RESUMO
Phosphorylated kininogen and some of its fragments containing serine phosphorylated bradykinin ([pS(6)]-Bk) were identified in human serum and plasma by a phosphoproteomic approach. We report the kininogenase ability of human tissue and plasma kallikreins and tryptase to generate [pS(6)]-Bk or Lys-[pS(6)]-Bk having as substrate the synthetic human kininogen fluorescent fragment Abz-MISLMKRPPGF[pS(386)]PFRSSRI-NH2. The pharmacological assays of [pS(6)]-Bk showed it as a full B2 bradykinin receptor agonist in smooth muscle, it produces a portal liver hypertensive response in rat and mouse paw edema that lasts longer than Bk. The rat hypotensive response to infusions of Bk is greater than that of [pS(6)]Bk, both if injected through femoral vein or aorta. [pS(6)]-Bk was more resistant than Bk to kininase digestion performed with angiotensin converting enzyme, neprilysin, thimet oligopeptidase, aminopeptidase P and carboxypeptidase M. (1)H-NMR experiments indicated that [pS(6)]-Bk has lower flexibility, with the pS(6)-P(7) bond restricted to the trans conformation, and can explain [pS(6)]-Bk resistance to hydrolysis. In conclusion, [pS(6)]-Bk presenting lower activity than Bk, with longer lasting effects and being slowly released by kininogenases from synthetic Abz-MISLMKRPPGF[pS(386)]PFRSSRI-NH2, suggests that phosphorylation of the kininogens can be an efficient kallikrein-kinin system regulator.
Assuntos
Bradicinina/análogos & derivados , Bradicinina/farmacologia , Peptídeo Hidrolases/farmacologia , Sequência de Aminoácidos , Animais , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Bradicinina/genética , Cobaias , Humanos , Hidrólise/efeitos dos fármacos , Camundongos , Dados de Sequência Molecular , Técnicas de Cultura de Órgãos , Peptídeo Hidrolases/genética , Coelhos , Ratos , Ratos Sprague-Dawley , Ratos WistarRESUMO
α-KTx toxin Tc32, from the Amazonian scorpion Tityus cambridgei, lacks the dyad motif, including Lys27, characteristic of the family and generally associated with channel blockage. The toxin has been cloned and expressed for the first time. Electrophysiological experiments, by showing that the recombinant form blocks Kv1.3 channels of olfactory bulb periglomerular cells like the natural Tc32 toxin, when tested on the Kv1.3 channel of human T lymphocytes, confirmed it is in an active fold. The nuclear magnetic resonance-derived structure revealed it exhibits an α/ß scaffold typical of the members of the α-KTx family. TdK2 and TdK3, all belonging to the same α-KTx 18 subfamily, share significant sequence identity with Tc32 but diverse selectivity and affinity for Kv1.3 and Kv1.1 channels. To gain insight into the structural features that may justify those differences, we used the recombinant Tc32 nuclear magnetic resonance-derived structure to model the other two toxins, for which no experimental structure is available. Their interaction with Kv1.3 and Kv1.1 has been investigated by means of docking simulations. The results suggest that differences in the electrostatic features of the toxins and channels, in their contact surfaces, and in their total dipole moment orientations govern the affinity and selectivity of toxins. In addition, we found that, regardless of whether the dyad motif is present, it is always a Lys side chain that physically blocks the channels, irrespective of its position in the toxin sequence.
Assuntos
Canal de Potássio Kv1.3/química , Venenos de Escorpião/química , Toxinas Biológicas/química , Sequência de Aminoácidos , Animais , Células Cultivadas , Humanos , Canal de Potássio Kv1.3/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Alinhamento de Sequência , Relação Estrutura-Atividade , Toxinas Biológicas/metabolismoRESUMO
Cerato-platanin (CP) is a secretion protein produced by the fungal pathogen Ceratocystis platani, the causal agent of the plane canker disease and the first member of the CP family. CP is considered a pathogen-associated molecular pattern because it induces various defense responses in the host, including production of phytoalexins and cell death. Although much is known about the properties of CP and related proteins as elicitors of plant defense mechanisms, its biochemical activity and host target(s) remain elusive. Here, we present the three-dimensional structure of CP. The protein, which exhibits a remarkable pH and thermal stability, has a double ψß-barrel fold quite similar to those found in expansins, endoglucanases, and the plant defense protein barwin. Interestingly, although CP lacks lytic activity against a variety of carbohydrates, it binds oligosaccharides. We identified the CP region responsible for binding as a shallow surface located at one side of the ß-barrel. Chemical shift perturbation of the protein amide protons, induced by oligo-N-acetylglucosamines of various size, showed that all the residues involved in oligosaccharide binding are conserved among the members of the CP family. Overall, the results suggest that CP might be involved in polysaccharide recognition and that the double ψß-barrel fold is widespread in distantly related organisms, where it is often involved in host-microbe interactions.
