RESUMEN

The primary virulence factor of Bacillus anthracis is a secreted zinc-dependent metalloprotease toxin known as lethal factor (LF) that is lethal to the host through disruption of signaling pathways, cell destruction, and circulatory shock. Inhibition of this proteolytic-based LF toxemia could be expected to provide therapeutic value in combination with an antibiotic during and immediately after an active anthrax infection. Herein is shown the crystal structure of an intimate complex between a hydroxamate, (2R)-2-[(4-fluoro-3-methylphenyl)sulfonylamino]-N-hydroxy-2-(tetrahydro-2H-pyran-4-yl)acetamide, and LF at the LF-active site. Most importantly, this molecular interaction between the hydroxamate and the LF active site resulted in (i) inhibited LF protease activity in an enzyme assay and protected macrophages against recombinant LF and protective antigen in a cell-based assay, (ii) 100% protection in a lethal mouse toxemia model against recombinant LF and protective antigen, (iii) approximately 50% survival advantage to mice given a lethal challenge of B. anthracis Sterne vegetative cells and to rabbits given a lethal challenge of B. anthracis Ames spores and doubled the mean time to death in those that died in both species, and (iv) 100% protection against B. anthracis spore challenge when used in combination therapy with ciprofloxacin in a rabbit "point of no return" model for which ciprofloxacin alone provided 50% protection. These results indicate that a small molecule, hydroxamate LF inhibitor, as revealed herein, can ameliorate the toxemia characteristic of an active B. anthracis infection and could be a vital adjunct to our ability to combat anthrax.

Asunto(s)

Carbunco/tratamiento farmacológico , Antígenos Bacterianos/toxicidad , Bacillus anthracis/patogenicidad , Toxinas Bacterianas/antagonistas & inhibidores , Toxinas Bacterianas/toxicidad , Ácidos Hidroxámicos/farmacología , Modelos Moleculares , Animales , Antígenos Bacterianos/metabolismo , Bacillus anthracis/metabolismo , Toxinas Bacterianas/metabolismo , Ciprofloxacina/uso terapéutico , Cristalografía , Pruebas Inmunológicas de Citotoxicidad , Cartilla de ADN , Quimioterapia Combinada , Ácidos Hidroxámicos/metabolismo , Ácidos Hidroxámicos/uso terapéutico , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Conejos

RESUMEN

Major histocompatabilty (MHC) proteins rely heavily on peptide backbone recognition for ligation. Nonetheless, modifications to the polyamide backbone of a tetrapeptide ligand can be made without abrogating binding.

Asunto(s)

Antígenos de Histocompatibilidad Clase II/efectos de los fármacos , Antígenos de Histocompatibilidad Clase II/metabolismo , Péptidos/química , Péptidos/farmacología , Sitios de Unión , Antígeno HLA-DR1/efectos de los fármacos , Antígeno HLA-DR1/metabolismo , Hemaglutininas/química , Concentración 50 Inhibidora , Lactamas/química , Ligandos , Imitación Molecular , Estructura Molecular , Fosfoproteínas/efectos de los fármacos , Fosfoproteínas/metabolismo , Relación Estructura-Actividad , Factores de Transcripción/efectos de los fármacos , Factores de Transcripción/metabolismo

RESUMEN

Tetrapeptide derived major histocompatability (MHC) II ligands have been developed that contain no unadulterated peptide bonds. These are the 'least peptidic' ligands for any MHC protein yet reported.

Asunto(s)

Antígenos de Histocompatibilidad Clase II/química , Antígenos de Histocompatibilidad Clase II/metabolismo , Péptidos/química , Diseño de Fármacos , Antígenos de Histocompatibilidad Clase II/efectos de los fármacos , Concentración 50 Inhibidora , Lactamas/química , Ligandos , Imitación Molecular , Relación Estructura-Actividad

