/farmacologia , Aminoácidos/farmacologia , Animais , Camundongos , Simulação de Acoplamento Molecular , Nucleosídeos , Receptor A1 de Adenosina/metabolismoRESUMO
Three-finger proteins (TFPs) are small proteins with characteristic three-finger ß-structural fold stabilized by the system of conserved disulfide bonds. These proteins have been found in organisms from different taxonomic groups and perform various important regulatory functions or act as components of snake venoms. Recently, four TFPs (Lystars 1-4) with unknown function were identified in the coelomic fluid proteome of starfish A. rubens. Here we analyzed the genomes of A. rubens and A. planci starfishes and predicted additional five and six proteins containing three-finger domains, respectively. One of them, named Lystar5, is expressed in A. rubens coelomocytes and has sequence homology to the human brain neuromodulator Lynx2. The three-finger structure of Lystar5 close to the structure of Lynx2 was confirmed by NMR. Similar to Lynx2, Lystar5 negatively modulated α4ß2 nicotinic acetylcholine receptors (nAChRs) expressed in X. laevis oocytes. Incubation with Lystar5 decreased the expression of acetylcholine esterase and α4 and α7 nAChR subunits in the hippocampal neurons. In summary, for the first time we reported modulator of the cholinergic system in starfish.
Assuntos
Asterias , Receptores Nicotínicos , Animais , Asterias/metabolismo , Encéfalo/metabolismo , Humanos , Neurotransmissores , Receptores Nicotínicos/metabolismo , Estrelas-do-Mar/metabolismo , Xenopus laevis/metabolismoRESUMO
Ly6/uPAR proteins regulate many essential functions in the nervous and immune systems and epithelium. Most of these proteins contain single ß-structural LU domains with three protruding loops and are glycosylphosphatidylinositol (GPI)-anchored to a membrane. The GPI-anchor role is currently poorly studied. Here, we investigated the positional and orientational preferences of six GPI-anchored proteins in the receptor-unbound state by molecular dynamics simulations. Regardless of the linker length between the LU domain and GPI-anchor, the proteins interacted with the membrane by polypeptide parts and N-/O-glycans. Lynx1, Lynx2, Lypd6B, and Ly6H contacted the membrane by the loop regions responsible for interactions with nicotinic acetylcholine receptors, while Lypd6 and CD59 demonstrated unique orientations with accessible receptor-binding sites. Thus, GPI-anchoring does not guarantee an optimal 'pre-orientation' of the LU domain for the receptor interaction.
Assuntos
Glicosilfosfatidilinositóis , Receptores Nicotínicos , Glicosilfosfatidilinositóis/metabolismo , Receptores Nicotínicos/metabolismo , Moléculas de Adesão Celular/metabolismo , Proteínas Ligadas por GPI/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Thionins are the family of small (â¼5 kDa) cationic cysteine-rich peptides involved in the immune response in plants. Viscotoxin A3 (VtA3) is the thionin from mistletoe (Viscum album) demonstrating antimicrobial and cytotoxic activity against cancer cells in vitro. VtA3 (charge +6) interacts with the membranes containing anionic lipids and forms cation-selective ion channels. Here we studied the VtA3 structure in membrane-mimicking media by NMR spectroscopy. Spatial structure of VtA3, consisting of a helical hairpin and a short ß-sheet, was stable and did not undergo significant changes during micelle binding. VtA3 molecule bound with high affinity to the surface of zwitterionic dodecylphosphocholine (DPC) micelle by hydrophobic patch in the helical hairpin. Oligomerization of VtA3 was observed in the anionic micelles of sodium dodecylsulphate (SDS). No direct contacts between the peptide molecules were observed and the possible interfaces of detergent-assisted oligomerization were revealed. The data obtained suggest that the VtA3 membrane activity, depending on the concentration, obeys the 'toroidal' pore model or the 'carpet' mechanism. The model of the membrane disrupting complex, which explains the ion channel formation in the partially anionic membranes, was proposed.
