RESUMO
BACKGROUND: Host defence peptides (HDPs) are evolutionarily conserved components of innate immunity. Human HDPs, produced by a variety of immune cells of hematopoietic and epithelial origin, are generally grouped into two families: beta structured defensins and variably-structured cathelicidins. We report the characterization of a very promising cryptic human HDP, here called GVF27, identified in 11-hydroxysteroid dehydrogenase-1 ß-like protein. METHODS: Conformational analysis of GVF27 and its propensity to bind endotoxins were performed by NMR, Circular Dichroism, Fluorescence and Dynamic Light Scattering experiments. Crystal violet and WST-1 assays, ATP leakage measurement and colony counting procedures were used to investigate antimicrobial, anti-biofilm, cytotoxicity and hemolytic activities. Anti-inflammatory properties were evaluated by ELISA. RESULTS: GVF27 possesses significant antibacterial properties on planktonic cells and sessile bacteria forming biofilm, as well as promising dose dependent abilities to inhibit attachment or eradicate existing mature biofilm. It is unstructured in aqueous buffer, whereas it tends to assume a helical conformation in mimic membrane environments as well as it is able to bind lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Notably it is not toxic towards human and murine cell lines and triggers a significant innate immune response by attenuating expression levels of pro-inflammatory interleukins and release of nitric oxide in LPS induced macrophages. CONCLUSION: Human GVF27 may offer significant advantages as leads for the design of human-specific therapeutics. GENERAL SIGNIFICANCE: Human cryptic host defence peptides are naturally no immunogenic and for this they are a real alternative for solving the lack of effective antibiotics to control bacterial infections.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/farmacologia , Anti-Infecciosos/farmacologia , Fragmentos de Peptídeos/farmacologia , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/química , Animais , Anti-Inflamatórios/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Humanos , Lipopolissacarídeos/farmacologia , Espectroscopia de Ressonância Magnética , Camundongos , Fragmentos de Peptídeos/químicaRESUMO
Interleukin 6 (IL-6) and its soluble receptor (IL-6sR) can markedly stimulate aromatase activity in cultured fibroblasts derived from normal or malignant breast tissues. IL-6 acts by binding to a low-affinity membrane-spanning receptor (IL-6R), which must associate with a high-affinity receptor (gp130) for signal transduction to occur. Sant 7 is a mutated form of IL-6 that can bind to the IL-6R, but inhibits its ability to interact with the gp130 signal transducing protein. In this study, we have used Sant 7 to examine its ability to inhibit IL-6+IL-6 soluble receptor (IL-6sR)-stimulated aromatase activity in breast tissue-derived fibroblasts. As previously observed, IL-6+IL-6sR markedly stimulated aromatase activity (7.7-20.8-fold) in fibroblasts derived from reduction mammoplasty tissue, tissue proximal to tumours and breast tumours. Sant 7 inhibited basal aromatase activity in some fibroblasts by 25-30% that had a high basal activity, but almost completely blocked the ability of IL-6+IL-6sR to stimulate aromatase activity. The IC(50) for the inhibition of IL-6+IL-6sR-stimulated aromatase activity by Sant 7 was 60 ng ml(-1). A comparison of the effects of prostaglandin E(2) (PGE(2)), which can also regulate aromatase activity, and IL-6+IL-6sR revealed a greater degree of aromatase stimulation by IL-6+IL-6sR. Sant 7, however, inhibited PGE(2)-stimulated aromatase activity by 70% suggesting that PGE(2) acts, in part, by stimulating IL-6 production. Much of the IL-6 and IL-6sR available to stimulate breast tumour aromatase activity may originate from infiltrating macrophages and lymphocytes. The ability to block aromatase stimulation by these factors may offer a novel therapeutic strategy for reducing oestrogen synthesis in breast tumours.
