RESUMEN
8-Membered cyclic ethers are found in a wide range of natural products; however, they are challenging synthetic targets due to enthalpic and entropic barriers. The gold(I)-catalyzed intramolecular dehydrative alkoxylation of ω-hydroxy allylic alcohols was explored to stereoselectively construct α,α'-cis-oxocenes and further applied in a formal synthesis of (+)-laurencin. The gold(I)-catalyzed intramolecular dehydrative alkoxylation may constitute an alternative method for the synthesis of molecular building blocks and natural products that contain highly functionalized 8-membered cyclic ethers.
Asunto(s)
Productos Biológicos/síntesis química , Éteres Cíclicos/síntesis química , Oro/química , Oxocinas/síntesis química , Productos Biológicos/química , Catálisis , Éteres Cíclicos/química , Oxocinas/química , Propanoles/síntesis química , Propanoles/química , EstereoisomerismoRESUMEN
The molybdenum cofactor (Moco) is a redox cofactor found in all kingdoms of life, and its biosynthesis is essential for survival of many organisms, including humans. The first step of Moco biosynthesis is a unique transformation of guanosine 5'-triphosphate (GTP) into cyclic pyranopterin monophosphate (cPMP). In bacteria, MoaA and MoaC catalyze this transformation, although the specific functions of these enzymes were not fully understood. Here, we report the first isolation and structural characterization of a product of MoaA. This molecule was isolated under anaerobic conditions from a solution of MoaA incubated with GTP, S-adenosyl-L-methionine, and sodium dithionite in the absence of MoaC. Structural characterization by chemical derivatization, MS, and NMR spectroscopy suggested the structure of this molecule to be (8S)-3',8-cyclo-7,8-dihydroguanosine 5'-triphosphate (3',8-cH2GTP). The isolated 3',8-cH2GTP was converted to cPMP by MoaC or its human homologue, MOCS1B, with high specificities (Km < 0.060 µM and 0.79 ± 0.24 µM for MoaC and MOCS1B, respectively), suggesting the physiological relevance of 3',8-cH2GTP. These observations, in combination with some mechanistic studies of MoaA, unambiguously demonstrate that MoaA catalyzes a unique radical C-C bond formation reaction and that, in contrast to previous proposals, MoaC plays a major role in the complex rearrangement to generate the pyranopterin ring.
Asunto(s)
Coenzimas/metabolismo , Metaloproteínas/metabolismo , Nucleótidos Cíclicos/biosíntesis , Pteridinas/metabolismo , Biocatálisis , Coenzimas/química , Cristalografía por Rayos X , Humanos , Hidrolasas/metabolismo , Metaloproteínas/química , Modelos Moleculares , Conformación Molecular , Cofactores de Molibdeno , Nucleótidos Cíclicos/química , Pteridinas/químicaRESUMEN
The Hedgehog signaling pathway plays an essential role in embryo development and adult tissue homeostasis, in regulating stem cells and is abnormally activated in many cancers. Given the importance of this signaling pathway, we developed a novel and versatile high-throughput, cell-based screening platform using confocal imaging, based on the role of ß-arrestin in Hedgehog signal transduction, that can identify agonists or antagonist of the pathway by a simple change to the screening protocol. Here we report the use of this assay in the antagonist mode to identify novel antagonists of Smoothened, including a compound (A8) with low nanomolar activity against wild-type Smo also capable of binding the Smo point mutant D473H associated with clinical resistance in medulloblastoma. Our data validate this novel screening approach in the further development of A8 and related congeners to treat Hedgehog related diseases, including the treatment of basal cell carcinoma and medulloblastoma.
