RESUMO
Evidence has emerged suggesting a role for the cannabinoid CB2 receptor in immune cell motility. This provides a rationale for a novel and generalized immunoregulatory role for cannabinoid CB2 receptor-specific compounds. In support of this possibility, we will review the biology of a class of cannabinoid CB2 receptor-specific inverse agonist, the triaryl bis-sulfones. We will show that one candidate, Sch.414319, is potent and selective for the cannabinoid CB2 receptor, based on profiling studies using biochemical assays for 45 enzymes and 80 G-protein coupled receptors and ion channels. We will describe initial mechanistic studies using this optimized triaryl bis-sulfone, showing that the compound exerts a broad effect on cellular protein phosphorylations in human monocytes. This profile includes the down regulation of a required phosphorylation of the monocyte-specific actin bundling protein L-plastin. We suggest that this observation may provide a mechanism for the observed activity of Sch.414319 in vivo. Our continued analysis of the in vivo efficacy of this compound in diverse disease models shows that Sch.414319 is a potent modulator of immune cell mobility in vivo, can modulate bone damage in antigen-induced mono-articular arthritis in the rat, and is uniquely potent at blocking experimental autoimmune encephalomyelitis in the rat.
Assuntos
Receptor CB2 de Canabinoide/agonistas , Sequência de Aminoácidos , Animais , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/fisiopatologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Humanos , Imunidade Celular/efeitos dos fármacos , Imunidade Celular/fisiologia , Dados de Sequência Molecular , Receptor CB2 de Canabinoide/genética , Sulfonas/química , Sulfonas/farmacologiaRESUMO
BACKGROUND AND PURPOSE: The peripheral cannabinoid receptor (CB(2)) is expressed on peripheral immune cells and is thought to have a role in the immunosuppressive effects of cannabinoids. Historically, there have been few potent, CB(2)-selective agonists to assess the contribution of CB(2) to this phenomenon. The studies presented here describe the synthesis of 8,10-bis[(2,2-dimethyl-1-oxopropyl)oxy]-11-methyl-1234-tetrahydro-6H-benzo[beta]quinolizin-6-one (Sch35966), which binds with low nanomolar potency to CB(2) in both primates and rodents. EXPERIMENTAL APPROACH: The affinity, potency and efficacy of Sch35966 and other cannabinoid ligands at CB(2) was assessed using competition binding assays vs [(3)H]CP55,940, [(35)S]GTPgammaS exchange, cAMP accumulation and cell chemotaxis assays. KEY RESULTS: We showed that Sch35966 has >450-fold selectivity for CB(2) binding vs the central cannabinoid receptor (CB(1)) in primates (humans and cynomolgus monkeys) and rodents (rats and mice). Sch35966 is an agonist as it effectively inhibited forskolin-stimulated cAMP synthesis in CHO-hCB(2) cells, stimulated [(35)S]GTPgammaS exchange and directed chemotaxis in cell membranes expressing CB(2). In all species examined, Sch35966 was more potent, more efficacious and more selective than JWH-015 (a commonly used CB(2)-selective agonist). CONCLUSIONS AND IMPLICATIONS: Taken together, the data show that Sch35966 is a potent and efficacious CB(2)-selective agonist in rodents and primates.
Assuntos
Ligação Competitiva , Quinolizinas/farmacologia , Receptor CB2 de Canabinoide/agonistas , Animais , Células CHO , Membrana Celular , Quimiotaxia , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Cicloexanóis , Dronabinol/análogos & derivados , Dronabinol/farmacologia , Guanosina 5'-O-(3-Tiotrifosfato) , Humanos , Indóis/farmacologia , Macaca fascicularis , Camundongos , Quinolizinas/síntese química , Ratos , Especificidade da EspécieRESUMO
Interferon-gamma (IFN-gamma) and its receptor complex are dimeric and bilaterally symmetric. We created mutants of IFN-gamma that bind only one IFN-gammaR1 chain per dimer molecule (called a monovalent IFN-gamma) to see if the interaction of IFN-gamma with one-half of the receptor complex is sufficient for bioactivity. Mutating a receptor-binding sequence in either AB loop of a covalent dimer of IFN-gamma yielded two monovalent IFN-gammas, gamma(m)-gamma and gamma-gamma(m), which cross-link to only a single soluble IFN-gammaR1 molecule in solution and on the cell surface. Monovalent IFN-gamma competes fully with wild type IFN-gamma for binding to U937 cells but only at a greater than 100-fold higher concentration than wild type IFN-gamma. Monovalent IFN-gamma had anti-vesicular stomatitis virus activity and antiproliferative activity, and it induced major histocompatibility complex class I and class II (HLA-DR) expression. In contrast, the maximal levels of activated Stat1alpha produced by monovalent IFN-gammas after 15 min were never more than half of those produced by either wild type or covalent IFN-gammas in human cell lines. These data indicate that while monovalent IFN-gamma activates only one-half of a four-chain receptor complex, this is sufficient for Stat1alpha activation, major histocompatibility complex class I surface antigen induction, and antiviral and antiproliferative activities. Thus, while interaction with both halves of the receptor complex is required for high affinity binding of IFN-gamma and efficient signal transduction, interaction with only one-half of the receptor complex is sufficient to initiate signal transduction.
