RESUMEN
(1) In the present study we have attempted to identify changes in gene expression which are associated with neuropathic pain using subtractive suppression hybridization analysis of the lumbar spinal cord of animals suffering streptozocin induced diabetic neuropathy. (2) Using this approach, we found a significant up-regulation of several key components of the extracellular signal-regulated kinase (ERK) cascade. These findings were confirmed by Western blot analysis, which demonstrated that the levels of active ERK1 and 2 correlated with the onset of streptozocin-induced hyperalgesia. (3) Intrathecal administration of the selective MAPK/ERK-kinase (MEK) inhibitor PD 198306 dose-dependently (1-30 micro g) blocked static allodynia in both the streptozocin and the chronic constriction injury (CCI) models of neuropathic pain. (4) The antihyperalgesic effects of PD 198306, in both the streptozocin and CCI models of neuropathic pain, correlated with a reduction in the elevated levels of active ERK1 and 2 in lumbar spinal cord. (5) Intraplantar administration of PD 198306 had no effect in either model of hyperalgesia, indicating that changes in the activation of ERKs and the effect of MEK inhibition are localized to the central nervous system. (6) In summary, we have demonstrated for the first time that the development of neuropathic pain is associated with an increase in the activity of the MAPK/ERK-kinase cascade within the spinal cord and that enzymes in this pathway represent potential targets for the treatment of this condition.
Asunto(s)
Neuropatías Diabéticas/tratamiento farmacológico , Neuropatías Diabéticas/enzimología , Sistemas de Liberación de Medicamentos/métodos , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Animales , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/enzimología , Neuropatías Diabéticas/inducido químicamente , Fluorobencenos/farmacología , Fluorobencenos/uso terapéutico , Regulación Enzimológica de la Expresión Génica/fisiología , MAP Quinasa Quinasa 1 , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Dimensión del Dolor/efectos de los fármacos , Dimensión del Dolor/métodos , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Phosphatidylinositol 5-phosphate (PtdIns5P), a novel inositol lipid, has been shown to be the major substrate for the type II PtdInsP kinases (PIPkins) ¿Rameh et al. (1997) Nature 390, 192-196. A PtdInsP fraction was prepared from cell extracts by neomycin chromatography, using a protocol devised to eliminate the interaction of acidic solvents with plasticware, since this was found to inhibit the enzyme. The PtdIns5P in this fraction was measured by incubating with ¿gamma-(32)PATP and recombinant PIPkin IIalpha, and quantifying the radiolabelled PtdInsP(2) formed. This assay was used on platelets to show that during 10 min stimulation with thrombin, the mass level of PtdIns5P increases, implying the existence of an agonist-stimulated synthetic mechanism.
Asunto(s)
Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Hemostáticos/farmacología , Fosfatos de Fosfatidilinositol/biosíntesis , Activación Plaquetaria , Trombina/farmacología , Cromatografía Líquida de Alta Presión , Humanos , Espectrometría de Masas , Activación Plaquetaria/efectos de los fármacosRESUMEN
Type II phosphatidylinositol phosphate kinases (PIPkins) have recently been found to be primarily phosphatidylinositol 5-phosphate 4-kinases, and their physiological role remains unclear. We have previously shown that a Type II PIPkin [isoform(s) unknown], is localized partly in the nucleus [Divecha, Rhee, Letcher and Irvine (1993) Biochem. J. 289, 617-620], and here we show, by transfection of HeLa cells with green-fluorescent-protein-tagged Type II PIPkins, that this is likely to be the Type IIbeta isoform. Type IIbeta PIPkin has no obvious nuclear localization sequence, and a detailed analysis of the localization of chimaeras and mutants of the alpha (cytosolic) and beta PIPkins shows that the nuclear localization requires the presence of a 17-amino-acid length of alpha-helix (alpha-helix 7) that is specific to the beta isoform, and that this helix must be present in its entirety, with a precise orientation. This resembles the nuclear targeting of the HIV protein Vpr, and Type IIbeta PIPkin is apparently therefore the first example of a eukaryotic protein that uses the same mechanism.