Assuntos
Aromatase/metabolismo , Ativação Enzimática/efeitos dos fármacos , Fibroblastos/enzimologia , Interleucina-6/análogos & derivados , Interleucina-6/antagonistas & inibidores , Interleucina-6/farmacologia , Receptores de Interleucina-6/antagonistas & inibidores , Mama/citologia , Fibroblastos/citologia , Regulação Enzimológica da Expressão Gênica , Humanos , Receptores de Interleucina-6/metabolismoRESUMO
Chemosensory proteins (CSPs) are a class of small, soluble proteins present at high concentrations in chemosensory organs of different insect species. Several pieces of evidence suggest their involvement in carrying chemical messages from the environment to chemosensory receptors. However, a structural description of the mechanism of delivery has not been reported. In order to provide the first detailed conformational characterization of these molecules, we cloned a specific isoform (CSP-sg4) from Schistocerca gregaria and expressed it in Escherichia coli. The product was obtained with yields of more than 20 mg per L of culture, all in its soluble form. The recombinant protein was identical to the native one with respect to pairing of the disulfide bridges, aggregative state and secondary structure elements. Structural investigations revealed a significantly stable polypeptide with respect to variations in temperature and acidity. CD analysis, preliminary NMR data and secondary structure prediction pointed to a correctly folded structure where helical regions and loops are alternated in a similar fashion as that observed for other classes of odorant- and pheromone-binding proteins presenting no sequence similarity to CSPs.
Assuntos
Gafanhotos/química , Proteínas de Insetos/química , Sequência de Aminoácidos , Animais , Dicroísmo Circular , Proteínas de Insetos/genética , Proteínas de Insetos/isolamento & purificação , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Dados de Sequência Molecular , Conformação Proteica , Proteínas Recombinantes/químicaAssuntos
Endorribonucleases/química , Mutação de Sentido Incorreto , Ressonância Magnética Nuclear Biomolecular , Proteínas de Plasma Seminal/química , Substituição de Aminoácidos , Animais , Bovinos , Códon/genética , Endorribonucleases/genética , Masculino , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas de Plasma Seminal/genéticaRESUMO
The design of safe sweeteners is very important for people who are affected by diabetes, hyperlipemia, and caries and other diseases that are linked to the consumption of sugars. Sweet proteins, which are found in several tropical plants, are many times sweeter than sucrose on a molar basis. A good understanding of their structure-function relationship can complement traditional SAR studies on small molecular weight sweeteners and thus help in the design of safe sweeteners. However, there is virtually no sequence homology and very little structural similarity among known sweet proteins. Studies on mutants of monellin, the best characterized of sweet proteins, proved not decisive in the localization of the main interaction points of monellin with its receptor. Accordingly, we resorted to an unbiased approach to restrict the search of likely areas of interaction on the surface of a typical sweet protein. It has been recently shown that an accurate survey of the surface of proteins by appropriate paramagnetic probes may locate interaction points on protein surface. Here we report the survey of the surface of MNEI, a single chain monellin, by means of a paramagnetic probe, and a direct assessment of bound water based on an application of ePHOGSY, an NMR experiment that is ideally suited to detect interactions of small ligands to a protein. Detailed surface mapping reveals the presence, on the surface of MNEI, of interaction points that include residues previously predicted by ELISA tests and by mutagenesis.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Proteínas de Plantas/química , Edulcorantes/química , Modelos Moleculares , Proteínas de Plantas/genética , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Água/químicaRESUMO
Peptide T (ASTTTNYT), a fragment corresponding to residues 185-192 of gp120, the coat protein of HIV, is endowed with several biological properties in vitro, notably inhibition of the binding of both isolated gp120 and HIV-1 to the CD4 receptor, and chemotactic activity. Based on previous nuclear magnetic resonance (NMR) studies performed in our laboratory, which were consistent with a regular conformation of the C-terminal pentapeptide, and SAR studies showing that the C-terminal pentapeptide retains most of the biological properties, we designed eight hexapeptides containing in the central part either the TNYT or the TTNY sequence, and charged residues (D/E/R) at the two ends. Conformational analysis based on NMR and torsion angle dynamics showed that all peptides assume folded conformations. albeit with different geometries and stabilities. In particular, peptides carrying an acidic residue at the N-terminus and a basic residue at the C-terminus are characterized by stable helical structures and retain full chemotactic activity. The solution conformation of peptide ETNYTR displays strong structural similarity to the region 19-26 of both bovine pancreatic and bovine seminal ribonuclease, which are endowed with anti-HIV activity. Moreover, the frequent occurrence, in many viral proteins, of TNYT and TTNY, the two core sequences employed in the design of the hexapeptides studied in the present work, hints that the sequence of the C-terminal pentapeptide TTNYT is probably representative of a widespread viral recognition motif.
