RESUMEN
Xyloglucan endotransglycosylase/hydrolase (XTH) enzymes play important roles in cell wall remodelling. Although previous studies have shown a pathway of evolution for XTH genes from bacterial licheninases, through plant endoglucanases (EG16), the order of development within the phylogenetic clades of true XTHs is yet to be elucidated. In addition, recent studies have revealed interesting and potentially useful patterns of transglycosylation beyond the standard xyloglucan-xyloglucan donor/acceptor substrate activities. To study evolutionary relationships and to search for enzymes with useful broad substrate specificities, genes from the 'ancestral' XTH clade of two monocots, Brachypodium distachyon and Triticum aestivum, and two eudicots, Arabidopsis thaliana and Populus tremula, were investigated. Specific activities of the heterologously produced enzymes showed remarkably broad substrate specificities. All the enzymes studied had high activity with the cellulose analogue HEC (hydroxyethyl cellulose) as well as with mixed-link ß-glucan as donor substrates, when compared with the standard xyloglucan. Even more surprising was the wide range of acceptor substrates that these enzymes were able to catalyse reactions with, opening a broad range of possible roles for these enzymes, both within plants and in industrial, pharmaceutical and medical fields. Genome screening and expression analyses unexpectedly revealed that genes from this clade were found only in angiosperm genomes and were predominantly or solely expressed in reproductive tissues. We therefore posit that this phylogenetic group is significantly different and should be renamed as the group-IV clade.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/fisiología , Regulación de la Expresión Génica de las Plantas/fisiología , Glucanos/metabolismo , Glicosiltransferasas/metabolismo , Proteínas de Plantas/metabolismo , Xilanos/metabolismo , Arabidopsis/enzimología , Arabidopsis/genética , Brachypodium/enzimología , Brachypodium/genética , Pared Celular/fisiología , Biología Computacional , Genoma de Planta , Glicosiltransferasas/clasificación , Glicosiltransferasas/genética , Filogenia , Células Vegetales/fisiología , Proteínas de Plantas/genética , Populus/enzimología , Populus/genética , Especificidad de la Especie , Especificidad por Sustrato , Triticum/enzimología , Triticum/genéticaRESUMEN
Treatments for cognitive deficits associated with central nervous system (CNS) disorders such as Alzheimer disease and schizophrenia remain significant unmet medical needs that incur substantial pressure on the health care system. The α7 nicotinic acetylcholine receptor (nAChR) has garnered substantial attention as a target for cognitive deficits based on receptor localization, robust preclinical effects, genetics implicating its involvement in cognitive disorders, and encouraging, albeit mixed, clinical data with α7 nAChR orthosteric agonists. Importantly, previous orthosteric agonists at this receptor suffered from off-target activity, receptor desensitization, and an inverted U-shaped dose-effect curve in preclinical assays that limit their clinical utility. To overcome the challenges with orthosteric agonists, we have identified a novel selective α7 positive allosteric modulator (PAM), BNC375. This compound is selective over related receptors and potentiates acetylcholine-evoked α7 currents with only marginal effect on the receptor desensitization kinetics. In addition, BNC375 enhances long-term potentiation of electrically evoked synaptic responses in rat hippocampal slices and in vivo. Systemic administration of BNC375 reverses scopolamine-induced cognitive deficits in rat novel object recognition and rhesus monkey object retrieval detour (ORD) task over a wide range of exposures, showing no evidence of an inverted U-shaped dose-effect curve. The compound also improves performance in the ORD task in aged African green monkeys. Moreover, ex vivo 13C-NMR analysis indicates that BNC375 treatment can enhance neurotransmitter release in rat medial prefrontal cortex. These findings suggest that α7 nAChR PAMs have multiple advantages over orthosteric α7 nAChR agonists for the treatment of cognitive dysfunction associated with CNS diseases. SIGNIFICANCE STATEMENT: BNC375 is a novel and selective α7 nicotinic acetylcholine receptor (nAChR) positive allosteric modulator (PAM) that potentiates acetylcholine-evoked α7 currents in in vitro assays with little to no effect on the desensitization kinetics. In vivo, BNC375 demonstrated robust procognitive effects in multiple preclinical models across a wide exposure range. These results suggest that α7 nAChR PAMs have therapeutic potential in central nervous system diseases with cognitive impairments.