Assuntos
Membrana Celular/química , Detergentes/química , Canais Iônicos/química , Micelas , Proteínas de Plantas/química , Dodecilsulfato de Sódio/química , Viscum album/química , Sequência de Aminoácidos , Membrana Celular/metabolismo , Canais Iônicos/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Folhas de Planta/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Caules de Planta/química , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Homologia de Sequência de AminoácidosRESUMO
Gating pore currents through the voltage-sensing domains (VSDs) of the skeletal muscle voltage-gated sodium channel NaV1.4 underlie hypokalemic periodic paralysis (HypoPP) type 2. Gating modifier toxins target ion channels by modifying the function of the VSDs. We tested the hypothesis that these toxins could function as blockers of the pathogenic gating pore currents. We report that a crab spider toxin Hm-3 from Heriaeus melloteei can inhibit gating pore currents due to mutations affecting the second arginine residue in the S4 helix of VSD-I that we have found in patients with HypoPP and describe here. NMR studies show that Hm-3 partitions into micelles through a hydrophobic cluster formed by aromatic residues and reveal complex formation with VSD-I through electrostatic and hydrophobic interactions with the S3b helix and the S3-S4 extracellular loop. Our data identify VSD-I as a specific binding site for neurotoxins on sodium channels. Gating modifier toxins may constitute useful hits for the treatment of HypoPP.
Assuntos
Mutação de Sentido Incorreto , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Neurotoxinas/toxicidade , Paralisia Periódica Hiperpotassêmica/metabolismo , Estrutura Secundária de Proteína , Venenos de Aranha/toxicidade , Substituição de Aminoácidos , Animais , Feminino , Células HEK293 , Humanos , Ativação do Canal Iônico , Canal de Sódio Disparado por Voltagem NAV1.4/química , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Paralisia Periódica Hiperpotassêmica/genética , Paralisia Periódica Hiperpotassêmica/patologia , Xenopus laevisRESUMO
We report a novel family of natural lipoglycopeptides produced by Streptomyces sp. INA-Ac-5812. Two major components of the mixture, named gausemycinsâ A and B, were isolated, and their structures were elucidated. The compounds are cyclic peptides with a unique peptide core and several remarkable structural features, including unusual positions of d-amino acids, lack of the Ca2+ -binding Asp-X-Asp-Gly (DXDG) motif, tyrosine glycosylation with arabinose, presence of 2-amino-4-hydroxy-4-phenylbutyric acid (Ahpb) and chlorinated kynurenine (ClKyn), and N-acylation of the ornithine side chain. Gausemycins have pronounced activity against Gram-positive bacteria. Mechanistic studies highlight significant differences compared to known glyco- and lipopeptides. Gausemycins exhibit only slight Ca2+ -dependence of activity and induce no pore formation at low concentrations. Moreover, there is no detectable accumulation of cell wall biosynthesis precursors under treatment with gausemycins.
Assuntos
Lipoglicopeptídeos/isolamento & purificação , Streptomyces/química , Lipoglicopeptídeos/química , Conformação MolecularRESUMO
Ly-6/uPAR or three-finger proteins (TFPs) contain a disulfide-stabilized ß-structural core and three protruding loops (fingers). In mammals, TFPs have been found in epithelium and the nervous, endocrine, reproductive, and immune systems. Here, using heteronuclear NMR, we determined the three-dimensional (3D) structure and backbone dynamics of the epithelial secreted protein SLURP-1 and soluble domains of GPI-anchored TFPs from the brain (Lynx2, Lypd6, Lypd6b) acting on nicotinic acetylcholine receptors (nAChRs). Results were compared with the data about human TFPs Lynx1 and SLURP-2 and snake α-neurotoxins WTX and NTII. Two different topologies of the ß-structure were revealed: one large antiparallel ß-sheet in Lypd6 and Lypd6b, and two ß-sheets in other proteins. α-Helical segments were found in the loops I/III of Lynx2, Lypd6, and Lypd6b. Differences in the surface distribution of charged and hydrophobic groups indicated significant differences in a mode of TFPs/nAChR interactions. TFPs showed significant conformational plasticity: the loops were highly mobile at picosecond-nanosecond timescale, while the ß-structural regions demonstrated microsecond-millisecond motions. SLURP-1 had the largest plasticity and characterized by the unordered loops II/III and cis-trans isomerization of the Tyr39-Pro40 bond. In conclusion, plasticity could be an important feature of TFPs adapting their structures for optimal interaction with the different conformational states of nAChRs.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Antígenos Ly/química , Proteínas Ligadas por GPI/química , Neuropeptídeos/química , Receptores Nicotínicos/química , Ativador de Plasminogênio Tipo Uroquinase/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Antígenos Ly/genética , Antígenos Ly/metabolismo , Sítios de Ligação , Clonagem Molecular , Venenos Elapídicos/química , Venenos Elapídicos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Endogenous antimicrobial peptides (AMPs) are among the earliest molecular factors in the evolution of animal innate immunity. In this study, novel AMPs named nicomicins were identified in the small marine polychaeta Nicomache minor in the Maldanidae family. Full-length mRNA sequences encoded 239-residue prepropeptides consisting of a putative signal sequence region, the BRICHOS domain within an acidic proregion, and 33-residue mature cationic peptides. Nicomicin-1 was expressed in the bacterial system, and its spatial structure was analyzed by circular dichroism and nuclear magnetic resonance spectroscopy. Nicomicins are unique among polychaeta AMPs scaffolds, combining an amphipathic N-terminal α-helix and C-terminal extended part with a six-residue loop stabilized by a disulfide bridge. This structural arrangement resembles the Rana-box motif observed in the α-helical host-defense peptides isolated from frog skin. Nicomicin-1 exhibited strong in vitro antimicrobial activity against Gram-positive bacteria at submicromolar concentrations. The main mechanism of nicomicin-1 action is based on membrane damage but not on the inhibition of bacterial translation. The peptide possessed cytotoxicity against cancer and normal adherent cells as well as toward human erythrocytes.
Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/farmacologia , Poliquetos/genética , Sequência de Aminoácidos , Animais , Anti-Infecciosos/química , Anti-Infecciosos/isolamento & purificação , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Células Sanguíneas/efeitos dos fármacos , Linhagem Celular , Células HeLa , Hemólise , Humanos , Fragmentos de Peptídeos/genética , Filogenia , Poliquetos/química , Poliquetos/metabolismo , Conformação Proteica , Domínios Proteicos , Homologia de Sequência de AminoácidosRESUMO
Determining the accurate chemical structures of synthesized compounds is essential for biomedical studies and computer-assisted drug design. The unequivocal determination of N-adamantylation or N-arylation site(s) in nitrogen-rich heterocycles, characterized by a low density of hydrogen atoms, using NMR methods at natural isotopic abundance is difficult. In these compounds, the heterocyclic moiety is covalently attached to the carbon atom of the substituent group that has no bound hydrogen atoms, and the connection between the two moieties of the compound cannot always be established via conventional 1H-1H and 1H-13C NMR correlation experiments (COSY and HMBC, respectively) or nuclear Overhauser effect spectroscopy (NOESY or ROESY). The selective incorporation of 15N-labelled atoms in different positions of the heterocyclic core allowed for the use of 1H-15N (JHN) and 13C-15N (JCN) coupling constants for the structure determinations of N-alkylated nitrogen-containing heterocycles in solution. This method was tested on the N-adamantylated products in a series of azolo-1,2,4-triazines and 1,2,4-triazolo[1,5-a]pyrimidine. The syntheses of adamantylated azolo-azines were based on the interactions of azolo-azines and 1-adamatanol in TFA solution. For azolo-1,2,4-triazinones, the formation of mixtures of N-adamantyl derivatives was observed. The JHN and JCN values were measured using amplitude-modulated 1D 1H spin-echo experiments with the selective inversion of the 15N nuclei and line-shape analysis in the 1D 13С spectra acquired with selective 15N decoupling, respectively. Additional spin-spin interactions were detected in the 15N-HMBC spectra. NMR data and DFT (density functional theory) calculations permitted to suggest a possible mechanism of isomerization for the adamantylated products of the azolo-1,2,4-triazines. The combined analysis of the JHN and JCN couplings in 15N-labelled compounds provides an efficient method for the structure determination of N-alkylated azolo-azines even in the case of isomer formation. The isomerization of adamantylated tetrazolo[1,5-b][1,2,4]triazin-7-ones in acidic conditions occurs through the formation of the adamantyl cation.