Assuntos
Fármacos Anti-HIV/química , Endorribonucleases/química , Epitopos/química , Oligopeptídeos/síntese química , Peptídeo T/química , Ribonuclease Pancreático/química , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/farmacologia , Sítios de Ligação/fisiologia , Antígenos CD4/química , Antígenos CD4/metabolismo , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/fisiologia , Desenho de Fármacos , Estabilidade de Medicamentos , Endorribonucleases/farmacologia , HIV/efeitos dos fármacos , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/metabolismo , Humanos , Conformação Molecular , Mimetismo Molecular/fisiologia , Monócitos/citologia , Monócitos/metabolismo , Oligopeptídeos/farmacologia , Peptídeo T/análogos & derivados , Peptídeo T/farmacologia , Ribonuclease Pancreático/farmacologiaRESUMO
Fish and mammalian metallothioneins (MTs) differ in the amino acid residues placed between their conserved cysteines. We have expressed the MT of an Antarctic fish, Notothenia coriiceps, and characterized it by means of multinuclear NMR spectroscopy. Overall, the architecture of the fish MT is very similar to that of mammalian MTs. However, NMR spectroscopy shows that the dynamic behaviour of the two domains is markedly different. With the aid of absorption and CD spectroscopies, we studied the conformational and electronic features of fish and mouse recombinant Cd-MT and the changes produced in these proteins by heating. When the temperature was increased from 20 to 90 degrees C, the Cd-thiolate chromophore absorbance at 254 nm of mouse MT was not modified up to 60 degrees C, whereas the absorbance of fish MT decreased significantly starting from 30 degrees C. The CD spectra also changed quite considerably with temperature, with a gradual decrease of the positive band at 260 nm that was more pronounced for fish than for mouse MT. The differential effect of temperature on fish and mouse MTs may reflect a different stability of metal-thiolate clusters of the two proteins. Such a conclusion is also corroborated by results showing differences in metal mobility between fish and mouse Zn-MT.
Assuntos
Metalotioneína/química , Perciformes/metabolismo , Sequência de Aminoácidos , Animais , Cádmio/metabolismo , Dicroísmo Circular , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Cinética , Espectroscopia de Ressonância Magnética , Metalotioneína/metabolismo , Camundongos , Dados de Sequência Molecular , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Resorcinóis/metabolismo , Alinhamento de Sequência , Espectrofotometria Atômica , Temperatura , Zinco/metabolismoRESUMO
The sweet protein MNEI is a construct of 96 amino acid residues engineered by linking, with a Gly-Phe dipeptide, chains B and A of monellin, a sweet protein isolated from Discoreophyllum cuminsii. Here, the solution structure of MNEI was determined on the basis of 1169 nuclear Overhauser enhancement derived distance restraints and 184 dihedral angle restraints obtained from direct measurement of three-bond spin coupling constants. The identification of hydrogen bonded NH groups was obtained by a combination of H/(2)H exchange data and NH resonance temperature coefficients derived from a series of HSQC spectra in the temperature range 278-328 K. The good resolution of the structure is reflected by the Z-score of the quality checking program in WHAT IF (-0.61). The topology of MNEI, like that of natural monellin and of SCM, another single-chain monellin, is typical of the cystatin superfamily: an alpha-helix cradled into the concave side of a five-strand anti-parallel beta-sheet. The high resolution (14 restraints/residue) 3D structure of MNEI shows close similarity to the crystal structures of natural monellin and of SCM but differs from the solution structure of SCM. The structures of SCM in the crystal and in solution differ in some of the secondary structure elements, but most of all in the relative arrangement of the elements: the four main beta-strands that surround the helix in the crystal structure of SCM, are displaced far from the helix in the solution structure of SCM. These differences were attributed to the fact that SCM is a monomer in solution and a dimer in the crystal. This result is at variance with the observation that our solution structure, like that of SCM, corresponds to a monomeric state of the protein, as demonstrated by the insensitivity of HSQC spectra to extreme dilution (down to 20 microM). On the basis of the solution structure of MNEI it is possible to propose that the main glucophores are hosted on loop L34, whereas the N-terminal and C-terminal regions host two other important interaction regions, centered around segments 6-9 and 94-96.