RESUMEN

Two homogeneous proximity assays for tyrosine kinases, scintillation proximity assay (SPA) and homogeneous time-resolved fluorescence (HTRF), have been developed and compared. In both formats, the kinase assay was performed using biotinylated peptide substrate, ATP ([33P]ATP in the case of SPA), and tyrosine kinase in a 96-well assay format. After the kinase reaction was stopped, streptavidin-coated SPA beads or europium cryptate-labeled anti-phosphotyrosine antibody and streptavidin-labeled allophycocyanin were added as detection reagents for SPA or HTRF assays, respectively. Since the assay signal was detected only when the energy donor (radioactivity for SPA, Eu for HTRF) and the energy acceptor molecules (SPA beads for SPA, allophycocyanin for HTRF) were in close proximity, both assays required no wash or liquid transfer steps. This homogeneous ("mix-and-measure") nature allows these assays to be much simpler, more robust, and easier to automate than traditional protein kinase assays, such as a filter binding assay or ELISA. Both assays have been miniaturized to a 384-well format to reduce the assay volume, thereby saving the valuable screening samples as well as assay reagents, and automated using automated pipetting stations to increase the assay throughput. Several advantages and disadvantages for each assay are described.

Asunto(s)

Proteínas Tirosina Quinasas/análisis , Secuencia de Aminoácidos , Animales , Técnicas de Química Analítica/métodos , Drosophila , Humanos , Indicadores y Reactivos , Células Jurkat , Miniaturización , Datos de Secuencia Molecular , Especificidad por Sustrato , Volumetría

RESUMEN

A homogeneous time-resolved fluorescence (HTRF) assay has been developed for human immunodeficiency viral (HIV) protease. The assay utilizes a peptide substrate, differentially labeled on either side of the scissile bond, to bring two detection components, streptavidin-cross-linked XL665 (SA/XL665) and a europium cryptate (Eu(K))-labeled antiphosphotyrosine antibody, into proximity allowing fluorescence resonance energy transfer (FRET) to occur. Cleavage of the doubly labeled substrate by HIV protease precludes complex formation, thereby decreasing FRET, and allowing enzyme activity to be measured. Potential substrates were evaluated by HTRF with the best results being obtained using (LCB)K4AVSQNbeta-NapPIVpYA(NH2) and Eu(K)-pY20 where the peptide titrated with an EC50 of 7.7 +/- 0.3 nM under optimized detection conditions. Using these HTRF detection conditions, HIV protease cleaved the substrate in 50 mM NaOAc, 150 mM KF, 0.05% Tween 20, pH 5.5, with apparent first-order kinetics with a Km of 37.8 +/- 8.7 microM and a kcat of 0.95 +/- 0.07 s-1. Examination of the first-order rate constant versus enzyme concentration suggested a Kd of 9.4 +/- 2.7 nM for the HIV protease monomer-dimer equilibrium. The HTRF assay was also utilized to measure the inhibition of the enzyme by two known inhibitors.

Asunto(s)

Técnica del Anticuerpo Fluorescente , Proteasa del VIH/análisis , Secuencia de Aminoácidos , Anticuerpos , Técnicas de Química Analítica/métodos , VIH/enzimología , Datos de Secuencia Molecular , Fosfotirosina/inmunología , Especificidad por Sustrato , Factores de Tiempo

RESUMEN

UNLABELLED: Ligand-dependent interactions between nuclear receptors and members of a family of nuclear receptor coactivators are associated with transcriptional activation. Here we used fluorescence resonance energy transfer (FRET) as an approach for detecting and quantitating such interactions. Using the ligand binding domain (LBD) of peroxisome proliferator-activated receptor (PPARgamma) as a model, known agonists (thiazolidinediones and delta12, 14-PGJ2) induced a specific interaction resulting in FRET between the fluorescently labeled LBD and fluorescently labeled coactivators [CREB-binding protein (CBP) or steroid receptor coactivator-1 (SRC-1)]. Specific energy transfer was dose dependent; individual ligands displayed distinct potency and maximal FRET profiles that were identical when results obtained using CBP vs. SRC-1 were compared. In addition, half-maximally effective agonist concentrations (EC59s) correlated well with reported results using cell-based assays. A site-directed AF2 mutant of PPARgamma (E471A) that abrogated ligand-stimulated transcription in transfected cells also failed to induce ligand-mediated FRET between PPARgamma LBD and CBP or SRC-1. Using estrogen receptor (ERalpha) as an alternative system, known agonists induced an interaction between ERalpha LBD and SRC-1, whereas ER antagonists disrupted agonist-induced interaction of ERalpha with SRC-1. In the presence of saturating agonist concentrations, unlabeled CBP or SRC-1 was used to compete with fluorescently labeled coactivators with saturation kinetics. Relative affinities for the individual receptor-coactivator pairs were determined as follows: PPARgamma-CBP = ERalpha-SRC-1 > PPARgamma-SRC-1 >> ERalpha-CBP. CONCLUSIONS: 1) FRET-based coactivator association is a novel approach for characterizing nuclear receptor agonists or antagonists; individual ligands display potencies that are predictive of in vivo effects and distinct profiles of maximal activity that are suggestive of alternative receptor conformations. 2) PPARgamma interacts with both CBP and SRC-1; transcriptional activation and coactivator association are AF2 dependent. 3) Nuclear receptor LBDs have distinct affinities for individual coactivators; thus, PPARgamma has a greater apparent affinity for CBP than for SRC-1, whereas ERalpha interacts preferentially with SRC-1 but very weakly with CBP.