Asunto(s)
Benzamidas/química , Proteínas Hedgehog/metabolismo , Piridinas/química , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Animales , Benzamidas/síntesis química , Benzamidas/farmacología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Femenino , Cabello/efectos de los fármacos , Cabello/crecimiento & desarrollo , Humanos , Ratones , Ratones Endogámicos C57BL , Mutación , Neuronas/citología , Piridinas/síntesis química , Piridinas/farmacología , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Receptor SmoothenedRESUMEN
The polar lipids of the anaerobic bacterium Clostridium tetani, the causative agent of tetanus, have been examined by two-dimensional thin layer chromatography, ESI mass spectrometry, and NMR spectroscopy. Plasmalogen and di- and tetra-acylated species of phosphatidylethanolamine, phosphatidylglycerol, cardiolipin, and N-acetylglucosaminyl diradylglycerol were the major lipids present in most strains examined except for strain ATCC 10779, the parent of strain E88, the first C. tetani strain to have its genome sequenced. This strain contained the same di- and tetra-acylated species but did not contain plasmalogens. All strains contained a novel derivative of N-acetylglucosaminyl diradylglycerol in which a phosphoethanolamine unit is attached to the 6'-position of the sugar, as judged by selective 31P-decoupled, 1H-detected NMR difference spectroscopy. The N-acetylglucosamine (GlcNAc) residue is presumably linked to the 3-positon of the diradylglycerol moiety, and it has the beta-anomeric configuration. Very little plasmalogen component was detected by mass spectrometry in the precursors phosphatidic acid and phosphatidylserine, consistent with the idea that plasmalogens are formed from diacylated phospholipids at a late stage of phospholipid assembly in anaerobic clostridia.
Asunto(s)
Clostridium tetani/química , Diglicéridos/análisis , Etanolaminas/análisis , Lípidos/análisis , Carbono/química , Cromatografía Liquida , Cromatografía en Capa Delgada , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Francisella tularensis is a highly infectious pathogen that causes tularemia. Francisella lipid A contains an unusual galactosamine (GalN) unit, attached to its 1-phosphate moiety. Two genes, flmF2 and flmK, are required for the addition of GalN to Francisella lipid A, but the relevant enzymes and the GalN donor substrate have not been characterized. We now report the purification and identification of a novel minor lipid from Francisella novicida that functions as the GalN donor. On the basis of electrospray ionization mass spectrometry (ESI/MS) and NMR spectroscopy, we propose that this compound is undecaprenyl phosphate-beta-d-GalN. Approximately 0.5 mg of pure lipid was obtained from 10 g of F. novicida by chloroform/methanol extraction, followed by DEAE-cellulose chromatography, mild alkaline hydrolysis, and thin-layer chromatography. ESI/MS in the negative mode revealed a molecular ion [M - H](-) at m/z 1006.699, consistent with undecaprenyl phosphate-GalN. (31)P NMR spectroscopy showed a single phosphorus atom in the phosphodiester linkage. Selective inverse decoupling difference spectroscopy demonstrated that the undecaprenyl phosphate group is attached to the anomeric carbon of the sugar. (1)H NMR studies showed the presence of a polyisoprene chain and a sugar consistent with a beta-d-GalN unit. Heteronuclear multiple-quantum coherence (HMQC) analysis confirmed that nitrogen is attached to C-2 of the sugar. Purified undecaprenyl phosphate-beta-d-GalN supports the in vitro modification of lipid IV(A) by membranes of Escherichia coli cells expressing FlmK, an orthologue of E. coli ArnT, the enzyme that transfers 4-amino-4-deoxy-l-arabinose to lipid A in polymyxin-resistant strains. The discovery of undecaprenyl phosphate-beta-d-GalN suggests Francisella modifies lipid A with GalN on the periplasmic surface of the inner membrane.