Assuntos
Ressonância Magnética Nuclear Biomolecular , Proteínas de Plantas/química , Engenharia de Proteínas , Edulcorantes/química , Sequência de Aminoácidos , Cistatinas/química , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Soluções , Propriedades de Superfície , TemperaturaRESUMO
H-IL-6 is a hybrid protein constructed to contain IL-6 and its soluble receptor linked by a flexible peptide chain. Here we show that H-IL-6 strongly enhances proliferation of human CD34(+)cells in serum-free liquid culture, and that the majority of the cells generated belong to the erythroid lineage, being positive for the marker Glycophorin A. Conversely, H-IL-6 does not increase the number of myeloid, CD13-positive cells. Comparable effects are observed on progenitors from cord blood and adult peripheral blood. Therefore, H-IL-6 triggers an erythroid-inducing signal in haematopoietic progenitor cells, independently from erythropoietin (EPO).
Assuntos
Antígenos CD34/biossíntese , Eritrócitos/citologia , Interleucina-6/metabolismo , Receptores de Interleucina-6/metabolismo , Antígenos CD13/metabolismo , Diferenciação Celular , Meios de Cultura Livres de Soro/metabolismo , Eritrócitos/metabolismo , Eritropoetina/farmacologia , Sangue Fetal/metabolismo , Citometria de Fluxo , Glicoforinas/biossíntese , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucócitos Mononucleares/metabolismo , Peptídeos/química , Proteínas Recombinantes de Fusão/metabolismo , Fator de Células-Tronco/farmacologia , Fatores de TempoRESUMO
Nocistatin, a new heptadecapeptide encoded in the bPNP-3 gene, has a powerful biological activity connected with the mechanisms of pain transmission. It does not bind to the opioid receptors but to another brain receptor with high affinity. In order to substantiate these novel biological data with a structural basis, we have undertaken a conformational study in solution. Proton nmr data in helicogenic solvents are consistent with a well-defined helical structure that is consistent with the nmr parameters of the C-terminal octapeptide, a shorter fragment that retains allodynia-blocking activity.
Assuntos
Analgésicos Opioides/química , Peptídeos Opioides/química , Sequência de Aminoácidos , Animais , Bovinos , Humanos , Camundongos , Dados de Sequência Molecular , Conformação Proteica , Ratos , Homologia de Sequência de Aminoácidos , SoluçõesRESUMO
Beta-endorphin is the largest natural opioid peptide. The knowledge of its bioactive conformation might be very important for the indirect mapping of the active site of opioid receptors. We have studied beta-endorphin in a variety of solution conditions with the goal of testing the intrinsic tendency of its sequence to assume a regular fold. We ran NMR experiments in water, dimethylsulfoxide and aqueous mixtures of methanol, ethylene glycol, trifluoroethanol, hexafluoracetone trihydrate and dimethylsulfoxide. The solvent in which the peptide is more ordered is the hexafluoracetone trihydrate/water mixture. The helical structure detected for beta-endorphin in this mixture at 300 K extends for the greater part of its address domain, hinting at a possible mechanism of interaction with opioid receptors: a two-point attachment involving an interaction of the helical part of the address domain (PLVTLFKNAIIKNAY) with one of the transmembrane helices and a classical interaction of the message domain (YGGF) with the receptor subsite common to all opioid receptors.
Assuntos
beta-Endorfina/química , Sequência de Aminoácidos , Animais , Dimetil Sulfóxido , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Soluções , Solventes , ÁguaRESUMO
Dynorphin A, the endogenous agonist for the kappa opioid receptor, has been studied by NMR spectroscopy in methanol, acetonitrile, DMSO and in mixtures of hexafluoroacetone/water and DMSO/water. NMR data in the DMSO/water cryomixture at 278 K are consistent with a conformer in which the N-terminal part, like the corresponding message domain of enkephalins, is poorly ordered, whereas the C-terminal part is folded in a loop centred around Pro10. The folded structure of the C-terminal part (address moiety) may shed light on the role of the essential residues Arg7, Lys11 and Lys13.