Asunto(s)

Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Estrógenos/metabolismo , Espectrometría de Fluorescencia/métodos , Tiazolidinedionas , Factores de Transcripción/metabolismo , Animales , Sitios de Unión , Proteína de Unión a CREB , Cricetinae , Transferencia de Energía , Receptor alfa de Estrógeno , Histona Acetiltransferasas , Proteínas Nucleares/metabolismo , Coactivador 1 de Receptor Nuclear , Pioglitazona , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores Citoplasmáticos y Nucleares/genética , Rosiglitazona , Tiazoles/farmacología , Transactivadores/metabolismo , Factores de Transcripción/agonistas , Factores de Transcripción/genética

RESUMEN

The calcium- and calmodulin-activated protein phosphatase calcineurin (CN) is the target for the immunosuppressive drugs FK506 and cyclosporin A (CsA) when bound to their intracellular receptor proteins, the immunophilins known as FK506-binding protein (FKBP) and cyclophilin A (CypA), respectively. Investigation of the reaction kinetics for inhibition of CN using progress curves of [33P]phosphopeptide hydrolysis revealed slow-binding inhibition by the FK506 . FKBP complex. Final steady-state velocities were extracted by curve fitting over a range of substrate and inhibitor concentrations; the data fit well to a simple competitive inhibition model with a Ki of 14 nM for the FK506 . FKBP complex. The FKBP complex with L-732,531, an analog of FK506 containing a hydroxyethylindole substituent, was significantly more potent than FK506 x FKBP and was investigated in greater detail. The hyperbolic dependencies of the initial velocities and the first-order rate constants for the approach to steady state upon the concentration of L-732,531 x FKBP were consistent with a two-step inhibition mechanism in which the initial E x I complex slowly isomerizes to a more stable E x I* form. The reverse isomerization rate constant with L-732,531 . FKBP was markedly slower than that with FK506 x FKBP and is likely responsible for the higher affinity of the former for CN. Inhibition of CN by the CsA x CypA complex was not time-dependent, but the data did conform to a competitive inhibition model like FK506 x FKBP. These results are consistent with the hypothesis that both classes of drug x immunophilin complexes interact with a common locus on CN which excludes phosphopeptide binding in the enzyme's active site.

Asunto(s)

Inhibidores de la Calcineurina , Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Choque Térmico/metabolismo , Inmunosupresores/metabolismo , Isomerasa de Peptidilprolil/metabolismo , Secuencia de Aminoácidos , Animales , Unión Competitiva/efectos de los fármacos , Calcineurina/metabolismo , Proteínas Portadoras/farmacología , Ciclosporina/metabolismo , Ciclosporina/farmacología , Proteínas de Unión al ADN/farmacología , Proteínas de Choque Térmico/farmacología , Humanos , Inmunosupresores/farmacología , Cinética , Modelos Químicos , Datos de Secuencia Molecular , Unión Proteica/efectos de los fármacos , Ratas , Tacrolimus/análogos & derivados , Tacrolimus/metabolismo , Tacrolimus/farmacología , Proteínas de Unión a Tacrolimus

