RESUMEN
Gonorrhoea infection rates and the risk of infection from opportunistic pathogens including P. aeruginosa have both risen globally, in part due to increasing broad-spectrum antibiotic resistance. Development of new antimicrobial drugs is necessary and urgent to counter infections from drug resistant bacteria. Aspartate-semialdehyde dehydrogenase (ASADH) is a key enzyme in the aspartate biosynthetic pathway, which is critical for amino acid and metabolite biosynthesis in most microorganisms including important human pathogens. Here we present the first structures of two ASADH proteins from N. gonorrhoeae and P. aeruginosa solved by X-ray crystallography. These high-resolution structures present an ideal platform for in silico drug design, offering potential targets for antimicrobial drug development as emerging multidrug resistant strains of bacteria become more prevalent.
Asunto(s)
Aspartato-Semialdehído Deshidrogenasa , Pseudomonas aeruginosa , Antibacterianos , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Neisseria gonorrhoeae/metabolismo , Pseudomonas aeruginosa/metabolismoRESUMEN
General control non-repressible 5 (GCN5)-related N-acetyltransferases (GNATs) catalyse the acetylation of a diverse range of substrates, thereby orchestrating a variety of biological processes within prokaryotes and eukaryotes. GNAT enzymes can catalyze the transfer of an acetyl group from acetyl coenzyme A to substrates such as aminoglycoside antibiotics, amino acids, polyamines, peptides, vitamins, catecholamines, and large macromolecules including proteins. Although GNATs generally exhibit low to moderate sequence identity, they share a conserved catalytic fold and conserved structural motifs. In this current study we characterize the high-resolution X-ray crystallographic structure of a GNAT enzyme bound with acetyl-CoA from Elizabethkingia anophelis, an important multi-drug resistant bacterium. The tertiary structure is comprised of six α-helices and nine ß-strands, and is similar with other GNATs. We identify a new and uncharacterized GNAT dimer interface, which is conserved in at least two other unpublished GNAT structures. This suggests that GNAT enzymes can form at least five different types of dimers, in addition to a range of other oligomers including trimer, tetramer, hexamer, and dodecamer assemblies. The high-resolution structure presented in this study is suitable for future in-silico docking and structure-activity relationship studies.
Asunto(s)
Acetilcoenzima A/metabolismo , Acetiltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Flavobacteriaceae/metabolismo , Acetiltransferasas/química , Proteínas Bacterianas/química , Cristalografía por Rayos X , Flavobacteriaceae/química , Modelos Moleculares , Unión Proteica , Conformación Proteica , Multimerización de ProteínaRESUMEN
Histone deacetylases (HDACs) are a growing family of enzymes implicated in transcriptional regulation by affecting the acetylation state of core histones in the nucleus of cells. HDACs are known to have key roles in the regulation of cell proliferation [Brehm, Miska, McCance, Reid, Bannister and Kouzarides (1998) Nature (London) 391, 597-600], and aberrant recruitment of an HDAC complex has been shown to be a key step in the mechanism of cell transformation in acute promyelocytic leukaemia [Grignani, De Matteis, Nervi, Tomassoni, Gelmetti, Cioce, Fanelli, Ruthardt, Ferrara, Zamir et al. (1998) Nature (London) 391, 815-818; Lin, Nagy, Inoue, Shao, Miller and Evans (1998), Nature (London) 391, 811-814]. Here we present the complete nucleotide sequence of a cDNA clone, termed HDAC8, that encodes a protein product with similarity to the RPD3 class (I) of HDACs. The predicted 377-residue HDAC8 product contains a shorter C-terminal extension relative to other members of its class. After expression in two cell systems, immunopurified HDAC8 is shown to possess trichostatin A- and sodium butyrate-inhibitable HDAC activity on histone H4 peptide substrates as well as on core histones. Expression profiling reveals the expression of HDAC8 to various degrees in every tissue tested and also in several tumour lines. Mutation of two adjacent histidine residues within the predicted active site severely decreases activity, confirming these residues as important for HDAC8 enzyme activity. Finally, linkage analysis after radiation hybrid mapping has localized HDAC8 to chromosomal position Xq21.2-Xq21.3. These results confirm HDAC8 as a new member of the HDAC family.