Assuntos
Dinorfinas/química , Sequência de Aminoácidos , Temperatura Baixa , Humanos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Conformação Proteica , Receptores Opioides/química , Receptores Opioides kappa/química , Receptores Opioides mu/química , Soluções , Solventes , Receptor de NociceptinaRESUMO
In order to assess the potential role of IL-6 in rheumatoid arthritis (RA), we have compared IL-6 deficient (IL-6 ko) mice and their wild-type (wt) counterpart for the capacity to develop methylated bovine serum albumin (mBSA)-induced arthritis. Our data show that IL-6 ko mice are not susceptible to antigen-induced arthritis (AIA). In fact, IL-6 ko mice treated by a standard protocol of immunization with mBSA did not develop joint swelling following intra-articular mBSA injection, nor revealed the characteristic joint lesions by histological examination. Conversely, wt mice treated according to the same protocol developed arthritis about 9 days after intra-articular injection, as detected by knee joint swelling and histological confirmation. We observed that the proliferative response of splenocytes to mBSA was impaired in ko mice following arthritis induction, as compared to the strong response observed in wt mice. Furthermore, anti-mBSA IgG levels were lower in ko mice as compared to wt mice. Finally, we show that sensitivity to AIA can be reconstituted in ko mice by subcutaneous injections of recombinant human IL-6 (rhIL-6). In addition, co-administration of IL-6 with mBSA by intra-articular injection into the joint was only partially effective in conferring sensitivity to AIA, suggesting the importance of a systemic effct for IL-6, but also that an additional role for this cytokine can be envisaged in the local inflammatory reaction during establishment of AIA.
Assuntos
Artrite Reumatoide/imunologia , Interleucina-6/imunologia , Soroalbumina Bovina/imunologia , Animais , Antígenos/imunologia , Artrite Reumatoide/genética , Bovinos , Predisposição Genética para Doença , Humanos , Interleucina-6/genética , Camundongos , Camundongos KnockoutRESUMO
Signal transducer and activator of transcription 3 (STAT3) is an important mediator of cytokine signaling, whose cDNA and protein sequences have been fully characterized. We sequenced the whole human STAT3 cDNA isolated from HepG2 cells. The new sequence determined contains 43 nucleotide changes overall, corresponding to six modifications at the amino-acid level. The revised amino-acid sequence of human STAT3 is now completely identical to the mouse sequence, except for a single amino-acid change at position 760. Thus STAT3 now results as one of the most evolutionarily conserved among known proteins. By using specific RT-PCR we could discriminate between the original sequence and the new variant. Amplification of regions within the src-homology domain 2 (SH2) of STAT3, from the RNAs of 11 different tissues or cells, revealed only the expression of the new SH2 variant. Besides, only this SH2 variant was amplified from human genomic DNA. We conclude that the new sequence we have determined in this study represents a revised sequence of hSTAT3 or, less likely, a new predominant allele.
Assuntos
Proteínas de Ligação a DNA/genética , Camundongos/genética , Transativadores/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , DNA Complementar/genética , Evolução Molecular , Genes , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , RNA Neoplásico/genética , Fator de Transcrição STAT3 , Transdução de SinaisRESUMO
BACKGROUND: Calcyclin is a member of the S100 subfamily of EF-hand Ca(2+)-binding proteins. This protein has implied roles in the regulation of cell growth and division, exhibits deregulated expression in association with cell transformation, and is found in high abundance in certain breast cancer cell lines. The novel homodimeric structural motif first identified for apo calcyclin raised the possibility that S100 proteins recognize their targets in a manner that is distinctly different from that of the prototypical EF-hand Ca2+ sensor, calmodulin. The NMR solution structure of Ca(2+)-bound calcyclin has been determined in order to identify Ca(2+)-induced structural changes and to obtain insights into the mechanism of Ca(2+)-triggered target protein recognition. RESULTS: The three-dimensional structure of Ca(2+)-bound calcyclin was calculated with 1372 experimental constraints, and is represented by an ensemble of 20 structures that have a backbone root mean square deviation of 1.9 A for the eight helices. Ca(2+)-bound calcyclin has the same symmetric homodimeric fold as observed for the apo protein. The helical packing within the globular domains and the subunit interface also change little upon Ca2+ binding. A distinct homology was found between the Ca(2+)-bound states of the calcyclin subunit and the monomeric S100 protein calbindin D9k. CONCLUSIONS: Only very modest Ca(2+)-induced changes are observed in the structure of calcyclin, in sharp contrast to the domain-opening that occurs in calmodulin and related Ca(2+)-sensor proteins. Thus, calcyclin, and by inference other members of the S100 family, must have a different mode for transducing Ca2+ signals and recognizing target proteins. This proposal raises significant questions concerning the purported roles of S100 proteins as Ca2+ sensors.