RESUMO
We present a structural and functional study of a sodium channel activation inhibitor from crab spider venom. Hm-3 is an insecticidal peptide toxin consisting of 35 amino acid residues from the spider Heriaeus melloteei (Thomisidae). We produced Hm-3 recombinantly in Escherichia coli and determined its structure by NMR spectroscopy. Typical for spider toxins, Hm-3 was found to adopt the so-called "inhibitor cystine knot" or "knottin" fold stabilized by three disulfide bonds. Its molecule is amphiphilic with a hydrophobic ridge on the surface enriched in aromatic residues and surrounded by positive charges. Correspondingly, Hm-3 binds to both neutral and negatively charged lipid vesicles. Electrophysiological studies showed that at a concentration of 1 µm Hm-3 effectively inhibited a number of mammalian and insect sodium channels. Importantly, Hm-3 shifted the dependence of channel activation to more positive voltages. Moreover, the inhibition was voltage-dependent, and strong depolarizing prepulses attenuated Hm-3 activity. The toxin is therefore concluded to represent the first sodium channel gating modifier from an araneomorph spider and features a "membrane access" mechanism of action. Its amino acid sequence and position of the hydrophobic cluster are notably different from other known gating modifiers from spider venom, all of which are described from mygalomorph species. We hypothesize parallel evolution of inhibitor cystine knot toxins from Araneomorphae and Mygalomorphae suborders.
Assuntos
Bloqueadores dos Canais de Sódio/química , Venenos de Aranha/química , Aranhas/química , Canais de Sódio Disparados por Voltagem/química , Sequência de Aminoácidos , Animais , Membrana Celular/química , Escherichia coli/genética , Escherichia coli/metabolismo , Evolução Molecular , Expressão Gênica , Interações Hidrofóbicas e Hidrofílicas , Ativação do Canal Iônico , Potenciais da Membrana , Modelos Moleculares , Dados de Sequência Molecular , Fosfatidilcolinas/química , Fosfatidilgliceróis/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Bloqueadores dos Canais de Sódio/isolamento & purificação , Venenos de Aranha/classificação , Venenos de Aranha/genética , Venenos de Aranha/isolamento & purificação , Aranhas/fisiologia , Lipossomas Unilamelares/química , Canais de Sódio Disparados por Voltagem/metabolismoRESUMO
Weak toxin from Naja kaouthia (WTX) belongs to the group of nonconventional "three-finger" snake neurotoxins. It irreversibly inhibits nicotinic acetylcholine receptors and allosterically interacts with muscarinic acetylcholine receptors (mAChRs). Using site-directed mutagenesis, NMR spectroscopy, and computer modeling, we investigated the recombinant mutant WTX analogue (rWTX) which, compared with the native toxin, has an additional N-terminal methionine residue. In comparison with the wild-type toxin, rWTX demonstrated an altered pharmacological profile, decreased binding of orthosteric antagonist N-methylscopolamine to human M1- and M2-mAChRs, and increased antagonist binding to M3-mAChR. Positively charged arginine residues located in the flexible loop II were found to be crucial for rWTX interactions with all types of mAChR. Computer modeling suggested that the rWTX loop II protrudes to the M1-mAChR allosteric ligand-binding site blocking the entrance to the orthosteric site. In contrast, toxin interacts with M3-mAChR by loop II without penetration into the allosteric site. Data obtained provide new structural insight into the target-specific allosteric regulation of mAChRs by "three-finger" snake neurotoxins.
Assuntos
Venenos Elapídicos/química , Neurotoxinas/metabolismo , Receptores Muscarínicos/metabolismo , Sequência de Aminoácidos , Animais , Elapidae , Dados de Sequência Molecular , Mutagênese Insercional , Neurotoxinas/química , Neurotoxinas/genética , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
Production of helical integral membrane proteins (IMPs) in a folded state is a necessary prerequisite for their functional and structural studies. In many cases large-scale expression of IMPs in cell-based and cell-free systems results in misfolded proteins, which should be refolded in vitro. Here using examples of the bacteriorhodopsin ESR from Exiguobacterium sibiricum and full-length homotetrameric K(+) channel KcsA from Streptomyces lividans we found that the efficient in vitro folding of the transmembrane domains of the polytopic and multimeric IMPs could be achieved during the protein encapsulation into the reconstructed high-density lipoprotein particles, also known as lipid-protein nanodiscs. In this case the self-assembly of the IMP/nanodisc complexes from a mixture containing apolipoprotein, lipids and the partially denatured protein solubilized in a harsh detergent induces the folding of the transmembrane domains. The obtained folding yields showed significant dependence on the properties of lipids used for nanodisc formation. The largest recovery of the spectroscopically active ESR (~60%) from the sodium dodecyl sulfate (SDS) was achieved in the nanodiscs containing anionic saturated lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPG) and was approximately twice lower in the zwitterionic DMPC lipid. The reassembly of tetrameric KcsA from the acid-dissociated monomer solubilized in SDS was the most efficient (~80%) in the nanodiscs containing zwitterionic unsaturated lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The charged and saturated lipids provided lower tetramer quantities, and the lowest yield (<20%) was observed in DMPC. The overall yield of the ESR and KcsA folding was mainly restricted by the efficiency of the protein encapsulation into the nanodiscs.