Asunto(s)
Histona Desacetilasas/genética , Proteínas Represoras/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Mapeo Cromosómico , ADN Complementario , Células HeLa , Histona Desacetilasas/química , Histona Desacetilasas/metabolismo , Humanos , Datos de Secuencia Molecular , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Homología de Secuencia de Aminoácido , Spodoptera , Cromosoma XRESUMEN
A murine leukemia retroviral vector was engineered to contain the DNA encoding either the wild-type, rat aorta 20-kDa myosin light chain (MLC20) or a mutant form of MLC20 in which Thr18 and Ser19 were mutated into alanines. These mutations result in a MLC20 that cannot be phosphorylated by myosin light chain kinase. An 11-amino acid epitope from c-myc was added to both MLC20 sequences to facilitate identification of these proteins. Madin-Darby canine kidney cells were stably transduced, and MLC20 expression was demonstrated by Western blot analysis using a myc-specific antibody. MLC20 exchange was demonstrated by purifying myosin from the transduced cells and repeating the Western blot analysis. Actin-activated adenosinetriphosphatase assays on the purified myosins demonstrated approximately 50% decrease in the rate of ATP hydrolysis by the myosin containing the mutant MLC20. Transepithelial electrical resistance was decreased and mannitol flux was increased across monolayers of cells expressing mutant MLC20. These data demonstrate that MLC20 phosphorylation is involved in regulating paracellular permeability and epithelial barrier function.
Asunto(s)
Riñón/metabolismo , Mutación , Cadenas Ligeras de Miosina/genética , Cadenas Ligeras de Miosina/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Línea Celular , Perros , Impedancia Eléctrica , Riñón/citología , Riñón/fisiología , Manitol/farmacocinética , Microscopía Fluorescente , Miosinas/metabolismo , Permeabilidad , Fosforilación , Ratas , Proteínas RecombinantesRESUMEN
Galanin is a neuroendocrine peptide that modulates many different normal physiological effects including memory, weight, and pain perception. To better understand galanin receptor function, we cloned and characterized the galanin-1 receptor (GALN1R) gene isolated from a human P1 library. We determined that this gene contains 2 introns of approximately 2.1 and 2.9 Kb, both of which are located in the 3rd intracellular loop. We identified transcriptional initiation sites 61 and 63 bp upstream of the translation initiation codon ATG. Sequencing of the GALN1R gene 5' flanking region revealed it to be GC rich and devoid of TATA or CCAAT boxes, features of housekeeping genes. To identify potential sites regulating promoter activity, variable lengths of the 5' flanking region were fused to a CAT gene and studied in Bowes human melanoma cells. Significant losses in CAT activity were observed only with the elimination of 2 NF-kappa B sites, located -269 and -809 bp upstream from the translational start site, respectively. These findings suggest the novel possibility that GALN1R gene expression may be regulated as a consequence of inflammatory conditions. Finally fluorescent in situ hybridization (FISH) assigned this gene to chromosome 15q24.