Asunto(s)
Núcleo Celular/enzimología , Isoenzimas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Secuencia de Aminoácidos , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Proteínas Luminiscentes/metabolismo , Antígenos de Histocompatibilidad Menor , Datos de Secuencia Molecular , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Estructura Secundaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Inositol lipid synthesis is regulated by several distinct families of enzymes [1]. Members of one of these families, the type II phosphatidylinositol phosphate kinases (PIP kinases), are 4-kinases and are thought to catalyse a minor route of synthesis of the multifunctional phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) from the inositide PI(5)P [2]. Here, we demonstrate the partial purification of a protein kinase that phosphorylates the type IIalpha PIP kinase at a single site unique to that isoform - Ser304. This kinase was identified as protein kinase CK2 (formerly casein kinase 2). Mutation of Ser304 to aspartate to mimic its phosphorylation had no effect on PIP kinase activity, but promoted both redistribution of the green fluorescent protein (GFP)-tagged enzyme in HeLa cells from the cytosol to the plasma membrane, and membrane ruffling. This effect was mimicked by mutation of Ser304 to alanine, although not to threonine, suggesting a mechanism involving the unmasking of a latent membrane localisation sequence in response to phosphorylation.
Asunto(s)
Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Isoformas de Proteínas/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Quinasa de la Caseína II , Membrana Celular/enzimología , Membrana Celular/ultraestructura , Citosol/metabolismo , Genes Reporteros , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas de la Membrana/metabolismo , Microscopía Confocal , Microscopía Fluorescente , Antígenos de Histocompatibilidad Menor , Datos de Secuencia Molecular , Proteínas de Neoplasias/fisiología , Fosforilación , Fosfoserina/química , Mutación Puntual , Isoformas de Proteínas/química , Isoformas de Proteínas/aislamiento & purificación , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
BACKGROUND: Many enzymes that digest polysaccharides contain separate polysaccharide-binding domains. Structures have been previously determined for a number of cellulose-binding domains (CBDs) from cellulases. RESULTS: The family IIb xylan-binding domain 1 (XBD1) from Cellulomonas fimi xylanase D is shown to bind xylan but not cellulose. Its structure is similar to that of the homologous family IIa CBD from C. fimi Cex, consisting of two four-stranded beta sheets that form a twisted 'beta sandwich'. The xylan-binding site is a groove made from two tryptophan residues that stack against the faces of the sugar rings, plus several hydrogen-bonding polar residues. CONCLUSIONS: The biggest difference between the family IIa and IIb domains is that in the former the solvent-exposed tryptophan sidechains are coplanar, whereas in the latter they are perpendicular, forming a twisted binding site. The binding sites are therefore complementary to the secondary structures of the ligands cellulose and xylan. XBD1 and CexCBD represent a striking example of two proteins that have high sequence similarity but a different function.
Asunto(s)
Celulosa/metabolismo , Xilanos/metabolismo , Xilosidasas/química , beta-Glucosidasa/química , Secuencia de Aminoácidos , Secuencia de Bases , Cristalografía por Rayos X , Cartilla de ADN , Endo-1,4-beta Xilanasas , Ligandos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Termodinámica , Xilosidasas/metabolismo , beta-Glucosidasa/metabolismoAsunto(s)
1-Fosfatidilinositol 4-Quinasa/química , 1-Fosfatidilinositol 4-Quinasa/metabolismo , 1-Fosfatidilinositol 4-Quinasa/fisiología , Secuencia de Aminoácidos , Animales , Núcleo Celular/metabolismo , Dimerización , Células HeLa , Humanos , Datos de Secuencia Molecular , Fosforilación , Isoformas de Proteínas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de AminoácidoRESUMEN
The phosphatidylinositolphosphate kinases (PIPkins) are a unique family of enzymes that catalyse the production of phosphorylated inositol lipids. Recent advances have revealed that, due to their ability to utilise a number of different lipid substrates (at least in vitro), this family is potentially able to generate several distinct, physiologically important inositol lipids. Despite their importance, however, our understanding of the regulation of the PIPkins and of their physiological role in cellular signalling and regulation is still poor. Here we describe in turn the diverse physiological functions of the known substrates and major products of the PIPkins. We then examine what is known about the members of the PIPkin family themselves, and their characteristics and regulation.