Asunto(s)
Bencetonio/farmacología , Clorobencenos/farmacología , Receptor Nicotínico de Acetilcolina alfa 7/agonistas , Regulación Alostérica , Animales , Encéfalo/efectos de los fármacos , Encéfalo/fisiología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cognición/efectos de los fármacos , Potenciación a Largo Plazo/efectos de los fármacos , Macaca mulatta , Masculino , Neurotransmisores/metabolismo , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Escopolamina/farmacologíaRESUMEN
Phylogenetic analyses of cellulose synthase (CesA) and cellulose synthase-like (Csl) families from the cellulose synthase gene superfamily were used to reconstruct their evolutionary origins and selection histories. Counterintuitively, genes encoding primary cell wall CesAs have undergone extensive expansion and diversification following an ancestral duplication from a secondary cell wall-associated CesA. Selection pressure across entire CesA and Csl clades appears to be low, but this conceals considerable variation within individual clades. Genes in the CslF clade are of particular interest because some mediate the synthesis of (1,3;1,4)-ß-glucan, a polysaccharide characteristic of the evolutionarily successful grasses that is not widely distributed elsewhere in the plant kingdom. The phylogeny suggests that duplication of either CslF6 and/or CslF7 produced the ancestor of a highly conserved cluster of CslF genes that remain located in syntenic regions of all the grass genomes examined. A CslF6-specific insert encoding approximately 55 amino acid residues has subsequently been incorporated into the gene, or possibly lost from other CslFs, and the CslF7 clade has undergone a significant long-term shift in selection pressure. Homology modeling and molecular dynamics of the CslF6 protein were used to define the three-dimensional dispositions of individual amino acids that are subject to strong ongoing selection, together with the position of the conserved 55-amino acid insert that is known to influence the amounts and fine structures of (1,3;1,4)-ß-glucans synthesized. These wall polysaccharides are attracting renewed interest because of their central roles as sources of dietary fiber in human health and for the generation of renewable liquid biofuels.
Asunto(s)
Evolución Molecular , Genes de Plantas , Glucosiltransferasas/genética , Familia de Multigenes , Poaceae/enzimología , Poaceae/genética , Sustitución de Aminoácidos , Aminoácidos/genética , Cromosomas de las Plantas/genética , Secuencia Conservada/genética , Modelos Moleculares , Filogenia , Selección Genética , Homología Estructural de ProteínaRESUMEN
We report the first UV laser photodissociation spectra of gas-phase I(-) â MI (M = Na, K, Cs) alkali halide anionic microclusters. The photodepletion spectra of these clusters display strong absorption bands just below the calculated vertical detachment energies, indicative of the presence of dipole-bound excited states. Photoexcitation at the peak of the transition to the dipole-bound excited state results in production of a primary [MI](-) photofragment along with a less intense I(-) ion. The photofragmentation mechanism of the excited state cluster is discussed in the context of an initial dipole-bound excited state that subsequently relaxes via a vibrational Feschbach resonance. The experiments described have been performed in an electrospray source laser-interfaced quadrupole ion-trap instrument and demonstrated for the first time that dipole-bound excited states can be identified in the relatively high-collision environment of a quadrupole ion-trap, in particular for systems with large dipole moments associated with the presence of charge separation. This indicates considerable potential for future experiments that identify dipole-bound excited states as a "low-resolution" structural probe of biomolecules and molecular charge separation using the instrumentation employed in this work.