Asunto(s)
Cromosomas Humanos Par 15 , Regulación de la Expresión Génica , Receptores de la Hormona Gastrointestinal/genética , Secuencia de Bases , Sitios de Unión , Mapeo Cromosómico , Clonación Molecular , Genes Reporteros , Humanos , Melanoma , Datos de Secuencia Molecular , FN-kappa B/metabolismo , Regiones Promotoras Genéticas , Receptores de Galanina , Transcripción Genética , Células Tumorales CultivadasRESUMEN
Contractile events resulting from phosphorylation of the 20-kDa myosin light chain (MLC20) have been implicated in the regulation of epithelial tight junction permeability. To address this question, Madin-Darby canine kidney cells were transfected with a murine leukemia retroviral vector containing DNA encoding either the catalytic domain of myosin light chain kinase (tMK) or the beta-galactosidase gene (beta-gal). Autoradiograms of sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of myosin immunoprecipitated from 32Pi-labeled transfected cells demonstrated that MLC20 phosphorylation was increased 3.1 +/- 0.9-fold in cells expressing tMK compared with cells expressing beta-gal. Phosphopeptide mapping confirmed that myosin light chain kinase was responsible for the increased MLC20 phosphorylation. Transepithelial electrical resistance, a measurement of barrier function, of tMK cell monolayers was consistently < 10% (123 +/- 20 omega.cm2) of that of monolayers comprised of wild-type cells (1,456 +/- 178 omega.cm2) or cells expressing beta-gal (1,452 +/- 174 omega.cm2). Dual 22Na+ and [3H]mannitol flux studies indicated that the decrease in resistance in tMK cells was attributable to increased paracellular flow. These data support the idea that MLC20 phosphorylation by myosin light chain kinase is involved in regulating epithelial tight junction permeability.
Asunto(s)
Permeabilidad de la Membrana Celular , Quinasa de Cadena Ligera de Miosina/biosíntesis , Animales , Calcio/farmacología , Línea Celular , Citoesqueleto/fisiología , Cartilla de ADN , Perros , Epitelio/fisiología , Riñón , Virus de la Leucemia Murina , Sustancias Macromoleculares , Manitol/metabolismo , Cadenas Ligeras de Miosina/química , Cadenas Ligeras de Miosina/metabolismo , Mapeo Peptídico , Fosfopéptidos/química , Fosfopéptidos/aislamiento & purificación , Fosforilación , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/biosíntesis , Uniones Estrechas/fisiología , Transfección , beta-Galactosidasa/biosíntesisRESUMEN
Recent studies suggest that galanin receptors may be expressed by mucosal epithelial cells lining the GI tract and play a role in regulating ion transport. However, no information exists as to which receptor subtype is expressed by these mucosal cells, or the relative distribution of such receptors within the GI tract. In this study we cloned the human galanin-1 receptor (huGal 1-R) from small intestinal RNA, and demonstrate its expression in endoscopically obtained mucosal pinch biopsies by RT-PCR from the esophagus to the rectum. Semi-quantitative PCR (SQ-PCR) was performed and revealed the largest amount of huGal 1-R message in the duodenum and the least amount in the gastric fundus. This study for the first time directly demonstrates the presence and quantity of huGal 1-R message in human G1 tract mucosal epithelial cells, and suggests that this subtype is of primary importance in regulating galanin-induced changes in intestinal absorption.
Asunto(s)
Sistema Digestivo/metabolismo , Mucosa Gástrica/metabolismo , Expresión Génica , Mucosa Intestinal/metabolismo , Receptores de la Hormona Gastrointestinal/biosíntesis , Secuencia de Bases , Biopsia , Clonación Molecular , Colon/metabolismo , Secuencia de Consenso , Cartilla de ADN , ADN Complementario , Sistema Digestivo/citología , Esófago/metabolismo , Histonas/biosíntesis , Humanos , Intestino Delgado , Melanoma/metabolismo , Datos de Secuencia Molecular , Membrana Mucosa/metabolismo , Especificidad de Órganos , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Receptores de Galanina , Recto/metabolismo , Homología de Secuencia de Ácido NucleicoRESUMEN
We investigated the role of myosin light chain (MLC20) phosphorylation (MLC-P) in non-muscle contractility by comparing MLC-P and the contractile properties of wild type 3T3 fibroblasts and 3T3 fibroblasts expressing the catalytic domain of myosin light chain kinase (tMK). MLC-P is 0.96 MOL of PO4/mol of MOL20 in cell expressing tMK compared to 0.20 mol of PO4/mol of MLC20 in control cells. Expressing tMK also results in a 2-fold increase in cortical stiffness compared to control cells. Contractile properties were quantified by growing wild type and transfected fibroblasts in collagen and attaching the ensuing fibers to an apparatus for performing mechanical measurements. Serum stimulation resulted in a dose-dependent increase in force with maximal force generated in the presence of 30% (v/v) serum. Surprisingly, MLC-P did not increase in wild type cells following stimulation with 30% serum, and tMK expression did not affect the contractile properties of fibers made from these cells. Moreover, the dose responses to serum, maximal force, force-velocity relationships, and dynamic stiffness were similar in the wild type cells and fibroblasts expressing tMK. These data demonstrate that non-muscle cells can generate force without an increase in MLC-P, and that an increase in MLC-P does not affect the contractile properties of fibroblast fibers.