RESUMEN

p38 has been shown to be a critical enzyme in the pro-inflammatory cytokine pathway and is a member of the mitogen-activated protein (MAP) kinase family. While the details for p38 activation and subsequent signal transduction have begun to be elucidated, little is known about the kinetic mechanism for p38. In this study, we have determined the kinetic mechanism for p38 MAP kinase. Data from initial velocity patterns in the presence and absence of a dead-end inhibitor and two triarylimidazole p38 inhibitors were consistent with an ordered sequential mechanism for p38 with protein substrate, glutathione S-transferase-activating transcription factor 2 (GST-ATF2), binding before ATP. The ATP analog, adenylyl methylenediphosphonate (AMP-PCP), and two triarylimidazoles were competitive inhibitors versus ATP and uncompetitive inhibitors versus GST-ATF2. Equilibrium binding studies utilizing a tritiated ATP-competitive inhibitor were also consistent with this mechanism and suggest an inability of ATP to bind to p38 in the absence of protein substrate. Moreover, the Michaelis constant for GST-ATF2 was 12-fold greater than the dissociation constant, indicating that the binding of ATP affected the binding of GST-ATF2. An ordered sequential mechanism with protein substrate binding first is unique to p38 compared to cyclic AMP-dependent protein kinase (cAPK) and most tyrosine kinases and helps to explain the interaction between enzyme, substrates, and inhibitors.

Asunto(s)

Adenosina Trifosfato/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Activadas por Mitógenos , Factores de Transcripción/metabolismo , Factor de Transcripción Activador 2 , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Clonación Molecular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Inhibidores Enzimáticos/farmacología , Glutatión Transferasa/genética , Humanos , Imidazoles/farmacología , Cinética , Estructura Molecular , Fosforilación , Unión Proteica , Piridinas/farmacología , Proteínas Recombinantes de Fusión/metabolismo , Linfocitos T/enzimología , Factores de Transcripción/genética , Proteínas Quinasas p38 Activadas por Mitógenos

RESUMEN

Methodology has been developed which gives a specific measure of the interaction of an SH2 domain with a phosphopeptide ligand using scintillation proximity assay (SPA) technology. Recombinant SH2 domains were expressed from a T7 RNA polymerase-based vector in Escherichia coli as fusions to the C-terminus of the FK506-binding protein (FKBP) and purified from freeze-thaw lysates in high yield by affinity chromatography using immobilized phosphopeptides. For binding assays the phosphopeptide ligands were synthesized with a biotin tag and the FKBP fusion proteins were noncovalently radiolabeled with commercially available [3H]dihydroFK506. Complexes of tritiated SH2 fusion protein and biotinyl-phosphopeptide were then captured on streptavidin-coated SPA beads and counted. The modular protocol is an equilibrium technique that does not employ washing steps or specialized radiochemical syntheses required in other binding assays. The utility of the assay has been demonstrated in an examination of the ligand specificity of the SH2 domains of the tyrosine kinases ZAP70, Syk, and Lck. The methodology is potentially generalizable to any receptor-ligand interaction in which one component can be expressed as a fusion partner with FKBP and the other component can be captured on a SPA bead.

Asunto(s)

Proteínas Portadoras/biosíntesis , Proteínas Portadoras/química , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/química , Proteínas de Choque Térmico/biosíntesis , Proteínas de Choque Térmico/química , Proteínas Recombinantes de Fusión/biosíntesis , Conteo por Cintilación , Dominios Homologos src , Secuencia de Aminoácidos , Biotina/química , ARN Polimerasas Dirigidas por ADN/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Vectores Genéticos , Ligandos , Datos de Secuencia Molecular , Fosfopéptidos/química , Proteínas Recombinantes de Fusión/química , Tacrolimus/química , Proteínas de Unión a Tacrolimus , Tritio , Proteínas Virales

RESUMEN

The proteolytic enzyme stromelysin-1 is a member of the family of matrix metalloproteinases and is believed to play a role in pathological conditions such as arthritis and tumor invasion. Stromelysin-1 is synthesized as a pro-enzyme that is activated by removal of an N-terminal prodomain. The active enzyme contains a catalytic domain and a C-terminal hemopexin domain believed to participate in macromolecular substrate recognition. We have determined the three-dimensional structures of both a C-truncated form of the proenzyme and an inhibited complex of the catalytic domain by X-ray diffraction analysis. The catalytic core is very similar in the two forms and is similar to the homologous domain in fibroblast and neutrophil collagenases, as well as to the stromelysin structure determined by NMR. The prodomain is a separate folding unit containing three alpha-helices and an extended peptide that lies in the active site of the enzyme. Surprisingly, the amino-to-carboxyl direction of this peptide chain is opposite to that adopted by the inhibitor and by previously reported inhibitors of collagenase. Comparison of the active site of stromelysin with that of thermolysin reveals that most of the residues proposed to play significant roles in the enzymatic mechanism of thermolysin have equivalents in stromelysin, but that three residues implicated in the catalytic mechanism of thermolysin are not represented in stromelysin.