Asunto(s)
Fosfatos de Fosfatidilinositol/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Humanos , Fosfatidilinositol 4,5-Difosfato/metabolismo , Transducción de Señal , Especificidad por SustratoRESUMEN
To investigate the mode of action of cellulose-binding domains (CBDs), the Type II CBD from Pseudomonas fluorescens subsp. cellulosa xylanase A (XYLACBD) and cellulase E (CELECBD) were expressed as individual entities or fused to the catalytic domain of a Clostridium thermocellum endoglucanase (EGE). The two CBDs exhibited similar Ka values for bacterial microcrystalline cellulose (CELECBD, 1.62x10(6) M-1; XYLACBD, 1.83x10(6) M-1) and acid-swollen cellulose (CELECBD, 1.66x10(6) M-1; XYLACBD, 1.73x10(6) M-1). NMR spectra of XYLACBD titrated with cello-oligosaccharides showed that the environment of three tryptophan residues was affected when the CBD bound cellohexaose, cellopentaose or cellotetraose. The Ka values of the XYLACBD for C6, C5 and C4 cello-oligosaccharides were estimated to be 3.3x10(2), 1.4x10(2) and 4.0x10(1) M-1 respectively, suggesting that the CBD can accommodate at least six glucose molecules and has a much higher affinity for insoluble cellulose than soluble oligosaccharides. Fusion of either the CELECBD or XYLACBD to the catalytic domain of EGE potentiated the activity of the enzyme against insoluble forms of cellulose but not against carboxymethylcellulose. The increase in cellulase activity was not observed when the CBDs were incubated with the catalytic domain of either EGE or XYLA, with insoluble cellulose and a cellulose/hemicellulose complex respectively as the substrates. Pseudomonas CBDs did not induce the extension of isolated plant cell walls nor weaken cellulose paper strips in the same way as a class of plant cell wall proteins called expansins. The XYLACBD and CELECBD did not release small particles from the surface of cotton. The significance of these results in relation to the mode of action of Type II CBDs is discussed.
Asunto(s)
Celulasa/química , Celulosa/metabolismo , Pseudomonas fluorescens/enzimología , Xilosidasas/química , Proteínas Bacterianas/química , Sitios de Unión/genética , Celulasa/genética , Clostridium/enzimología , Endo-1,4-beta Xilanasas , Cinética , Espectroscopía de Resonancia Magnética , Oligosacáridos/metabolismo , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Activity of a cellulosomal endoglucanase (endoglucanase E; EGE) from Clostridium thermocellum against two crystalline forms of cellulose was enhanced by combination with the cellulosome integrating protein (CipA), but CipA did not enhance EGE activity against amorphous cellulose, even though it was able to bind to it. Similarly, CipA added in trans to genetically truncated EGE that was unable to combine with it nevertheless enhanced EGE activity against crystalline cellulose. These results indicate that the CipA cellulose binding domain does not mediate an increase in activity solely by bringing the catalytic subunits of the cellulosome complex into intimate contact with the substrate.
Asunto(s)
Proteínas Bacterianas/metabolismo , Celulasa/metabolismo , Clostridium/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/aislamiento & purificación , Celulasa/biosíntesis , Celulasa/aislamiento & purificación , Celulosa/química , Celulosa/metabolismo , Clonación Molecular , Escherichia coli , Cinética , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/aislamiento & purificación , Orgánulos/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Enzyme-aided bleaching of softwood and hardwood kraft pulps by glycosyl hydrolase family-10 and -11 xylanases and a family-26 mannanase was investigated. The ability to release reducing sugar from pulp xylan and to enhance bleachability is not a characteristic shared by all xylanases. Of the six enzymes tested, two xylanases belonging to family 11 were most effective at increasing bleachability and improving final paper brightness. None of the enzymes had a deleterious effect on pulp fibre integrity. The efficiency of individual xylanases as bleach enhancers was not dependent on the source microorganism, and could not be predicted solely on the basis of the quantity or nature of products released from pulp xylan. Cooperative interactions between xylanase/xylanase and xylanase/mannanase combinations, during the pretreatment of softwood and hardwood pulps, were investigated. Synergistic effects on reducing-sugar release and kappa number reduction were elicited by a combination of two family-10 xylanases. Pretreatment of kraft pulp with mannanase A from Pseudomonas fluorescens subsp. cellulosa and any one of a number of xylanases resulted in increased release of reducing sugar and a larger reduction in kappa number than obtained with the xylanases alone, confirming the beneficial effects of family-26 mannanases on enzyme-aided bleaching of paper pulp.