Assuntos
Ascomicetos/química , Proteínas Fúngicas/química , Ascomicetos/genética , Ascomicetos/metabolismo , Sítios de Ligação , Carboidratos/química , Carboidratos/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Doenças das Plantas/microbiologia , Ligação Proteica , Estrutura Secundária de ProteínaRESUMO
The Schistosoma mansoni fatty acid binding protein (FABP), Sm14, is a vaccine candidate against, S. mansoni and F. hepatica. Previously, we demonstrated the importance of a correct fold to achieve protection in immunized animals after cercariae challenge [[10]. C.R.R. Ramos, R.C.R. Figueredo, T.A. Pertinhez, M.M. Vilar, A.L.T.O. Nascimento, M. Tendler, I. Raw, A. Spisni, P.L. Ho, Gene structure and M20T polymorphism of the Schistosoma mansoni Sm14 fatty acid-binding protein: structural, functional and immunoprotection analysis. J. Biol. Chem. 278 (2003) 12745-12751.]. Here we show that the reduction of vaccine efficacy over time is due to protein dimerization and subsequent aggregation. We produced the mutants Sm14-M20(C62S) and Sm14-M20(C62V) that, as expected, did not dimerize in SDS-PAGE. Molecular dynamics calculations and unfolding experiments highlighted a higher structural stability of these mutants with respect to the wild-type. In addition, we found that the mutated proteins, after thermal denaturation, refolded to their active native molecular architecture as proved by the recovery of the fatty acid binding ability. Sm14-M20(C62V) turned out to be the more stable form over time, providing the basis to determine the first 3D solution structure of a Sm14 protein in its apo-form. Overall, Sm14-M20(C62V) possesses an improved structural stability over time, an essential feature to preserve its immunization capability and, in experimentally immunized animals, it exhibits a protection effect against S. mansoni cercariae infections comparable to the one obtained with the wild-type protein. These facts indicate this protein as a good lead molecule for large-scale production and for developing an effective Sm14 based anti-helminthes vaccine.
Assuntos
Proteínas de Transporte de Ácido Graxo/química , Proteínas de Transporte de Ácido Graxo/imunologia , Proteínas de Helminto/química , Proteínas de Helminto/imunologia , Schistosoma mansoni/química , Animais , Simulação por Computador , Proteínas de Transporte de Ácido Graxo/genética , Feminino , Proteínas de Helminto/genética , Camundongos , Modelos Moleculares , Mutação , Dobramento de Proteína , Multimerização Proteica , Estabilidade Proteica , Schistosoma mansoni/genética , Schistosoma mansoni/crescimento & desenvolvimento , Schistosoma mansoni/imunologia , Esquistossomose mansoni/parasitologia , Esquistossomose mansoni/prevenção & controle , Vacinas/administração & dosagem , Vacinas/químicaRESUMO
Peptides derived from endogenous hemoglobin play important biological roles in a variety of living systems. In previous works we showed that the fragment 33-61 of bovine alpha-hemoglobin (Hb33-61) and its C-terminus amidated analogue (Hb33-61a) exhibit antimicrobial activity and we determined the 3D structure of Hb33-61a bound to sodium dodecyl sulfate micelles. Here we report that Hb33-61a is lethal to Candida albicans at 6.25 microM probably through disruption of its plasma membrane. In addition, we show that, even when used at 50 microM, Hb33- 61a produces low hemolysis (16% +/- 3.0%). Recognizing that one of the key steps to study new compounds with potential pharmaceutical application is to identify the structural elements essential to express biological activity, we also investigated the anticandidal activity of Hb33- 61a fragments. The results indicated that Hb40-61a exhibits the same minimal inhibitory concentration as Hb33-61a, whereas Hb33-52a and Hb48-61a are significantly less active. Noteworthy, for all the peptides tested, we observed that C-terminus amidation produces a potentiation of their anticandidal activity and we associate that increased biological activity to a preferred structural and spatial organization of the C-terminal region favored by amidation. Finally, the data show that the most active peptides (Hb33-61a and Hb40-61a) are characterized by a central hinge joining the C-terminal region that presents, containing a beta-turn, followed by and a helical element, to the N-terminal region that presents only a beta-turn. We hypothesize that these two structured regions, by fluctuating independently in the lipid environment, may act in a coordinated fashion disrupting the yeast plasma membrane.