Assuntos
Proteínas de Bactérias/química , Lipídeos/química , Nanoestruturas/química , Canais de Potássio/química , Proteínas/química , Bactérias/metabolismo , Bacteriorodopsinas/metabolismo , Membrana Celular/metabolismo , Detergentes/química , Dimerização , Dimiristoilfosfatidilcolina/química , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Nanotecnologia/métodos , Fosfatidilcolinas/química , Fosfatidilgliceróis/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Dodecilsulfato de Sódio/química , Streptomyces lividans/metabolismoRESUMO
Capitellacin is the ß-hairpin membrane-active cationic antimicrobial peptide from the marine polychaeta Capitella teleta. Capitellacin exhibits antibacterial activity, including against drug-resistant strains. To gain insight into the mechanism of capitellacin action, we investigated the structure of the peptide in the membrane-mimicking environment of dodecylphosphocholine (DPC) micelles using high-resolution NMR spectroscopy. In DPC solution, two structural forms of capitellacin were observed: a monomeric ß-hairpin was in equilibrium with a dimer formed by the antiparallel association of the N-terminal ß-strands and stabilized by intermonomer hydrogen bonds and Van der Waals interactions. The thermodynamics of the enthalpy-driven dimerization process was studied by varying the temperature and molar ratios of the peptide to detergent. Cooling the peptide/detergent system promoted capitellacin dimerization. Paramagnetic relaxation enhancement induced by lipid-soluble 12-doxylstearate showed that monomeric and dimeric capitellacin interacted with the surface of the micelle and did not penetrate into the micelle interior, which is consistent with the "carpet" mode of membrane activity. An analysis of the known structures of ß-hairpin AMP dimers showed that their dimerization in a membrane-like environment occurs through the association of polar or weakly hydrophobic surfaces. A comparative analysis of the physicochemical properties of ß-hairpin AMPs revealed that dimer stability and hemolytic activity are positively correlated with surface hydrophobicity. An additional positive correlation was observed between hemolytic activity and AMP charge. The data obtained allowed for the provision of a more accurate description of the mechanism of the oligomerization of ß-structural peptides in biological membranes.
Assuntos
Peptídeos Catiônicos Antimicrobianos , Poliquetos , Animais , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Dimerização , Micelas , Detergentes , Espectroscopia de Ressonância Magnética , TermodinâmicaRESUMO
1,2,4-Triazole derivatives have a wide range of biological activities. The most well-known drug that contains 1,2,4-triazole as part of its structure is the nucleoside analogue ribavirin, an antiviral drug. Finding new nucleosides based on 1,2,4-triazole is a topical task. The aim of this study was to synthesize ribosides and deoxyribosides of 1,2,4-triazole-3-thione derivatives and test their antiviral activity against herpes simplex viruses. Three compounds from a series of synthesized mono- and disubstituted 1,2,4-triazole-3-thione derivatives were found to be substrates for E. coli purine nucleoside phosphorylase. Of six prepared nucleosides, the riboside and deoxyriboside of 3-phenacylthio-1,2,4-triazole were obtained at good yields. The yields of the disubstituted 1,2,4-triazol-3-thiones were low due to the effect of bulky substituents at the C3 and C5 positions on the selectivity of enzymatic glycosylation for one particular nitrogen atom in the triazole ring. The results of cytotoxic and antiviral studies on acyclovir-sensitive wild-type strain HSV-1/L2(TK+) and acyclovir-resistant strain (HSV-1/L2/RACV) in Vero E6 cell culture showed that the incorporation of a thiobutyl substituent into the C5 position of 3-phenyl-1,2,4-triazole results in a significant increase in the cytotoxicity of the base and antiviral activity. The highest antiviral activity was observed in the 3-phenacylthio-1-(ß-D-ribofuranosyl)-1,2,4-triazole and 5-butylthio-1-(2-deoxy-ß-D-ribofuranosyl)-3-phenyl-1,2,4-triazole nucleosides, with their selectivity indexes being significantly higher than that of ribavirin. It was also found that with the increasing lipophilicity of the nucleosides, the activity and toxicity of the tested compounds increased.