Assuntos
Proteínas de Ligação ao Cálcio/química , Cálcio/química , Conformação Proteica , Proteínas S100 , Transdução de Sinais/fisiologia , Animais , Apoproteínas/química , Calbindinas , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Dimerização , Pulmão , Modelos Moleculares , Fatores de Crescimento Neural/química , Ressonância Magnética Nuclear Biomolecular/métodos , Coelhos , Proteína G de Ligação ao Cálcio S100/química , Subunidade beta da Proteína Ligante de Cálcio S100RESUMO
To discriminate between two general models of antagonism (participation and allosteric), an opioid antagonist lacking the basic nitrogen of tyramine was designed and characterized. Cyclo-[Tyr(Me)2-Tic-], the diketopiperazine of 2,6-dimethyltyrosyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, is a partially rigid opioid antagonist; its pA2 (5.8) is one smaller than that of N,N-bisallyl-enkephalin but it has a very high binding affinity (10 nM) and has a delta selectivity (66 with respect to the binding to mu receptors) higher than that of naltrindole. The conformational state of this diketopiperazine, studied under a variety of solvent and temperature conditions by NMR and molecular dynamics, can be described in terms of only three conformers whose relative populations vary widely with solvent. Only one of the three conformers, characterized by a 90 degree arrangement of the aromatic rings of Tyr(Me)2 and Tic similar to those of rigid agonists and of the bioactive conformation of the corresponding linear antagonist, is consistent with the antagonist activity. This finding favors the participation model among the general mechanisms proposed to explain antagonism. Due to the simple composition of the conformational mixture and to the rigidity of the molecule, it is possible to propose a quantitative explanation for the discrepancy between the very high binding affinity (10 nM) and the fairly small in mouse vas deferens value (1.5 microM).
Assuntos
Desenho de Fármacos , Antagonistas de Entorpecentes/química , Animais , Cobaias , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Conformação Molecular , Antagonistas de Entorpecentes/farmacologiaRESUMO
We describe the setting up and validation of a reporter gene assay for type I IFN based on monkey Vero cells transfected with pMx-Luc, a plasmid carrying the luciferase gene under the control of the type I IFN inducible mouse Mx1 promoter. Vero cells were stably transfected with pMx-Luc and clone 3-143/5 was selected on the basis of luciferase inducibility by IFN-beta. A linear dose-response relationship was found between 1 and 16 IU/ml IFN-beta. The assay was shown to be specific for IFN-alpha and -beta as no effect by a number of other cytokines including IFN-gamma could be detected. In order to render the IFN-beta reporter gene assay protocol more suitable for routine assays, a 3 x 3 balanced parallel line assay design was applied using a 96-well luminometer for luminescence measurement. The assay was shown to be precise with a coefficient of variation of less than 9%. This assay is characterized by high precision coupled to high efficiency, as reflected by a very short assay duration (1 day), when compared to the classical cytopathic effect assays for IFNs and the previously published IFN reporter gene assay based on growth hormone measurement (Lleonart, R., Näf, D., Browning, H. and Weissmann, C. (1990) A novel, quantitative bioassay for type 1 interferon using a recombinant indicator cell line. Biotechnology 8, 1263-1267).