RESUMEN
We present a comparative assessment of the performance of the M06 suite of density functionals (M06, M06-2X, and M06-HF) against an MP2 benchmark for calculating the relative energies and geometric structures of the Cl(-)·arginine and Br(-)·arginine halide ion-amino acid clusters. Additional results are presented for the popular B3LYP density functional. The Cl(-)·arginine and Br(-)·arginine complexes are important prototypes for the phenomenon of anion-induced zwitterion formation. Results are presented for the canonical (noncharge separated) and zwitterionic (charge separated) tautomers of the clusters, as well as the numerous conformational isomers of the clusters. We find that all of the M06 functions perform well in terms of predicting the general trends in the conformer relative energies and identifying the global minimum conformer. This is in contrast to the B3LYP functional, which performed significantly less well for the canonical tautomers of the clusters where dispersion interactions contribute more significantly to the conformer energetics. We find that the M06 functional gave the lowest mean unsigned error for the relative energies of the canonical conformers (2.10 and 2.36 kJ/mol for Br(-)·arginine and Cl(-)·arginine), while M06-2X gave the lowest mean unsigned error for the zwitterionic conformers (0.85 and 1.23 kJ/mol for Br(-)·arginine and Cl(-)·arginine), thus providing insight into the types of physical systems where each of these functionals should perform best.
Asunto(s)
Arginina/química , Bromo/química , Cloro/química , Teoría Cuántica , Ácido Fluorhídrico , Enlace de Hidrógeno , Iones/química , Modelos Moleculares , Estructura MolecularRESUMEN
The barley (Hordeum vulgare) brittle stem mutants, fs2, designated X054 and M245, have reduced levels of crystalline cellulose compared with their parental lines Ohichi and Shiroseto. A custom-designed microarray, based on long oligonucleotide technology and including genes involved in cell wall metabolism, revealed that transcript levels of very few genes were altered in the elongation zone of stem internodes, but these included a marked decrease in mRNA for the HvCesA4 cellulose synthase gene of both mutants. In contrast, the abundance of several hundred transcripts changed in the upper, maturation zones of stem internodes, which presumably reflected pleiotropic responses to a weakened cell wall that resulted from the primary genetic lesion. Sequencing of the HvCesA4 genes revealed the presence of a 964-bp solo long terminal repeat of a Copia-like retroelement in the first intron of the HvCesA4 genes of both mutant lines. The retroelement appears to interfere with transcription of the HvCesA4 gene or with processing of the mRNA, and this is likely to account for the lower crystalline cellulose content and lower stem strength of the mutants. The HvCesA4 gene maps to a position on chromosome 1H of barley that coincides with the previously reported position of fs2.
Asunto(s)
Glucosiltransferasas/genética , Hordeum/genética , Proteínas de Plantas/genética , Retroelementos , Pared Celular/química , Celulosa/análisis , Mapeo Cromosómico , Perfilación de la Expresión Génica , Genes de Plantas , Glucosiltransferasas/metabolismo , Hordeum/enzimología , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Proteínas de Plantas/metabolismo , ARN de Planta/genéticaRESUMEN
Plant beta-D-mannosidases and a rice beta-D-glucosidase, Os3BGlu7, with weak beta-D-mannosidase activity, cluster together in phylogenetic analysis. To investigate the relationship between substrate specificity and amino acid sequence similarity in family GH1 glycoside hydrolases, Os3BGlu8 and Os7BGlu26, putative rice beta-D-glucosidases from this cluster, and a beta-D-mannosidase from barley (rHvBII), were expressed in Escherichia coli and characterized. Os3BGlu8, the amino acid sequence and molecular model of which are most similar to Os3BGlu7, hydrolysed 4-nitrophenyl-beta-D-glucopyranoside (4NPGlc) faster than 4-nitrophenyl-beta-D-mannopyranoside (4NPMan), while Os7BGlu26, which is most similar to rHvBII by these criteria, hydrolysed 4NPMan faster than 4NPGlc. All the enzymes hydrolyzed cellooligosaccharides with increased hydrolytic rates as the degree of polymerization increased from 3-6, but only rHvBII hydrolyzed cellobiose with a higher k(cat)/K(m) value than cellotriose. This was primarily due to strong binding of glucosyl residues at the+2 subsite for the rice enzymes, and unfavorable interactions at this subsite with rHvBII.