Asunto(s)

Precursores Enzimáticos/química , Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/química , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Sitios de Unión , Colagenasas/química , Cristalografía por Rayos X , Fibroblastos/enzimología , Humanos , Enlace de Hidrógeno , Metaloproteinasa 3 de la Matriz , Modelos Moleculares , Neutrófilos/enzimología , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química

RESUMEN

A cDNA for the human cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase (EC 4.1.3.5) was subcloned and expressed from a T7-based vector in Escherichia coli. The over-produced enzyme was purified using a three-step protocol that generated 20 to 30 mg protein/liter cell culture. The physical and catalytic properties of the recombinant synthase are similar to those reported for the nonrecombinant enzymes from chicken liver [Clinkenbeard et al. (1975a) J. Biol. Chem. 250, 3124-3135] and rat liver [Mehrabian et al. (1986) J. Biol. Chem. 261, 16249-16255]. Mutation of Cys129 to serine or alanine destroys HMG-CoA synthase activity by disrupting the first catalytic step in HMG-CoA synthesis, enzyme acetylation by acetyl coenzyme A. Furthermore, unlike the wild-type enzyme, neither mutant was capable of covalent modification by the beta-lactone inhibitor, L-659,699 [Greenspan et al. (1987) Proc. Natl. Acad. Sci. USA 84, 7488-7492]. Kinetic analysis of the inhibition by L-659,699 revealed that this compound is a potent inhibitor of the recombinant human synthase, with an inhibition constant of 53.7 nM and an inactivation rate constant of 1.06 min-1.

Asunto(s)

Cisteína/genética , Hidroximetilglutaril-CoA Sintasa/genética , Hidroximetilglutaril-CoA Sintasa/metabolismo , Mutación , Acetilcoenzima A/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Escherichia coli/genética , Ácidos Grasos Insaturados/química , Ácidos Grasos Insaturados/farmacología , Colorantes Fluorescentes/química , Colorantes Fluorescentes/farmacología , Biblioteca de Genes , Humanos , Hidroximetilglutaril-CoA Sintasa/antagonistas & inhibidores , Hidroximetilglutaril-CoA Sintasa/aislamiento & purificación , Lactonas/química , Lactonas/farmacología , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad

RESUMEN

The three-dimensional structure of the catalytic domain of stromelysin-1 complexed with an N-carboxyl alkyl inhibitor has been determined by NMR methods. The global fold consists of three helices, a five stranded beta-sheet and a methionine located in a turn near the catalytic histidines, classifying stromelysin-1 as a metzincin. Stromelysin-1 is unique in having two independent zinc binding sites: a catalytic site and a structural site. The inhibitor binds in an extended conformation. The S1' subsite is a deep hydrophobic pocket, whereas S2' appears shallow and S3' open.

Asunto(s)

Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/química , Secuencia de Aminoácidos , Sitios de Unión , Catálisis , Humanos , Técnicas In Vitro , Espectroscopía de Resonancia Magnética , Metaloproteinasa 3 de la Matriz , Metaloendopeptidasas/genética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Conformación Proteica , Pliegue de Proteína , Homología de Secuencia de Aminoácido , Zinc/química