Asunto(s)
Acetilgalactosamina/metabolismo , Galactosamina/análogos & derivados , Lípido A/metabolismo , Fosfatos de Poliisoprenilo/metabolismo , Acetilgalactosamina/química , Acetilgalactosamina/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Biocatálisis , Francisella , Galactosamina/química , Galactosamina/aislamiento & purificación , Galactosamina/metabolismo , Lípido A/química , Espectroscopía de Resonancia Magnética , Fosfatos de Poliisoprenilo/química , Fosfatos de Poliisoprenilo/aislamiento & purificación , Espectrometría de Masa por Ionización de Electrospray , Especificidad por SustratoRESUMEN
We report the synthesis of α-trifluoromethyl benzylic amines through the vicinal fluoroarylation of gem-difluoro-2-azadienes. Our studies indicate that XPhos plays an important role as a phase transfer catalyst that promotes the addition of AgF to the difluoroazadiene, generating an α-trifluoromethyl azaallyl-silver intermediate that we have characterized by NMR spectroscopy. This intermediate likely transmetallates to Pd, coupling several aryl iodides to deliver products in up to 90% yield. Modification of the azadiene's activating group facilitates challenging cross-couplings.
RESUMEN
The karlotoxins (KmTxs) are a family of compounds produced by the dinoflagellate Karlodinium veneficum that cause membrane permeabilization. The structure of KmTx 1, determined using extensive 2D NMR spectroscopy, is very similar to the amphidinols and related compounds, though KmTx 1 features unique structural modifications of the conserved core region. The structure of KmTx 1 differs from that reported for KmTx 2, the only other reported karlotoxin to date, in lacking chlorination at its terminal alkene and possessing a hydrophobic arm that is two carbons longer.
RESUMEN
Lipid-derived inositol phosphates (IPs) are a complex group of second messengers generated by the sequential phosphorylation of inositol 1,4,5-trisphosphate (IP(3)). Synthetic pathways leading from IP(3) to the formation of inositol tetrakisphosphate IP(4), inositol pentakisphosphate IP(5), inositol hexakisphosphate IP(6), and inositol pyrophosphates PP-IPs have been elucidated in eukaryotes from yeast to human. Studies have attributed a variety of cellular functions to IPs, highlighting the importance of understanding how the pathways for their synthesis are regulated. This chapter summarizes experimental techniques for the biochemical characterization of the key inositol phosphate kinases IPKs necessary for producing the diverse array of IP species.
Asunto(s)
Inositol 1,4,5-Trifosfato/metabolismo , Fosfatos de Inositol/metabolismo , Fosfotransferasas/metabolismo , Arabidopsis/enzimología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cromatografía Líquida de Alta Presión/métodos , Cromatografía en Capa Delgada/métodos , Inositol 1,4,5-Trifosfato/química , Inositol 1,4,5-Trifosfato/genética , Espectroscopía de Resonancia Magnética/métodos , Fosforilación , Fosfotransferasas/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
A stereoselective radical cascade cyclization to 5alpha-pregnanes is presented. Oxidative free radical cyclization of an appropriately substituted chloro cyano ester polyene was used to introduce the all trans stereochemistry in the steroid nucleus. The cyano group was utilized to introduce a C-8beta angular hydrogen, while the chloro ester moiety served as an entry to the geminal hydrogens at C-4.
RESUMEN
A series of monoacyl and diacyl 1,2,4-triazolidine-3,5-diones substituted at position 4 with either a phenyl or a tert-butyl group was prepared. Both acetyl and benzoyl groups were utilized as the acyl substituents. The diacylated compounds containing one or two acetyl groups were somewhat unstable to moisture. The acylated compounds were studied by (1)H, (13)C and (15)N NMR spectroscopy and X-ray crystallography to determine if they were acylated on nitrogen or ring carbonyl oxygen. The results indicated that the acylations occurred on nitrogen. The NMR spectra and molecular modeling computations were used to assign conformations to several of the diacylated compounds.
RESUMEN
A stereoselective oxidative free-radical cyclization of beta-keto ester polyenes 7 and 19 has been accomplished as a one-step entry to the tricarbocyclic synthons 8and 21 which contain five and six stereogenic centers, respectively. These key synthons possessing an axial carboethoxy group at C-4 were ultimately converted to the spongian skeleton (8--> 14 and 21 --> 25 -->14). The synthesis of d,l-isospongiadiol (3) from the common intermediate 14 was realized after introduction of the 2alpha-hydroxy group in the spongian A-ring via epoxidation of silyl enol ether 28 and subsequent desilylation.