Assuntos
Bioensaio/métodos , Genes Reporter/imunologia , Interferon beta/genética , Animais , Chlorocebus aethiops , Células Clonais , Humanos , Luciferases/análise , Células VeroRESUMO
The sequence of deltorphin I, a delta-selective opioid agonist, has been systematically modified by inserting conformationally constrained C alpha, alpha disubstituted apolar residues in the third position. As expected, substitution of Phe with Ac6c, Ac5c and Ac3c yields analogues with decreasing but sizeable affinity. Surprisingly, substitution with Aib yields an analogue with almost the same binding affinity of the parent compound but with a greatly increased selectivity. This is the first case of a potent and very selective opioid peptide containing a single aromatic residue in the message domain, that is, only Tyr1. Here we report a detailed conformational analysis of [Aib3]deltorphin I and [Ac6c3]deltorphin I in DMSO at room temperature and in a DMSO/water cryomixture at low temperature, based on NMR spectroscopy and energy calculations. The peptides are highly structured in both solvents, as indicated by the exceptional finding of a nearly zero temperature coefficient of Val5 NH resonance. NMR data cannot be explained on the basis of a single structure but it was possible to interpret all NMR data on the basis of a few structural families. The conformational averaging was analysed by means of an original computer program that yields qualitative and quantitative composition of the mixture. Comparison of the preferred solution conformation with two rigid delta-selective agonists shows that the shapes of [Aib3]deltorphin I and [Ac6c3]deltorphin I are consistent with those of rigid agonists and that the message domain of opioid peptides can be defined only in conformational terms.
Assuntos
Oligopeptídeos/síntese química , Oligopeptídeos/metabolismo , Conformação Proteica , Receptores Opioides delta/agonistas , Animais , Bioensaio , Cobaias , Íleo , Ligantes , Espectroscopia de Ressonância Magnética , Oligopeptídeos/farmacologia , Ligação Proteica , Estrutura Terciária de Proteína , TermodinâmicaRESUMO
The pentapeptide Boc-Val-delta Phe-Gly-delta Phe-Val-OME, containing two dehydro-phenylalanine (delta Phe) residues, has been synthesized and its structure investigated. In the crystalline state, the molecule adopts a right-handed 3(10)-helical conformation stabilized by two intramolecular hydrogen bonds between CO of Val1 and NH of delta Phe4, and between CO of delta Phe2 and NH of Val5, respectively. NMR measurements are consistent with the presence of 3(10)-helical structures also in acetonitrile and dimethylsulphoxide solution: the distances between backbone protons estimated from NOE connectivities are in overall agreement with those observed in the solid state; the chemical shifts of the amide protons show the smaller temperature coefficients for the NHs that in solid state are involved in intramolecular hydrogen bonds. The CD spectra in acetonitrile, chloroform, methanol and dimethylsulphoxide display exciton couplets of bands corresponding to the delta Phe electronic transition at 280 nm; the sign of the bands is consistent with the presence of helical structures having a prevalent left-handed screw sense. Addition of 1,1,1,3,3,3-hexafluoro-propan-2-ol gives rise to the gradual appearance of a couplet of opposite sign, suggesting the helix reversal from left-handed sense to right-handed sense. The conformational behaviour is discussed on the basis of the specific sequence of the peptide.
Assuntos
Oligopeptídeos/química , Estrutura Secundária de Proteína , Dicroísmo Circular , Cristalografia por Raios X , Espectroscopia de Ressonância Magnética , Modelos Químicos , Modelos Moleculares , Conformação ProteicaRESUMO
The delta selectivity and antagonism of peptides containing L-tetrahydro-3-isoquinoline carboxylic acid (Tic) in second position can be attributed mainly to the Tyr-Tic unit. These properties can be further enhanced by substituting Tyr1 with 2,6-dimethyl-L-tyrosyl (Dmt). Dmt-Tic-NH2, Dmt-Tic-OH, Dmt-Tic-Ala-NH2 and Dmt-Tic-Ala-OH are all more active and/or selective than the corresponding [Tyr1]-parent peptides. In fact the selectivities of Dmt-Tic-OH and Dmt-Tic-Ala-OH are the highest ever recorded for opioid molecules. 1H NMR spectra in a DMSO/water mixture at 278 K reveal the presence of two similar conformers, characterised by a cis or trans Dmt-Tic bond, in all four peptides. A detailed conformational analysis in solution of Dmt-Tic-NH2 shows that these conformers have a shape very similar to that of the bioactive conformation of Tyr-Tic-NH2 and to that of naltrindole.