Assuntos
Hemoglobinas/química , Hemoglobinas/farmacologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Antifúngicos/síntese química , Antifúngicos/química , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Bovinos , Desenho de Fármacos , Hemoglobinas/genética , Hemólise/efeitos dos fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Técnicas In Vitro , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/toxicidade , Estrutura Secundária de Proteína , Eletricidade EstáticaRESUMO
The peptide pIV/S4-S5 encompasses the cytoplasmic linker between helices S4-S5 in domain IV of the voltage-gated Na+ channel, residues 1644-1664. The interaction of two local anesthetics (LA), lidocaine and benzocaine, with pIV/S4-S5 has been studied by DOSY, heteronuclear NMR 1H-15N-HSQC spectroscopy and computational methods. DOSY indicates that benzocaine, a neutral ester, exhibits stronger interaction with pIV/S4-S5 than lidocaine, a charged amine-amide. Weighted average chemical shifts, Deltadelta(1H-15N), show that benzocaine affects residues L1653, M1655 and S1656 while lidocaine slightly perturbs residues I1646, L1649 and A1659, L1660, near the N- and C-terminus, respectively. Computational methods confirmed the stability of the benzocaine binding and the existence of two binding sites for lidocaine. Even considering that the approach of studying the peptide in the presence of a co-solvent (TFE/H2O, 30%/70% v/v) has an inherently limited implication, our data strongly support the existence of multiple LA binding sites in the IV/S4-S5 linker, as suggested in the literature. In addition, we consider that LA can bind to the S4-S5 linker with diverse binding modes and strength since this linker is part of the receptor for the "inactivation gate particle". Conditions for devising new functional studies, aiming to better understand Na+ channel functionality as well as the various facets of LA pharmacological activity are proposed in this work.
Assuntos
Anestésicos Locais/química , Benzocaína/química , Lidocaína/química , Peptídeos/química , Canais de Sódio/química , Sequência de Aminoácidos , Dicroísmo Circular , Modelos Moleculares , Dados de Sequência MolecularRESUMO
The PCI domain comprises approx 200 amino acids and is found in subunits of the eukaryotic translation initiation factor 3 (eIF3), the 26S proteasome and the COP9/signalosome complexes. The PCI domain is involved in protein-protein interaction, and mouse INT6 truncated proteins lacking the PCI domain show cell malignanttransforming activity. In this work, the Arabidopsis thaliana INT6/eIF3e (AtINT6) protein was dissected using limited proteolysis, and a protease-resistant fragment containing the PCI domain was identified. Based on mass spectrometry analyses of the protease-resistant fragments and on secondary structure prediction, AtINT6-truncated proteins were cloned and expressed in Escherichia coli. Stability studies using thermal unfolding followed by circular dichroism revealed a midpoint transition temperature of 44 degrees C for the full-length AtINT6 protein, whereas the truncated proteins comprising residues 125-415 (AtINT6TR2) and 172-415 (AtINT6TR3) showed transition temperatures of 49 and 58 degrees C, respectively. AtINT6TR3 contains the PCI domain with additional amino acids at the N and C termini. It shows high solubility, and together with the high thermal stability, should facilitate further characterization of the PCI domain structure, which is important to understand its function in protein- protein interaction.