Asunto(s)
Glicósido Hidrolasas/metabolismo , Oryza/enzimología , beta-Glucosidasa/metabolismo , beta-Manosidasa/metabolismo , Secuencia de Aminoácidos , Dominio Catalítico , Clonación Molecular , Glicósido Hidrolasas/química , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/aislamiento & purificación , Glicósidos/química , Glicósidos/metabolismo , Hordeum/enzimología , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Oligosacáridos/química , Oligosacáridos/metabolismo , Filogenia , Alineación de Secuencia , Análisis de Secuencia de ADN , Especificidad por SustratoRESUMEN
Positive allosteric modulators (PAMs) of α7 nAChRs can have different properties with respect to their effects on channel kinetics. Type I PAMs amplify peak channel response to acetylcholine but do not appear to influence channel desensitization kinetics, whereas Type II PAMs both increase channel response and delay receptor desensitization. Both Type I and Type II PAMs are reported in literature, but there are limited reports describing their structure-kinetic profile relationships. Here, we report a novel class of compounds with either Type I or Type II behavior that can be tuned by the relative stereochemistry around the central cyclopropyl ring: for example, (R,R)-13 (BNC375) and its analogues with RR stereochemistry around the central cyclopropyl ring are Type I PAMs, whereas compounds in the same series with SS stereochemistry (e.g., (S,S)-13) are Type II PAMs as measured using patch-clamp electrophysiology. Further fine control over the kinetics has been achieved by changing the substitutions on the aniline ring: generally the substitution of aniline with strong electron withdrawing groups reduces the Type II character of these compounds. Our structure-activity optimization efforts have led to the discovery of BNC375, a small molecule with good CNS-drug like properties and clinical candidate potential.
RESUMEN
Chalcone derivatives of the natural product khellinone were synthesised and screened for bioactivity against the voltage-gated potassium channel Kv1.3. X-ray crystallography was employed to investigate relationships between the structure and function of a selection of the reported chalcones.
Asunto(s)
Chalconas/síntesis química , Chalconas/farmacología , Canal de Potasio Kv1.3/antagonistas & inhibidores , Bloqueadores de los Canales de Potasio/síntesis química , Bloqueadores de los Canales de Potasio/farmacología , Benzofuranos/farmacología , Chalconas/química , Cristalografía por Rayos X , Electrofisiología , Humanos , Canal de Potasio Kv1.3/metabolismo , Modelos Químicos , Estructura Molecular , Bloqueadores de los Canales de Potasio/química , Relación Estructura-ActividadRESUMEN
The voltage-gated potassium channel Kv1.3 constitutes an attractive target for the selective suppression of effector memory T cells in autoimmune diseases. We have previously reported the natural product khellinone, 1a, as a versatile lead molecule and identified two new classes of Kv1.3 blockers: (i) chalcone derivatives of khellinone, and (ii) khellinone dimers linked through the 6-position. Here we describe the multiple parallel synthesis of a new class of khellinone derivatives selectively alkylated at either the 4- or 7-position via the phenolic OH and show that several chloro, bromo, methoxy, and nitro substituted benzyl derivatives inhibit Kv1.3 with submicromolar potencies. Representative examples of the most potent compounds from each subclass, 11m (5-acetyl-4-(4'-chloro)benzyloxy-6-hydroxy-7-methoxybenzofuran) and 14m (5-acetyl-7-(4'-bromo)benzyloxy-6-hydroxy-4-methoxybenzofuran), block Kv1.3 with EC50 values of 480 and 400 nM, respectively. Both compounds exhibit moderate selectivity over other Kv1-family channels and HERG, are not cytotoxic, and suppress human T cell proliferation at low micromolar concentrations.