Asunto(s)
Papel , Madera , Xilosidasas/farmacología , Glicósido Hidrolasas/farmacología , Manosidasas/farmacología , Xilano Endo-1,3-beta-Xilosidasa , beta-ManosidasaRESUMEN
The groESL operon from Clostridium thermocellum (Ct) has been isolated and sequenced, revealing two ORFs of 285 and 1626 nt, separated by 48 nt. The first ORF encoded a 94-aa 10.6-kDa GroES homologue; the second encoded a 541-aa polypeptide of 57.6 kDa, that exhibited 61% and 77% sequence identity with GroEL from Escherichia coli (Ec) and Clostridium acetobutylicum (Ca), respectively. A putative tsp, preceded by -10 and -35 consensus promoters, was identified upstream of groES. This was followed by an inverted repeat observed previously in bacterial heat shock genes. A 15-nt palindrome characteristic of a Rho-independent transcription terminator, was located downstream of groEL. The first nt of the groES translational start codon was preceded (7 nt) by a putative RBS (AGGAGG); a second RBS sequence was located 8 nt upstream of the groEL start. Production of GroE homologues by Ct was constitutive, but was enhanced significantly during a temperature upshift from 60 degrees C to 70 degrees C. The Ct GroEL, expressed in Ec as a fusion protein with GST, was purified, free of contaminating Ec GroEL.
Asunto(s)
Chaperonina 10/genética , Chaperonina 60/genética , Clostridium/genética , Transcripción Genética , Northern Blotting , Chaperonina 10/biosíntesis , Chaperonina 60/biosíntesis , Clonación Molecular , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido NucleicoRESUMEN
Homologues of the chaperonins Cpn60 and Cpn10 have been purified from the Gram-positive cellulolytic thermophile Clostridium thermocellum. The Cpn60 protein was purified by ATP-affinity chromatography and the Cpn10 protein was purified by gel-filtration, ion-exchange and hydrophobic interaction chromatographies. The identities of the proteins were confirmed by N-terminal sequence analysis and antigenic cross-reactivity. The Cpn60 homologue is a weak, thermostable ATPase (t1/2 at 70 decrees C more than 90 min) with optimum activity (Kcat 0.07 S-1) between 60 degrees C and 70 degrees C. The ATPase activity of the authentic Cpn60 was inhibited by Escherichia coli GroES. The catalytic properties of a recombinant C. thermocellum Cpn60 purified from a GST-Cpn60 fusion protein expressed in E. coli [Ciruela (1995) Ph.D. Thesis, University of Kent] were identical with those of the authentic C. thermocellum Cpn60. Gel-filtration studies show that at room temperature the Cpn60 migrates mainly as a heptamer. Electron microscopy confirms the presence of complexes showing 7-fold rotational symmetry and also reveals a small number of particles that seem to be tetradecamers with a similar structure to E. coli GroEL complexes.
Asunto(s)
Chaperonina 10/química , Chaperonina 60/química , Clostridium/química , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Western Blotting , Chaperonina 10/aislamiento & purificación , Chaperonina 10/farmacología , Chaperonina 60/antagonistas & inhibidores , Chaperonina 60/aislamiento & purificación , Chaperonina 60/ultraestructura , Cromatografía de Afinidad , Electroforesis en Gel de Poliacrilamida , Estabilidad de Enzimas , Escherichia coli/química , Microscopía Electrónica , Datos de Secuencia Molecular , Peso Molecular , Homología de Secuencia de Aminoácido , TemperaturaRESUMEN
Short antigenic peptides bound in the groove of class I major histocompatibility complex molecules enable T cells to detect intracellular pathogens. It has been assumed that structural features of the class I molecule alone select which peptides are bound. It is now demonstrated that a complex polymorphism in one of the major histocompatibility complex-encoded putative peptide-transporter genes is associated with an altered spectrum of bound peptides.