Assuntos
Proteínas de Arabidopsis/biossíntese , Arabidopsis/genética , Fator de Iniciação 3 em Eucariotos/química , Fator de Iniciação 3 em Eucariotos/isolamento & purificação , Peptídeo Hidrolases/química , Proteínas Recombinantes de Fusão/metabolismo , Motivos de Aminoácidos , Animais , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Complexo do Signalossomo COP9 , Clonagem Molecular/métodos , Bases de Dados de Proteínas , Escherichia coli/citologia , Escherichia coli/genética , Escherichia coli/metabolismo , Fator de Iniciação 3 em Eucariotos/biossíntese , Fator de Iniciação 3 em Eucariotos/genética , Camundongos , Dados de Sequência Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Fatores de Iniciação de Peptídeos , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/genética , Ligação Proteica/fisiologia , Homologia de Sequência , Solubilidade , Fatores de Transcrição/metabolismo , Temperatura de TransiçãoRESUMO
Trialysin is a pore-forming protein found in the saliva of Triatoma infestans (Hemiptera, Reduviidae), the insect vector of Chagas' disease. The protein is active against a broad range of cell types from bacteria to eukaryotic cells. Recognizing that the N-terminus of trialysin harbors the lytic motif [Amino, R., Martins, R. M., Procopio, J., Hirata, I. Y., Juliano, M. A., and Schenkman, S. (2002) J. Biol. Chem. 277, 6207-6213], we designed a set of peptides scanning this region to investigate the structural basis of its biological function. Peptides encompassing residues 1-32 (P6), 1-27 (P7), and 6-32 (P5) efficiently induced lysis of the protozoan parasite Trypanosoma cruzi and Escherichia coli in the 0.4-9.0 microM range, while much higher concentrations were required to cause hemolysis. Other more internal peptides, including peptide P2 (residues 21-47) and others up to residue 52, were less effective. P6 turned out to be the most active of all. P7 has a significantly higher activity than P5 against E. coli, while P5 has a hemolytic activity comparable to that of P6. CD spectroscopy showed that all tested peptides acquire a comparable helical content in solvent mixtures or in detergent micelles. The solution structure of P2 and P5-P7 was determined in a 30% trifluoroethanol/water mixture by nuclear magnetic resonance. All peptides exhibit a structure characterized by a central helical fold, and except for P2, which does not show a continuous hydrophobic surface, they are amphipathic. The structural models show that P5 and P7 extend their structural similarities with the most active peptide, P6, in either the C-terminus or the N-terminus. Amino acid substitutions in the N-terminus of P6 improved hemolysis but did not change the activity against T. cruzi. These results suggest that while amphipathicity is essential for the lytic activity, the selectivity of the active peptides for specific organisms appears to be associated with the structural features of their N- and C-termini.
Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Proteínas e Peptídeos Salivares/metabolismo , Triatoma/química , Trypanosoma cruzi/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Doença de Chagas/tratamento farmacológico , Dicroísmo Circular , Espectroscopia de Ressonância Magnética , Micelas , Peptídeos/química , Peptídeos/farmacologia , Conformação Proteica , Proteínas e Peptídeos Salivares/química , Proteínas e Peptídeos Salivares/farmacologia , Solventes/químicaRESUMO
To investigate the role of the N-terminal region in the lytic mechanism of the pore-forming toxin sticholysin II (St II), we studied the conformational and functional properties of peptides encompassing the first 30 residues of the protein. Peptides containing residues 1-30 (P1-30) and 11-30 (P11-30) were synthesized and their conformational properties were examined in aqueous solution as a function of peptide concentration, pH, ionic strength, and addition of the secondary structure-inducing solvent trifluoroethanol (TFE). CD spectra showed that increasing concentration, pH, and ionic strength led to aggregation of P1-30; as a consequence, the peptide acquired beta-sheet conformation. In contrast, P11-30 exhibited practically no conformational changes under the same conditions, remaining essentially structureless. Moreover, this peptide did not undergo aggregation. These differences clearly point to the modulating effect of the first 10 hydrophobic residues on the peptides aggregation and conformational properties. In TFE both the first ten hydrophobic peptides acquired alpha-helical conformation, albeit to a different extent, P11-30 displayed lower alpha-helical content. P1-30 presented a larger fraction of residues in alpha-helical conformation in TFE than that found in St II's crystal structure for that portion of the protein. Since TFE mimics the membrane environment, such increase in helical content could also occur upon toxin binding to membranes and represent a step in the mechanism of pore formation. The peptides conformational properties correlated well with their functional behavior. Thus, P1-30 exhibited much higher hemolytic activity than P11-30. In addition, P11-30 was able to block the toxin's hemolytic activity. The size of pores formed in red blood cells by P1-30 was estimated by measuring the permeability to PEGs of different molecular mass. The pore radius (0.95 +/- 0.01 nm) was very similar to that of the pore formed by the toxin. The results demonstrate that the synthetic peptide P1-30 is a good model of St II conformation and function and emphasize the contribution of the toxin's N-terminal region, and, in particular, the hydrophobic residues 1-10 to pore formation.