Asunto(s)
Benzofuranos/síntesis química , Inmunosupresores/síntesis química , Canal de Potasio Kv1.3/antagonistas & inhibidores , Bloqueadores de los Canales de Potasio/síntesis química , Benzofuranos/química , Benzofuranos/farmacología , Línea Celular , Proliferación Celular/efectos de los fármacos , Humanos , Inmunosupresores/química , Inmunosupresores/farmacología , Técnicas In Vitro , Canal de Potasio Kv1.3/fisiología , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio/química , Bloqueadores de los Canales de Potasio/farmacología , Relación Estructura-Actividad , Linfocitos T/citología , Linfocitos T/efectos de los fármacosRESUMEN
Family GH16 glycoside hydrolases can be assigned to five subgroups according to their substrate specificities, including xyloglucan transglucosylases/hydrolases (XTHs), (1,3)-beta-galactanases, (1,4)-beta-galactanases/kappa-carrageenases, "nonspecific" (1,3/1,3;1,4)-beta-D-glucan endohydrolases, and (1,3;1,4)-beta-D-glucan endohydrolases. A structured family GH16 glycoside hydrolase database has been constructed (http://www.ghdb.uni-stuttgart.de) and provides multiple sequence alignments with functionally annotated amino acid residues and phylogenetic trees. The database has been used for homology modeling of seven glycoside hydrolases from the GH16 family with various substrate specificities, based on structural coordinates for (1,3;1,4)-beta-D-glucan endohydrolases and a kappa-carrageenase. In combination with multiple sequence alignments, the models predict the three-dimensional (3D) dispositions of amino acid residues in the substrate-binding and catalytic sites of XTHs and (1,3/1,3;1,4)-beta-d-glucan endohydrolases; there is no structural information available in the databases for the latter group of enzymes. Models of the XTHs, compared with the recently determined structure of a Populus tremulos x tremuloides XTH, reveal similarities with the active sites of family GH11 (1,4)-beta-D-xylan endohydrolases. From a biological viewpoint, the classification, molecular modeling and a new 3D structure of the P. tremulos x tremuloides XTH establish structural and evolutionary connections between XTHs, (1,3;1,4)-beta-D-glucan endohydrolases and xylan endohydrolases. These findings raise the possibility that XTHs from higher plants could be active not only on cell wall xyloglucans, but also on (1,3;1,4)-beta-D-glucans and arabinoxylans, which are major components of walls in grasses. A role for XTHs in (1,3;1,4)-beta-D-glucan and arabinoxylan modification would be consistent with the apparent overrepresentation of XTH sequences in cereal expressed sequence tags databases.
Asunto(s)
Glicósido Hidrolasas/química , Glicosiltransferasas/química , Glicosiltransferasas/genética , Glicosiltransferasas/fisiología , Modelos Moleculares , Poaceae/enzimología , Secuencia de Aminoácidos , Pared Celular/química , Pared Celular/enzimología , Endo-1,4-beta Xilanasas/química , Glucanos/metabolismo , Glicósido Hidrolasas/genética , Glicosiltransferasas/clasificación , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Filogenia , Estructuras de las Plantas/química , Homología de Secuencia , Homología de Secuencia de Aminoácido , Xilanos/metabolismoRESUMEN
The voltage-gated potassium channel Kv1.3 constitutes a promising new target for the treatment of T-cell-mediated autoimmune diseases such as multiple sclerosis. In this study, we report the discovery of two new classes of Kv1.3 blockers based on the naturally occurring compound khellinone, 5-acetyl-4,7-dimethoxy-6-hydroxybenzofuran: (1) khellinone dimers linked via the alkylation of the 6-hydroxy groups and (2) chalcone derivatives of khellinone formed by Claisen-Schmidt condensation of the 5-acetyl group with aryl aldehydes. In particular, the chalcone 3-(4,7-dimethoxy-6-hydroxybenzofuran-5-yl)-1-phenyl-3-oxopropene (16) and several of its derivatives inhibited Kv1.3 with K(d) values of 300-800 nM and a Hill coefficient of 2, displayed moderate selectivity over other Kv1-family K(+) channels, suppressed T-lymphocyte proliferation at submicromolar concentrations, and showed no signs of acute toxicity in mice. Because of their relatively low molecular weight and lipophilicity and their high affinity to Kv1.3, aryl-substituted khellinone derivatives represent attractive lead compounds for the development of more potent and selective Kv1.3 blocking immunosuppressants.