RESUMEN
The accuracy, precision, and reproducibility of a rapid method for determination of fluoride in wine, using a fluoride selective ion electrode, were established by a collaborative study involving 12 laboratories, 5 in Europe and 7 in the United States. The laboratories assayed 6 Youden pairs of fluoride-fortified, red and white wine samples with fluoride concentrations ranging from 0.2 to 3.0 mg/L. The relative standard deviations of repeatability ranged from 1.94 to 4.88%; relative standard deviations of reproducibility ranged from 4.15 to 18.40%. HORRAT values ranged from 0.30 to 0.97. The average recovery was 99.97%. Based on the statistical results of this collaborative study, the Study Director recommends that this method be adopted First Action.
Asunto(s)
Fluoruros/análisis , Vino/análisis , Algoritmos , Tampones (Química) , Calibración , Electroquímica , Electrodos , Concentración de Iones de Hidrógeno , Indicadores y Reactivos , Estándares de Referencia , Reproducibilidad de los Resultados , SolucionesRESUMEN
Lactate accumulation in tumors has been associated with metastases and poor overall survival in cancer patients. Lactate promotes angiogenesis and metastasis, providing rationale for understanding how it is processed by cells. The concentration of lactate in tumors is a balance between the amount produced, amount carried away by vasculature and if/how it is catabolized by aerobic tumor or stromal cells. We examined lactate metabolism in human normal and breast tumor cell lines and rat breast cancer: 1. at relevant concentrations, 2. under aerobic vs. hypoxic conditions, 3. under conditions of normo vs. hypoglucosis. We also compared the avidity of tumors for lactate vs. glucose and identified key lactate catabolites to reveal how breast cancer cells process it. Lactate was non-toxic at clinically relevant concentrations. It was taken up and catabolized to alanine and glutamate by all cell lines. Kinetic uptake rates of lactate in vivo surpassed that of glucose in R3230Ac mammary carcinomas. The uptake appeared specific to aerobic tumor regions, consistent with the proposed "metabolic symbiont" model; here lactate produced by hypoxic cells is used by aerobic cells. We investigated whether treatment with alpha-cyano-4-hydroxycinnamate (CHC), a MCT1 inhibitor, would kill cells in the presence of high lactate. Both 0.1 mM and 5 mM CHC prevented lactate uptake in R3230Ac cells at lactate concentrations at ≤ 20 mM but not at 40 mM. 0.1 mM CHC was well-tolerated by R3230Ac and MCF7 cells, but 5 mM CHC killed both cell lines ± lactate, indicating off-target effects. This study showed that breast cancer cells tolerate and use lactate at clinically relevant concentrations in vitro (± glucose) and in vivo. We provided additional support for the metabolic symbiont model and discovered that breast cells prevailingly take up and catabolize lactate, providing rationale for future studies on manipulation of lactate catabolism pathways for therapy.