RESUMEN

Experiments were carried out to determine whether complexes between MHC class I molecules and synthetic peptides are representative of those formed under more physiologically relevant conditions, with peptides derived intracellularly from processed antigens. Lysis of cells sensitized with exogenously provided and endogenously generated peptide analogues of the optimal nonameric peptide 58-66 (GILGFVFTL; derived from the influenza virus matrix protein) was compared. Endogenous loading was accomplished by expressing minigene DNA coding for alanine-substituted analogues of peptide 58-66 in HLA-A2-positive cells. Susceptibility to lysis by HLA-A2-restricted, peptide-specific cytotoxic lymphocytes was compared with lysis of cells sensitized with the same synthetic peptides. Although results were quite comparable, differences were observed. The endogenously presented analogues 58-66L60A, G61A, T65A, and L66A were recognized more efficiently than the corresponding exogenously presented analogues. This difference in recognition was most striking for peptide 58-66G61A. These results indicate the need for caution in using synthetic peptides in defining peptide binding motifs. Additional experiments with endogenously expressed analogues of 58-66 with substitutions other than alanine were carried out to define the interaction between this peptide and HLA-A2. Results are compatible with the interpretation that residues 58, 59, and 60 interact with pockets A, B, and D, respectively, in the HLA-A2 binding groove and that these interactions contribute to peptide binding.

Asunto(s)

Antígeno HLA-A2/inmunología , Oligopéptidos/inmunología , Linfocitos T Citotóxicos/inmunología , Proteínas de la Matriz Viral/inmunología , Secuencia de Aminoácidos , Células Cultivadas , Citotoxicidad Inmunológica/inmunología , Humanos , Virus de la Influenza A/inmunología , Datos de Secuencia Molecular , Oligopéptidos/síntesis química , Oligopéptidos/genética , Plásmidos , Relación Estructura-Actividad , Transfección , Proteínas de la Matriz Viral/genética

RESUMEN

L-685,818 differs only slightly in structure from the immunosuppressive drug FK-506, and both compounds bind with comparable affinity to the 12-kDa FK-506-binding protein (FKBP12), the major intracellular receptor for the drug. Despite these similarities, L-685,818 is a potent antagonist of both the immunosuppressive and toxic effects of the drug. Here, we present a structural analysis of this problem. Although FK-506 and L-685,818 differ greatly in pharmacology, we have found that the three-dimensional structures of their complexes with FKBP12 are essentially identical. Approximately half of each ligand is in contact with the receptor protein, and half is exposed to solvent; the exposed region includes the two sites where the compounds differ. These results indicate that the profound differences in the pharmacology of these two compounds are not caused by any difference in their interaction with FKBP12. Rather, these effects arise because relatively minor changes in the exposed part of a bound ligand have a strong effect on how FKBP12-ligand complexes interact with calcineurin, their putative intracellular target. In addition, FK-506 complexes with FKBP12 proteins from several species all inhibit mammalian calcineurin. Analysis of the three-dimensional structure of the complex with respect to residues conserved among these proteins suggests a small number of surface residues near the bound ligands that may play a critical role in interactions between the protein-drug complex and calcineurin.

Asunto(s)

Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Conformación Proteica , Tacrolimus/análogos & derivados , Tacrolimus/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Bovinos , Escherichia coli/genética , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , Tacrolimus/metabolismo , Proteínas de Unión a Tacrolimus , Difracción de Rayos X

RESUMEN

The ability of minigene-encoded viral peptide epitopes to be presented by class I molecules in the absence of MHC-encoded transporters has been evaluated in mutant T2 cells. These cells have a large deletion in the class II MHC region that includes the known transporter protein for antigenic peptides and proteasome genes and they are defective in presenting viral epitopes to CTL. T2 cells that express minigenes encoding the influenza virus matrix peptide 58-66 (GILGFVFTL) and two HTLV 1 Tax peptides 11-19 (LLFGYPVYV) and 12-19 were lysed by HLA-A2-restricted peptide-specific CTL. Minigene expression of a HLA-A2-restricted HIV reverse transcriptase peptide 476-484 (ILKEPVHGV) with three charged residues sensitized T2 cells poorly for lysis by HIV-specific CTL unless the peptide was preceded by an endoplasmic reticulum translocation signal sequence. Expression of an influenza virus nucleoprotein peptide 383-391 (SRYWAIRTR) with three charged arginine residues did sensitize HLA-B27+ T2 cells for lysis by peptide-specific CTL. These and other results with endogenously expressed peptide analogs in which hydrophobic and charged amino acids were interchanged demonstrate that antigenic peptides can be translocated from the cytoplasm into the class I Ag presentation pathway independent of MHC-encoded transporters; and that peptide hydrophobicity appears not to be a major determinant in selecting peptides for this alternate pathway.