Asunto(s)
Benzofuranos/síntesis química , Chalcona/síntesis química , Inmunosupresores/síntesis química , Bloqueadores de los Canales de Potasio/síntesis química , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/efectos de los fármacos , Animales , Benzofuranos/química , Benzofuranos/farmacología , División Celular , Línea Celular , Chalcona/análogos & derivados , Chalcona/química , Chalcona/farmacología , Chalconas , Humanos , Inmunosupresores/química , Inmunosupresores/farmacología , Activación del Canal Iónico , Canal de Potasio Kv1.3 , Ratones , Estructura Molecular , Bloqueadores de los Canales de Potasio/química , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio/fisiología , Relación Estructura-Actividad , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/fisiología , Pruebas de Toxicidad AgudaRESUMEN
A family 16 glycoside hydrolase, xyloglucan xyloglucosyl transferase (EC 2.4.1.207), also known as xyloglucan endotransglycosylase (XET), and designated isoenzyme HvXET6, was purified approximately 400-fold from extracts of young barley seedlings. The complete amino acid sequence of HvXET6 was deduced from the nucleotide sequence of a near full-length cDNA, in combination with tryptic peptide mapping. An additional five to six isoforms or post-translationally modified XET enzymes were detected in crude seedling extracts of barley. The HvXET6 isoenzyme was expressed in Pichia pastoris, characterized and compared with the previously purified native HvXET5 isoform. Barley HvXET6 has a similar apparent molecular mass of 33-35 kDa to the previously purified HvXET5 isoenzyme, but the two isoenzymes differ in their isoelectric points, pH optima, kinetic properties and substrate specificities. The HvXET6 isoenzyme catalyses transfer reactions between xyloglucans and soluble cellulosic substrates, using oligo-xyloglucosides as acceptors, but at rates that are significantly different from those observed for HvXET5. No hydrolytic activity could be detected with either isoenzyme. Comparisons of the reaction rates using xyloglucan or hydroxyethyl cellulose as donors and a series of cellodextrins as acceptors indicated that the acceptor site of HvXET can accommodate five glucosyl residues. Molecular modelling supported this conclusion and further confirmed the ability of the enzyme's active site to accommodate xyloglucan and cellulosic substrates. The two HvXETs followed a ping-pong (Bi, Bi) rather than a sequential reaction mechanism.
Asunto(s)
Biocatálisis , Glicosiltransferasas/metabolismo , Hordeum/enzimología , Secuencia de Aminoácidos , Electroforesis , Glicosiltransferasas/química , Glicosiltransferasas/genética , Glicosiltransferasas/aislamiento & purificación , Hordeum/genética , Isoenzimas/química , Isoenzimas/clasificación , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Ionización de Electrospray , Especificidad por Sustrato , Factores de TiempoRESUMEN
Cellulose synthase-like CslF genes have been implicated in the biosynthesis of (1,3;1,4)-beta-d-glucans, which are major cell wall constituents in grasses and cereals. Seven CslF genes from barley (Hordeum vulgare) can be divided into two classes on the basis of intron-exon arrangements. Four of the HvCslF genes have been mapped to a single locus on barley chromosome 2H, in a region corresponding to a major quantitative trait locus for grain (1,3;1,4)-beta-d-glucan content. The other HvCslF genes map to chromosomes 1H, 5H, and 7H, and in two cases the genes are close to other quantitative trait loci for grain (1,3;1,4)-beta-d-glucan content. Spatial and temporal patterns of transcription of the seven genes have been defined through quantitative polymerase chain reaction. In developing barley coleoptiles HvCslF6 mRNA is most abundant. Transcript levels are maximal in 4- to 5-d coleoptiles, at a time when (1,3;1,4)-beta-d-glucan content of coleoptile cell walls also reaches maximal levels. In the starchy endosperm of developing grain, HvCslF6 and HvCslF9 transcripts predominate. Two peaks of transcription are apparent. One occurs just after endosperm cellularization, 4 to 8 d after pollination, while the second occurs much later in grain development, more than 20 d after pollination. Marked varietal differences in transcription of the HvCslF genes are observed during endosperm development. Given the commercial importance of cereal (1,3;1,4)-beta-d-glucans in human nutrition, in stock feed, and in malting and brewing, the observation that only two genes, HvCslF6 and HvCslF9, are transcribed at high levels in developing grain is of potential relevance for the future manipulation of grain (1,3;1,4)-beta-d-glucan levels.