Assuntos
Interferon gama/metabolismo , Receptores de Interferon/metabolismo , Transdução de Sinais , Sequência de Bases , Biopolímeros , Linhagem Celular , Cromatografia em Gel , Primers do DNA , Dimerização , Humanos , Interferon gama/química , Ligação Proteica , Receptor de Interferon gamaRESUMO
Crystal structures of human endothelial nitric oxide synthase (eNOS) and human inducible NOS (iNOS) catalytic domains were solved in complex with the arginine substrate and an inhibitor S-ethylisothiourea (SEITU), respectively. The small molecules bind in a narrow cleft within the larger active-site cavity containing heme and tetrahydrobiopterin. Both are hydrogen-bonded to a conserved glutamate (eNOS E361, iNOS E377). The active-site residues of iNOS and eNOS are nearly identical. Nevertheless, structural comparisons provide a basis for design of isozyme-selective inhibitors. The high-resolution, refined structures of eNOS (2.4 A resolution) and iNOS (2.25 A resolution) reveal an unexpected structural zinc situated at the intermolecular interface and coordinated by four cysteines, two from each monomer.
Assuntos
Sequência Conservada , Óxido Nítrico Sintase/química , Sequência de Aminoácidos , Animais , Arginina/química , Sítios de Ligação , Biopterinas/análogos & derivados , Biopterinas/metabolismo , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Heme/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , Estrutura Secundária de Proteína , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
We have purified a protease with characteristics of TNFalpha convertase from bovine spleen membranes. Peptide sequencing of the purified protein identified it as ADAM 10 (Genbank accession no. Z21961). This metalloprotease cleaves a recombinant proTNFalpha substrate to mature TNFalpha, and can cleave a synthetic peptide substrate to yield the mature TNFalpha amino terminus in vitro. The enzyme is sensitive to a hydroxamate inhibitor of MMPs, but insensitive to phosphoramidon. In addition, cloned ADAM 10 mediates proTNFalpha processing in a processing-incompetent cell line.
Assuntos
Proteínas de Membrana/isolamento & purificação , Metaloendopeptidases/isolamento & purificação , Baço/enzimologia , Proteínas ADAM , Proteína ADAM10 , Proteína ADAM17 , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide , Animais , Bovinos , Linhagem Celular , Clonagem Molecular , Ácido Edético/farmacologia , Inibidores Enzimáticos/farmacologia , Glicopeptídeos/farmacologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Dados de Sequência Molecular , Precursores de Proteínas/metabolismo , Proteínas Recombinantes/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
We have expressed active full-length human inducible nitric oxide synthase (iNOS) in E. coli. Expression required co-expression with calmodulin, a particularly tight-binding cofactor. The extracts also required tetrahydrobiopterin to display activity. Specific activity of the purified recombinant iNOS was similar to iNOS purified from murine macrophages. This result indicates that no special processing events unique to eucaryotic cells are necessary for iNOS activity.
Assuntos
Encéfalo/enzimologia , Isoenzimas/biossíntese , Óxido Nítrico Sintase/biossíntese , Sequência de Bases , Biopterinas/análogos & derivados , Biopterinas/farmacologia , Calmodulina/biossíntese , Calmodulina/fisiologia , Cromatografia em Gel , Cromatografia por Troca Iônica , Clonagem Molecular , Primers do DNA , DNA Complementar , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Expressão Gênica/efeitos dos fármacos , Humanos , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Cinética , Dados de Sequência Molecular , Peso Molecular , Óxido Nítrico Sintase/isolamento & purificação , Óxido Nítrico Sintase/metabolismo , Reação em Cadeia da Polimerase , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
The crystal structure of interferon-gamma bound to the extracellular fragment of its high-affinity cell-surface receptor reveals the first view of a class-2 cytokine receptor-ligand complex. In the complex, one interferon-gamma homodimer binds two receptor molecules. Unlike the class-1 growth hormone receptor complex, the two interferon-gamma receptors do not interact with one another and are separated by 27 A. Upon receptor binding, the flexible AB loop of interferon-gamma undergoes a conformational change that includes the formation of a 3(10) helix.