Asunto(s)
Quinasa de Cadena Ligera de Miosina/fisiología , Miosinas/metabolismo , Células 3T3 , Animales , Fibroblastos/fisiología , Ratones , FosforilaciónRESUMEN
A series of deletion mutants of the wild-type Escherichia coli lactose promoter, with endpoints at +25, +19, +14, +1 and -6 (relative to the start of transcription at +1), was constructed and the deleted DNA replaced with non-lac DNA. These mutants were used to show that no specific DNA sequences downstream from -6 are required for efficient promoter utilization in vitro. In all cases transcription is dependent on the presence of the catabolite activator protein (CAP) and cAMP, and begins at +1 at a level indistinguishable from that at the wild-type promoter. A set of lac DNA fragments deleted to -6 was constructed, having an A, C, G or T residue at +1 and heterologous DNA downstream. These synthetic promoters allow systematic testing of the effect of the initiating nucleotide on the transcription process. Again, transcription occurs mainly from +1, at a level similar to the normal wild-type level. No substantial differences between these promoters are observed in the rates of formation of stable complexes, in the degree of complex formation, in the rate at which polymerase "escapes" from the complex or in abortive transcription products. Equivalent results are seen with a related set of constructs based on the CAP-insensitive lac UV5 promoter. Thus, lac promoter sequences including consensus hexamers at -10 and -35, plus the spacer region between them, provide specificity and efficiency both in initiation of transcription by RNA polymerase and in CAP-polymerase interactions. A question as to whether there is a third RNA polymerase binding site at lac, in addition to the known overlapping P1 and P2 regions, was not unambiguously answered. However, if a "P3" site does exist, it must lie between P1 and P2. Alternatively, the variety of polymerase interactions at wild-type lac may reflect different structural states of the enzyme. The results presented here indicate that DNA downstream from -6 plays little part in determining the conformation of the enzyme at the lactose promoter.
Asunto(s)
Escherichia coli/genética , Genes Bacterianos , Lactosa/metabolismo , Regiones Promotoras Genéticas , Transcripción Genética , Secuencia de Bases , Deleción Cromosómica , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Cinética , Datos de Secuencia Molecular , Mutación , Mapeo RestrictivoRESUMEN
The lac promoter is known to have overlapping, mutually exclusive, binding sites for RNA polymerase. A number of techniques have been used to probe solutions of polymerase and linear lac DNA fragments, including gel electrophoresis binding assays, transcription experiments, and exonuclease III digestions. The data indicate that mixing RNA polymerase with the wild type lac promoter leads to formation of more than one kind of complex; a typical solution contains enzyme in heparin resistant, "open" complexes at the P2 site, while other DNA molecules have polymerase bound in a heparin sensitive, "closed" complex at P1. There may be other rather stable complexes as well. The presence of more than one type of complex has obvious implications for in vitro physical studies of this system. The data suggest that using truncated DNA fragments which eliminate the P2 site may allow isolation and study of P1 closed complexes. Quantitative analysis of the fractions of polymerase found at P1 and P2 implies that P2 can have only a limited effect on lac transcription in the cell.