Asunto(s)

Antígeno HLA-A2/inmunología , Antígeno HLA-B27/inmunología , Péptidos/inmunología , Proteínas de Unión al ARN , Linfocitos T Citotóxicos/inmunología , Secuencia de Aminoácidos , Transporte Biológico , Eliminación de Gen , Expresión Génica , Productos del Gen tax/inmunología , Antígeno HLA-A2/análisis , Antígeno HLA-B27/análisis , Humanos , Datos de Secuencia Molecular , Mutación , Proteínas de la Nucleocápside , Nucleoproteínas/inmunología , Fragmentos de Péptidos/inmunología , Péptidos/genética , ADN Polimerasa Dirigida por ARN/inmunología , Transfección , Proteínas del Núcleo Viral/inmunología , Proteínas de la Matriz Viral/inmunología

RESUMEN

A determination of the zinc stoichiometry of the catalytic domain of the human matrix metalloproteinase stromelysin-1 has been carried out using enzyme purified from recombinant Escherichia coli that express C-terminally truncated protein. Atomic absorption spectrometry revealed that both the proenzyme (prostrom255) and the mature active form (strom255) contained nearly 2 mol of Zn/mol of protein. Full-length prostromelysin purified from a mammalian cell culture line also contained zinc in excess of 1 equiv. While zinc in prostrom255 could not be removed by dialysis against o-phenanthroline, similar treatment of mature strom255 resulted in the loss of one-half of the original zinc content. The peptidase activity of the zinc-depleted protein was reduced by greater than 85% but could be restored upon addition of Zn2+ or Co2+. Addition of a thiol-containing inhibitor to a CoZn hybrid enzyme resulted in marked spectral changes in both the visible and ultraviolet regions characteristic of sulfur ligation to Co2+. This direct evidence for an integral role in catalysis and inhibitor binding confirms the location of the exchangeable metal at the active site. To examine the environment of zinc in the proenzyme, a fully cobalt-substituted proenzyme was prepared by in vivo metal replacement. The absorbance features of dicobalt prostrom255 were consistent with metal coordination by the single cysteine present in the propeptide, although the data do not allow assignment to a particular zinc site.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Proteínas Portadoras/química , Cisteína/química , Precursores Enzimáticos/química , Metaloendopeptidasas/química , Zinc/química , Secuencia de Aminoácidos , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Catálisis , Cobalto/química , Precursores Enzimáticos/genética , Humanos , Metaloproteinasa 3 de la Matriz , Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/genética , Unión Proteica , Proteínas Recombinantes/química , Especificidad por Sustrato

RESUMEN

Episomal plasmids (p8901) with minigenes coding for the influenza virus matrix peptide amino acids 57-68 (KGILGFVFTLTV; referred to as M57-68) or coding for a modified peptide were introduced into HLA-A2-positive target cells. The association of these peptides, synthesized in the cytoplasm, with HLA-A2 and the expression of this complex at the cell surface was evaluated with HLA-A2-restricted CTL specific for the influenza virus matrix peptide M57-68. Cells expressing M57-68 were lysed effectively, as were cells expressing a peptide that retained residues 60-64 with seven flanking alanine residues (AAALGFVFAAAA). An exogenously added synthetic analog of peptide M57-68 that inhibited sensitization of targets with synthetic peptide M57-68 also inhibited lysis of cells expressing the minigene coding for the peptide with seven alanine substitutions. These results demonstrate the utility of minigene DNA constructs in creating experimental systems to develop agents to diminish the severity of CTL-mediated tissue damage in autoimmune diseases and graft rejection.

Asunto(s)

Antígenos Virales/metabolismo , Antígeno HLA-A2/metabolismo , Virus de la Influenza A/inmunología , Péptidos/inmunología , Proteínas de la Matriz Viral/inmunología , Secuencia de Aminoácidos , Células Presentadoras de Antígenos/inmunología , Antígenos Virales/química , Secuencia de Bases , Unión Competitiva , Clonación Molecular , Citotoxicidad Inmunológica , Relación Dosis-Respuesta Inmunológica , Vectores Genéticos , Humanos , Inmunidad Celular , Técnicas In Vitro , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Péptidos/metabolismo , Plásmidos , Linfocitos T Citotóxicos/inmunología , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/metabolismo