Assuntos
Interferon gama/química , Receptores de Interferon/química , Gráficos por Computador , Cristalografia por Raios X , Citocinas/química , Escherichia coli , Glicosilação , Hormônio do Crescimento/química , Humanos , Conformação Proteica , Receptores da Somatotropina/química , Proteínas Recombinantes/química , Solubilidade , Receptor de Interferon gamaRESUMO
Characterization of murine-human hybrid interferon-gamma (IFN-gamma) molecules suggests that substitution of the peptide connecting the A and B helices in human IFN-gamma with the murine sequence significantly blocks the protein's binding to the human interferon-gamma receptor. Mutagenesis showed that this effect is localized to the central part of this A-B loop peptide, particularly Ser20, Asp21, Val22, and Ala23. One mutant, IFN-gamma/A23E,D24E,N25K, was examined by NMR. This "EEK" mutation does not significantly alter the conformation of interferon-gamma, suggesting that the effects of these mutations are not the result of global conformational changes. The A-B loop is near histidine 111, a residue previously shown to be important in receptor-ligand interaction (Lunn, C. A., Fossetta, J., Dalgarno, D., Murgolo, N., Windsor, W., Zavodny, P. J., Narula, S. K., and Lundell, D. (1992) Protein Eng. 5, 253-257). We show that copper forms a complex between histidine 19 in the A-B loop and histidine 111. This metal complex lacks the ability to interact with the interferon-gamma receptor. These results suggest that the A-B loop contains important structural information needed for receptor-ligand binding and hence biological activity of human interferon-gamma.
Assuntos
Interferon gama/metabolismo , Receptores de Interferon/metabolismo , Sequência de Aminoácidos , Animais , Análise Mutacional de DNA , Relação Dose-Resposta a Droga , Humanos , Interferon gama/genética , Metais/farmacologia , Camundongos , Dados de Sequência Molecular , Ligação Proteica/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Especificidade da Espécie , Relação Estrutura-Atividade , Receptor de Interferon gamaRESUMO
We have constructed and expressed a covalently linked head to tail dimer of human interferon-gamma (IFN-gamma) in which two monomers are joined head to tail via a rigid peptide hinge using genetic engineering techniques. The hinge was derived from the human immunoglobin IgA1 sequence (Hallewell, R.A., Laria, I., Tabrizi, A., Carlin, G., Getzoff, E.D., Tainer, J.A., Cousens, L.S., and Mullenbach, G.T. (1989) J. Biol. Chem. 264, 5260-5268). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the polypeptide produced by this construction migrates as a 30,000 polypeptide species. The protein elutes as a single species by molecular sieve chromatography under native conditions. The covalently linked dimer exhibits one-half the antiviral activity of native dimeric IFN-gamma; receptor binding assays show the covalently linked dimer binds to the IFN-gamma receptor with one-half the avidity of native IFN-gamma. This difference is not due to conformational differences between the two molecules, as the aromatic region of the NMR spectrum of the purified covalently linked dimer is identical with that of the wild type protein. From these data, we suggest that human IFN-gamma associates in a head to tail dimer in its active configuration. Regions of IFN-gamma are contiguous with the amino and carboxyl termini and are obscured by the hinge peptide in the covalently linked dimer. Our studies demonstrate that these regions may be important for receptor-ligand interaction.