Asunto(s)
Perfilación de la Expresión Génica , Glucosiltransferasas/genética , Hordeum/enzimología , Familia de Multigenes , Transcripción Genética , Secuencia de Bases , Cromosomas Artificiales Bacterianos , Clonación Molecular , Cartilla de ADN , ADN Complementario , Hordeum/genética , Datos de Secuencia Molecular , Reacción en Cadena de la PolimerasaRESUMEN
A characteristic feature of grasses and commercially important cereals is the presence of (1,3;1,4)-beta-d-glucans in their cell walls. We have used comparative genomics to link a major quantitative trait locus for (1,3;1,4)-beta-d-glucan content in barley grain to a cluster of cellulose synthase-like CslF genes in rice. After insertion of rice CslF genes into Arabidopsis, we detected (1,3;1,4)-beta-d-glucan in walls of transgenic plants using specific monoclonal antibodies and enzymatic analysis. Because wild-type Arabidopsis does not contain CslF genes or have (1,3;1,4)-beta-d-glucans in its walls, these experiments provide direct, gain-of-function evidence for the participation of rice CslF genes in (1,3;1,4)-beta-d-glucan biosynthesis.
Asunto(s)
Genes de Plantas , Glucosiltransferasas/genética , Oryza/genética , beta-Glucanos/metabolismo , Anticuerpos Monoclonales , Arabidopsis/química , Arabidopsis/genética , Arabidopsis/metabolismo , Pared Celular/química , Genoma de Planta , Genómica , Glucosiltransferasas/metabolismo , Hordeum/química , Hordeum/genética , Oryza/enzimología , Hojas de la Planta/química , Plantas Modificadas Genéticamente , Sitios de Carácter Cuantitativo , Sintenía , Transformación Genética , beta-Glucanos/análisis , beta-Glucanos/químicaRESUMEN
The ShK toxin is a polypeptide that blocks the Kv1.3 potassium channel in T-lymphocytes and has been identified as a potential therapeutic for multiple sclerosis. ShK is well characterised in terms of structure and binding, offering an attractive target for the design of structural and functional mimetics. Building on our previous success in developing rationally designed peptidomimetics of ShK, we report a novel mimetic of the K22-Y23-R24 residues of the peptide. The mimetic was shown to inhibit the Kv1.3 channel with moderate activity.
Asunto(s)
Venenos de Cnidarios/química , Oligopéptidos/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio con Entrada de Voltaje/antagonistas & inhibidores , Animales , Línea Celular , Epítopos , Canal de Potasio Kv1.3 , Ratones , Imitación Molecular , Oligopéptidos/química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Bloqueadores de los Canales de Potasio/química , Canales de Potasio con Entrada de Voltaje/genética , TransfecciónRESUMEN
ShK toxin is a structurally defined, 35-residue polypeptide which blocks the voltage-gated Kv1.3 potassium channel in T-lymphocytes and has been identified as a possible immunosuppressant. Our interest lies in the rational design and synthesis of type-III mimetics of protein and polypeptide structure and function. ShK toxin is a challenging target for mimetic design as its binding epitope consists of relatively weakly binding residues, some of which are discontinuous. We discuss here our investigations into the design and synthesis of 1st generation, small molecule mimetics of ShK toxin and highlight any principles relevant to the generic design of type-III mimetics of continuous and discontinuous binding epitopes. We complement our approach with attempted pharmacophore-based database mining.