Asunto(s)
Neoplasias de la Mama/metabolismo , Ácido Láctico/metabolismo , Adulto , Anciano , Alanina/biosíntesis , Animales , Neoplasias de la Mama/patología , Muerte Celular/efectos de los fármacos , Hipoxia de la Célula , Línea Celular Tumoral , Ácidos Cumáricos/farmacología , Modelos Animales de Enfermedad , Femenino , Glucosa/metabolismo , Ácido Glutámico/biosíntesis , Humanos , Cinética , Redes y Vías Metabólicas/efectos de los fármacos , Metabolómica , Persona de Mediana Edad , Estadificación de Neoplasias , RatasRESUMEN
Switching to a glycolytic metabolism is a rapid adaptation of tumor cells to hypoxia. Although this metabolic conversion may primarily represent a rescue pathway to meet the bioenergetic and biosynthetic demands of proliferating tumor cells, it also creates a gradient of lactate that mirrors the gradient of oxygen in tumors. More than a metabolic waste, the lactate anion is known to participate to cancer aggressiveness, in part through activation of the hypoxia-inducible factor-1 (HIF-1) pathway in tumor cells. Whether lactate may also directly favor HIF-1 activation in endothelial cells (ECs) thereby offering a new druggable option to block angiogenesis is however an unanswered question. In this study, we therefore focused on the role in ECs of monocarboxylate transporter 1 (MCT1) that we previously identified to be the main facilitator of lactate uptake in cancer cells. We found that blockade of lactate influx into ECs led to inhibition of HIF-1-dependent angiogenesis. Our demonstration is based on the unprecedented characterization of lactate-induced HIF-1 activation in normoxic ECs and the consecutive increase in vascular endothelial growth factor receptor 2 (VEGFR2) and basic fibroblast growth factor (bFGF) expression. Furthermore, using a variety of functional assays including endothelial cell migration and tubulogenesis together with in vivo imaging of tumor angiogenesis through intravital microscopy and immunohistochemistry, we documented that MCT1 blockers could act as bona fide HIF-1 inhibitors leading to anti-angiogenic effects. Together with the previous demonstration of MCT1 being a key regulator of lactate exchange between tumor cells, the current study identifies MCT1 inhibition as a therapeutic modality combining antimetabolic and anti-angiogenic activities.
Asunto(s)
Células Endoteliales/metabolismo , Factor 1 Inducible por Hipoxia/metabolismo , Ácido Láctico/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Neoplasias/irrigación sanguínea , Neovascularización Patológica/metabolismo , Simportadores/metabolismo , Análisis de Varianza , Animales , Western Blotting , Movimiento Celular/fisiología , Células Endoteliales/fisiología , Ensayo de Inmunoadsorción Enzimática , Factores de Crecimiento de Fibroblastos/metabolismo , Humanos , Inmunohistoquímica , Ácido Láctico/farmacología , Luciferasas , Ratones , Resonancia Magnética Nuclear Biomolecular , Reacción en Cadena de la Polimerasa , Interferencia de ARN , ARN Interferente Pequeño/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
We have characterized the positional specificity of the mammalian and yeast VIP/diphosphoinositol pentakisphosphate kinase (PPIP5K) family of inositol phosphate kinases. We deployed a microscale metal dye detection protocol coupled to a high performance liquid chromatography system that was calibrated with synthetic and biologically synthesized standards of inositol pyrophosphates. In addition, we have directly analyzed the structures of biological inositol pyrophosphates using two-dimensional 1H-1H and 1H-31P nuclear magnetic resonance spectroscopy. Using these tools, we have determined that the mammalian and yeast VIP/PPIP5K family phosphorylates the 1/3-position of the inositol ring in vitro and in vivo. For example, the VIP/PPIP5K enzymes convert inositol hexakisphosphate to 1/3-diphosphoinositol pentakisphosphate. The latter compound has not previously been identified in any organism. We have also unequivocally determined that 1/3,5-(PP)2-IP4 is the isomeric structure of the bis-diphosphoinositol tetrakisphosphate that is synthesized by yeasts and mammals, through a collaboration between the inositol hexakisphosphate kinase and VIP/PPIP5K enzymes. These data uncover phylogenetic variability within the crown taxa in the structures of inositol pyrophosphates. For example, in the Dictyostelids, the major bis-diphosphoinositol tetrakisphosphate is 5,6-(PP)2-IP4 ( Laussmann, T., Eujen, R., Weisshuhn, C. M., Thiel, U., Falck, J. R., and Vogel, G. (1996) Biochem. J. 315, 715-725 ). Our study brings us closer to the goal of understanding the structure/function relationships that control specificity in the synthesis and biological actions of inositol pyrophosphates.