Assuntos
Interferon gama/química , Receptores Imunológicos/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Escherichia coli/genética , Genes Sintéticos , Humanos , Imunoglobulina A/química , Interferon gama/genética , Interferon gama/metabolismo , Interferon gama/farmacologia , Substâncias Macromoleculares , Espectroscopia de Ressonância Magnética/métodos , Dados de Sequência Molecular , Conformação Proteica , Receptores de Interferon , Proteínas RecombinantesRESUMO
We have identified a mutation of human gamma-interferon (IFN gamma) causing a temperature-sensitive phenotype. We used a randomized oligonucleotide to mutagenize a synthetic human IFN gamma gene, then screened the resulting mutants produced in Escherichia coli for proteins with altered biological activity. One mutant protein selected for detailed characterization exhibited less than 0.3% of the specific biological activity of native IFN gamma in an antiviral activity assay performed at 37 degrees C. However, the protein bound the human IFN gamma receptor with native efficiency at 4 degrees C. Sequencing the plasmid DNA encoding this protein showed that the mutation changed the lysine residue at amino acid 43 to glutamic acid (IFN gamma/K43E). Site-specific mutagenesis at amino acid 43 showed that this protein's phenotype resulted from positioning a negative charge at position 43. Structural characterization of IFN gamma/K43E using CD demonstrated that the protein had native conformation at 25 degrees C, but assumed an altered conformation at 37 degrees C. IFN gamma/K43E in this altered conformation bound poorly to the IFN gamma receptor at 37 degrees C, providing a rationale for the mutant's decreased antiviral activity.
Assuntos
Interferon gama/química , Sequência de Aminoácidos , Dicroísmo Circular , Efeito Citopatogênico Viral , Escherichia coli/genética , Humanos , Interferon gama/genética , Interferon gama/metabolismo , Interferon gama/farmacologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligonucleotídeos/genética , Fenótipo , Conformação Proteica , Engenharia de Proteínas , Receptores de Interferon/metabolismo , Proteínas Recombinantes , TemperaturaRESUMO
We identified a single amino acid mutation that abolished the bioactivity of human IFN gamma. The mutation was identified by screening a mutagenized IFN gamma expression library for molecules with altered biological activity. The mutant protein was expressed at high levels in Escherichia coli, and remained soluble upon purification. However, the protein was completely inactive in all IFN gamma assays investigated, exhibiting less than 0.0006% of the specific activity of native IFN gamma antiviral activity. Sequencing the plasmid DNA encoding this mutant protein showed that the histidine at position 111 of native human IFN gamma is changed to aspartic acid (IFN gamma/H111D). Other mutations at this site showed that only hydrophobic amino acids at position 111 maintain significant, though low, biological activity. Structural characterization of the IFN gamma/H111D protein by NMR as well as CD spectroscopy demonstrated that the protein has limited conformational differences from native IFN gamma. Models of the X-ray crystal structure of human IFN gamma [Ealick, P.E., W.J. Cook, S. Vijay-Kumar, M. Carson, T.L. Nagabhushan, P.P. Trotta and C.E. Bugg (1991) Science, 252, 698-702] suggest that this histidine residue is located at a severe 55 degrees bend in the C-terminal F helix. We conclude that H111 lies within or affects the receptor binding domain of human IFN gamma.
Assuntos
Interferon gama/química , Sequência de Aminoácidos , Dicroísmo Circular , Efeito Citopatogênico Viral , Escherichia coli/genética , Humanos , Interferon gama/genética , Interferon gama/metabolismo , Interferon gama/farmacologia , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Mutação , Plasmídeos/genética , Conformação Proteica , Engenharia de Proteínas , Receptores de Interferon/metabolismo , Proteínas RecombinantesRESUMO
Human IL-4 (hIL-4) has been cloned from a human T cell line based on its homology to the murine IL-4 cDNA sequence. We have compared cytoplasmic and extra-cytoplasmic expression of this basic protein in Escherichia coli using various combinations of promoters, replicons and host strains. Strains producing a cytoplasmic product were most successful at heterologous protein expression, producing up to 500 mg/l of an inactive aggregated form of the protein. The biological activity of the protein could be restored by refolding the protein with guanidine hydrochloride and glutathione giving a specific activity identical to that of IL-4 derived from CHO cell lines stably transformed with an hIL-4 expression plasmid. Strains designed to secrete human IL-4 into the periplasmic space produced far less protein (approximately 5 mg/l). However, a significant fraction of this protein was detected in the culture medium. This fraction appeared to be soluble after ultracentrifugation, and demonstrated high specific activity without refolding. Leakage of heterologous protein into the culture medium may be a viable way to recover biologically active products without relying on the denaturation and refolding in vitro that can, at times, yield incorrectly folded gene product.