Asunto(s)
Venenos de Cnidarios/química , Venenos de Cnidarios/síntesis química , Diseño de Fármacos , Animales , Sitios de Unión , Venenos de Cnidarios/farmacología , Diseño Asistido por Computadora , Humanos , Inmunosupresores/síntesis química , Inmunosupresores/química , Inmunosupresores/farmacología , Técnicas In Vitro , Modelos Moleculares , Imitación Molecular , Estructura Molecular , Bloqueadores de los Canales de Potasio/síntesis química , Bloqueadores de los Canales de Potasio/química , Bloqueadores de los Canales de Potasio/farmacología , Conformación Proteica , TermodinámicaRESUMEN
Sequence data from cDNA and genomic clones, coupled with analyses of expressed sequence tag databases, indicate that the CesA (cellulose synthase) gene family from barley (Hordeum vulgare) has at least eight members, which are distributed across the genome. Quantitative polymerase chain reaction has been used to determine the relative abundance of mRNA transcripts for individual HvCesA genes in vegetative and floral tissues, at different stages of development. To ensure accurate expression profiling, geometric averaging of multiple internal control gene transcripts has been applied for the normalization of transcript abundance. Total HvCesA mRNA levels are highest in coleoptiles, roots, and stems and much lower in floral tissues, early developing grain, and in the elongation zone of leaves. In most tissues, HvCesA1, HvCesA2, and HvCesA6 predominate, and their relative abundance is very similar; these genes appear to be coordinately transcribed. A second group, comprising HvCesA4, HvCesA7, and HvCesA8, also appears to be coordinately transcribed, most obviously in maturing stem and root tissues. The HvCesA3 expression pattern does not fall into either of these two groups, and HvCesA5 transcript levels are extremely low in all tissues. Thus, the HvCesA genes fall into two general groups of three genes with respect to mRNA abundance, and the co-expression of the groups identifies their products as candidates for the rosettes that are involved in cellulose biosynthesis at the plasma membrane. Phylogenetic analysis allows the two groups of genes to be linked with orthologous Arabidopsis CesA genes that have been implicated in primary and secondary wall synthesis.
Asunto(s)
Genes de Plantas , Glucosiltransferasas/genética , Hordeum/enzimología , Hordeum/genética , Familia de Multigenes , Secuencia de Bases , Mapeo Cromosómico , ADN Complementario/genética , ADN de Plantas/genética , Etiquetas de Secuencia Expresada , Expresión Génica , Filogenia , Estructuras de las Plantas/metabolismo , Plantas/enzimología , Plantas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Planta/genética , ARN de Planta/metabolismoRESUMEN
A putative barley (1 --> 3)-beta-D-glucan synthase cDNA of 6.1 kb, which is homologous to the yeast FKS gene, was assembled from DNA fragments obtained through screening of barley cDNA and BAC libraries, and by PCR amplification. The corresponding gene, designated HvGSL1, is a member of a family of at least six genes in barley. Gene transcripts are detected at relatively high levels in early developing grain, florets, coleoptiles and roots, but not in leaves infected with a fungal pathogen. A (1 --> 3)-beta-D-glucan synthase has been purified more than 60-fold from barley suspension-cultured cells by detergent extraction, CaCl2 treatment, sucrose density gradient centrifugation and non-denaturing gel electrophoresis. The enzyme synthesizes (1 --> 3)-beta-D-glucan in vitro and is recognized by antibodies raised against a 17 kDa protein generated by heterologous expression of a fragment of the HvGSL1 cDNA. Furthermore, mass spectrometric analyses show that tryptic peptides produced by in-gel digestion of the active enzyme match peptides predicted from the gene sequence. Thus, the amino acid sequence predicted from the HvGSL1 gene has been linked with the actual amino acid sequence of an active (1 --> 3)-beta-D-glucan synthase fraction from barley.