RESUMEN

Stromelysin-1 is a member of a tissue metalloproteinase family whose members are all capable of degrading extracellular matrix components. A truncated form of human fibroblast prostromelysin 1 lacking the C-terminal, hemopexin-like domain has been expressed in Escherichia coli and purified to homogeneity. Treatment of this short form of prostromelysin with (aminophenyl)mercuric acetate resulted in activation and loss of the propeptide in a manner identical with the wild-type, full-length protein. Kinetic comparisons using Nle11-substance P as a substrate showed that the wild-type stromelysin and the truncated form of the enzyme had similar kcat and Km values. Likewise, both enzymes displayed similar Ki values for a hydroxamate-containing peptide inhibitor. Taken together, these results indicate that the C-terminal portion of stromelysin is not required for proper folding of the catalytic domain, maintenance of the enzyme in a latent form, activation with an organomercurial, cleavage of a peptide substrate, or interaction with an inhibitor. Moreover, the active short form of stromelysin displayed a reduction in the C-terminal heterogeneity, a characteristic degradation of the full-length stromelysin, and thereby provides a more suitable protein for future structural studies.

Asunto(s)

Metaloendopeptidasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Precursores Enzimáticos/aislamiento & purificación , Precursores Enzimáticos/metabolismo , Escherichia coli/genética , Fibroblastos/enzimología , Humanos , Ácidos Hidroxámicos/farmacología , Cinética , Sustancias Macromoleculares , Metaloproteinasa 3 de la Matriz , Metaloendopeptidasas/aislamiento & purificación , Metaloendopeptidasas/metabolismo , Colagenasa Microbiana/genética , Modelos Estructurales , Datos de Secuencia Molecular , Peso Molecular , Sondas de Oligonucleótidos , Plásmidos , Reacción en Cadena de la Polimerasa/métodos , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico

RESUMEN

How easy is it to improve the catalytic power of an enzyme? To address this question, the gene encoding a sluggish mutant triose-phosphate isomerase (D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1) has been subjected to random mutagenesis over its whole length by using "spiked" oligonucleotide primers. Transformation of an isomerase-minus strain of Escherichia coli was followed by selection of those colonies harboring an enzyme of higher catalytic potency. Six amino acid changes in the Glu-165----Asp mutant of triosephosphate isomerase improve the specific catalytic activity of this enzyme (from 1.3-fold to 19-fold). The suppressor sites are scattered across the sequence (at positions 10, 96, 97, 167, and 233), but each of them is very close to the active site. These experiments show both that there are relatively few single amino acid changes that increase the catalytic potency of this enzyme and that all of these improvements derive from alterations that are in, or very close to, the active site.

Asunto(s)

Carbohidrato Epimerasas/genética , Escherichia coli/genética , Genes Bacterianos , Mutación , Triosa-Fosfato Isomerasa/genética , Ácido Aspártico , Escherichia coli/enzimología , Glutamatos , Ácido Glutámico , Cinética , Modelos Moleculares , Sondas de Oligonucleótidos , Plásmidos , Conformación Proteica , Triosa-Fosfato Isomerasa/metabolismo

RESUMEN

A new procedure for the production of a defined library of random mutants is described. Long spiked oligodeoxyribonucleotides (oligos), in which a predetermined level of the three 'wrong' phosphoramidites are used at each position, are made as primers for a standard oligo-directed mutagenesis protocol. Spiked oligo synthesis on a DNA synthesizer is achieved using an in-line mixing procedure that only requires five phosphoramidite reservoirs and which avoids contamination of any of the pure phosphoramidite reagents. Immutable positions (i.e., positions in the oligo for which pure reagents are used) can be specified, and a silent 'marker' base can be included that allows an early estimate of the mutagenesis efficiency. The randomness of the library in respect to the number, type, and position of the altered bases, is easily verified by DNA sequencing. This procedure has been used to generate a random mutant library of the gene encoding a sluggish triosephosphate isomerase. Among the transformants from this library, a number of second-site suppressor mutations have been found that increase the specific catalytic activity of the starting isomerase. This approach provides a more complete library than a method using chemical mutagenic reagents.

Asunto(s)

Escherichia coli/genética , Biblioteca de Genes , Mutación , Sondas de Oligonucleótidos/síntesis química , Secuencia de Bases , Clonación Molecular , Datos de Secuencia Molecular , Plásmidos , Moldes Genéticos