Assuntos
Escherichia coli/genética , Interleucina-4/biossíntese , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Citoplasma/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Humanos , Interleucina-4/genética , Interleucina-4/metabolismo , Dados de Sequência Molecular , Plasmídeos , Conformação Proteica , Solubilidade , Especificidade da EspécieRESUMO
To investigate structural characteristics important for eukaryotic signal peptide function in vivo, a hybrid gene with interchangeable signal peptides was cloned into yeast. The hybrid gene encoded nine residues from the amino terminus of the major Escherichia coli lipoprotein, attached to the amino terminus of the entire mature E. coli beta-lactamase sequence. To this sequence were attached sequences encoding the nonmutant E. coli lipoprotein signal peptide, or lipoprotein signal peptide mutants lacking an amino-terminal cationic charge, with shortened hydrophobic core, with altered potential helicity, or with an altered signal-peptide cleavage site. These signal-peptide mutants exhibited altered processing and secretion in E. coli. Using the GAL10 promoter, production of all hybrid proteins was induced to constitute 4-5% of the total yeast protein. Hybrid proteins with mutant signal peptides that show altered processing and secretion in E. coli, were processed and translocated to a similar degree as the non-mutant hybrid protein in yeast (approximately 36% of the total hybrid protein). Both non-mutant and mutant signal peptides appeared to be removed at the same unique site between cysteine 21 and serine 22, one residue from the E. coli signal peptidase II processing site. The mature lipo-beta-lactamase was translocated across the cytoplasmic membrane into the yeast periplasm. Thus the protein secretion apparatus in yeast recognizes the lipoprotein signal sequence in vivo but displays a specificity towards altered signal sequences which differs from that of E. coli.
Assuntos
Clonagem Molecular , Escherichia coli/genética , Genes Bacterianos , Genes , Sinais Direcionadores de Proteínas/genética , Saccharomyces cerevisiae/genética , beta-Lactamases/genética , Sequência de Aminoácidos , Dados de Sequência Molecular , Plasmídeos , Sinais Direcionadores de Proteínas/metabolismoRESUMO
Hybrid proteins were constructed by coupling beta-lactamase to the signal sequence (plus nine amino acids) of selected mutant prolipoproteins of Escherichia coli. The mutant prolipoprotein signal peptides contained lesions in two structural domains of the signal peptide, the basic amino-terminal domain and the hydrophobic core domain. We then compared the processing and localization of the mutant prolipo-beta-lactamases to the processing and localization of the comparable mutant prolipoproteins. We show that a mutant signal sequence with an anionic amino terminus exhibits similar limitations in the processing of prolipo-beta-lactamase as previously observed in prolipoprotein. Deletion of four hydrophobic residues from hydrophobic core results in a signal peptide which slowly translocates a fraction of the total mutant hybrid protein synthesized. This signal peptide was previously shown to translocate lipoprotein efficiently. Alteration of this hydrophobic core, which stimulated synthesis of mutant prolipoproteins, does not stimulate synthesis of prolipo-beta-lactamase. Finally mutations that slowed processing of prolipoprotein by affecting the proposed helical structure of the signal peptide had no significant effect on the processing of prolipo-beta-lactamase. These results suggest that the positively charged amino-terminal domain of the signal peptide has a common role in protein secretion regardless of the secretory protein. On the other hand, other domains of the signal peptide exhibit different phenotypes when the secretory protein is changed.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Lipoproteínas , Mutação , Precursores de Proteínas/genética , Sinais Direcionadores de Proteínas/genética , beta-Lactamases/genética , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/biossíntese , Proteínas da Membrana Bacteriana Externa/metabolismo , Enzimas de Restrição do DNA , Escherichia coli/enzimologia , Plasmídeos , Conformação Proteica , Precursores de Proteínas/biossíntese , Sinais Direcionadores de Proteínas/metabolismo , beta-Lactamases/biossínteseRESUMO
The intracellular localization of thioredoxin in Escherichia coli was determined by immunoelectron microscopy and correlated to previous biochemical data which had suggested that thioredoxin resides at inner-outer membrane adhesion sites. Since a considerable amount of thioredoxin was lost during preparation of cells for electron microscopy, we immobilized the protein with the heterobifunctional photoactivatable cross-linker p-azidophenacylbromide before the cells were fixed with aldehyde and embedded in Lowicryl K4M. Thin sections were labeled with affinity-purified antithioredoxin antiserum and protein A-gold complexes. Densities of immunolabel in a designated membrane-associated area and in the rest of the cytoplasm were compared and the data were statistically evaluated. Wild-type strain W3110 and strain SK3981, an overproducer of thioredoxin, exhibited increased labeling at the inner membrane and its adjacent cytoplasmic area. In contrast, the more centrally located cytoplasm of both strains showed much lower label density. This label distribution did not change with cell growth or in the stationary phase. Immunolabel was often found at bridges between the inner and outer membranes; this result is consistent with a model which places at least a portion of the thioredoxin at membrane adhesion sites, corresponding to an osmotically sensitive cytoplasmic compartment bounded by a hybrid inner-outer membrane (C.A. Lunn and V. Pigiet, J. Biol. Chem. 257:11424-11430, 1982; C.A. Lunn and V. Pigiet, J. Biol. Chem. 261:832-838, 1986). Specific label was absent in the periplasmic space.
Assuntos
Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Tiorredoxinas/metabolismo , Azidas , Sítios de Ligação , Compartimento Celular , Membrana Celular/metabolismo , Reagentes de Ligações Cruzadas , Escherichia coli/ultraestrutura , Técnicas de Imunoadsorção , Microscopia Eletrônica , FotoquímicaRESUMO
The ability of Escherichia coli thioredoxin to protect cells from lethal amounts of gamma radiation was tested using bacterial strains engineered to contain different amounts of thioredoxin per cell. Cells grown to late stationary phase demonstrated a decreasing sensitivity to gamma-radiation with increasing amounts of thioredoxin per cell. Exponentially growing cells were equally sensitive to the gamma-radiation regardless of the intracellular concentration of thioredoxin. Cells exhibiting the radiation-resistant phenotype in the stationary phase reverted to the radiation-sensitive phenotype when diluted into fresh growth medium. These results suggest that thioredoxin can protect cells from gamma-radiation under certain metabolic conditions.
Assuntos
Bactérias/efeitos da radiação , Proteínas de Bactérias/fisiologia , Tiorredoxinas/fisiologia , Tolerância a RadiaçãoRESUMO
Rabbit polyclonal antibody was raised to a chemically synthesized nonapeptide (Trp-Ala-Glu-Trp-Cys-Gly-Pro-Cys-Lys) corresponding to the active-site sequence of Escherichia coli thioredoxin. The antiserum efficiently inhibited thioredoxin activity in the standard thioredoxin reductase/NADPH coupled assay. This inhibition was blocked by preincubation of the antiserum with the nonapeptide. Tight association of the E. coli thioredoxin to the active-site antibody required SDS denaturation. These results suggest that thioredoxin reductase (NADPH: oxidized-thioredoxin oxidoreductase, EC 1.6.4.5) alters the conformation of thioredoxin sufficiently to permit binding to the antibody. The antiserum bound to plant and liver thioredoxins. Bovine pancreatic trypsin inhibitor, whose active site (Gly-Pro-Cys-Lys) is homologous to that of thioredoxin, also competes for the active-site antibody. This result led to experiments showing that thioredoxin can inhibit the digestion of cytochrome c by trypsin. The ability of thioredoxin to act as a trypsin inhibitor analogue provides a rationale for thioredoxin's resistance to digestion by trypsin.
Assuntos
Proteínas de Bactérias , Tiorredoxinas , Inibidores da Tripsina , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/imunologia , Ligação Competitiva , Bovinos , Pâncreas/enzimologia , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Relação Estrutura-Atividade , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxinas/imunologiaAssuntos
Bactérias/genética , Proteínas de Bactérias/genética , Clonagem Molecular , Genes Bacterianos , Genes , Vetores Genéticos , Sequência de Aminoácidos , Bacillus subtilis/genética , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Conformação Proteica , Processamento de Proteína Pós-TraducionalRESUMO
Thioredoxin was cross-linked to a membrane fraction in vivo using the heterobifunctional photoreactive cross-linking reagent p-azidophenacyl bromide, chosen to couple thioredoxin via its highly reactive thiol. Under mild reaction conditions, a significant amount of thioredoxin (30%) was rapidly cross-linked to the crude membrane fraction. The cross-linking reaction was selective, with thioredoxin purified 15-fold in the cross-linked membrane fraction. Membrane fractionation studies showed that thioredoxin associated with the inner membrane and with a hybrid membrane fraction. This hybrid membrane fraction banded at a density between the inner and outer membranes. This result is consistent with the localization of thioredoxin in association with the bacterial membrane adhesion sites first described by Bayer (Bayer, M. (1968) J. Gen. Microbiol. 53, 395-404). Association of thioredoxin with the membrane adhesion sites defines a structure corresponding to the osmotically sensitive cytoplasmic compartment (Lunn, C. A., and Pigiet, V. (1982) J. Biol. Chem. 257, 11424-11430).