Asunto(s)
Fosfatos de Inositol/química , Fosfotransferasas (Aceptor del Grupo Fosfato)/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimología , Animales , Dictyosteliida/enzimología , Humanos , Fosfatos de Inositol/metabolismo , Ratones , Estructura Molecular , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Filogenia , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidad de la Especie , Especificidad por Sustrato/fisiología , Células 3T3 SwissRESUMEN
BACKGROUND AND PURPOSE: Dietary intake of citrate in the form of citrus juices (eg, lemonade, orange juice) will enhance urinary citrate excretion, a valuable benefit for patients with hypocitraturic calcium oxalate nephrolithiasis. While information on citrate concentrations in select citrus juices is available, data on citrate concentrations of commercially available beverages (juice and otherwise) are limited. Using nuclear magnetic resonance spectroscopy (1H NMR), we report citrate concentrations of several beverages to help guide dietary recommendations aimed at increasing urinary citrate excretion and correcting hypocitraturia. METHODS: Citrate concentrations of a squeezed lemon, several fruit juices, and common beverages were measured using 1H NMR. Spectra for each sample were obtained in duplicate; citrate peak was identified, measured, and quantified and compared with the citrate concentration in the juice of 1 medium lemon. RESULTS: Quantitative analysis revealed the highest concentration of citrate was in grapefruit juice (64.7 mmol/L), followed in decreasing concentrations by lemon juice (47.66 mmol/L), orange juice (47.36 mmol/L), pineapple juice (41.57 mmol/L), reconstituted lemonade (38.65 mmol/L), lemonade flavored Crystal Light (38.39 mmol/L), ready to consume not from concentrate lemonade (38.24 mmol/L), cranberry juice (19.87 mmol/L), lemon-flavored Gatorade (19.82 mmol/L), homemade lemonade (17.42 mmol/L), Mountain Dew (8.84 mmol/L), and Diet 7Up (7.98 mmol/L), respectively. CONCLUSIONS: According to 1H NMR, all of the tested "natural" citrus juices have high concentrations of citrate (38.3-67.4 mmol/L), with grapefruit juice having the highest concentration of the beverages chosen. Lemonade flavored Crystal Light had the highest concentration of citrate in the nonjuice category of tested beverages. In patients with mild to moderate hypocitraturia, dietary supplementation with citrus-based juices may be an effective alternative to medical management while not requiring large serving sizes. Further prospective studies are warranted to evaluate the clinical significance of these findings.
Asunto(s)
Bebidas/análisis , Ácido Cítrico/análisis , Citrus/química , Frutas/química , Nefrolitiasis/terapia , Humanos , Espectroscopía de Resonancia Magnética , Evaluación NutricionalRESUMEN
Francisella tularensis causes tularemia, a highly contagious disease of animals and humans, but the virulence features of F. tularensis are poorly defined. F. tularensis and the related mouse pathogen Francisella novicida synthesize unusual lipid A molecules lacking the 4'-monophosphate group typically found in the lipid A of Gram-negative bacteria. LpxF, a selective phosphatase located on the periplasmic surface of the inner membrane, removes the 4'-phosphate moiety in the late stages of F. novicida lipid A assembly. To evaluate the relevance of the 4'-phosphatase to pathogenesis, we constructed a deletion mutant of lpxF and compared its virulence with wild-type F. novicida. Intradermal injection of 10(6) wild-type but not 10(8) mutant F. novicida cells is lethal to mice. The rapid clearance of the lpxF mutant is associated with a stronger local cytokine response and a greater influx of neutrophils compared with wild-type. The F. novicida mutant was highly susceptible to the cationic antimicrobial peptide polymyxin. LpxF therefore represents a kind of virulence factor that confers a distinct lipid A phenotype, preventing Francisella from activating the host innate immune response and preventing the bactericidal actions of cationic peptides. Francisella lpxF mutants may be useful for immunization against tularemia.
Asunto(s)
Francisella tularensis/genética , Francisella tularensis/patogenicidad , Mutación , Monoéster Fosfórico Hidrolasas/genética , Animales , Proteínas de la Membrana Bacteriana Externa/metabolismo , Secuencia de Carbohidratos , Francisella tularensis/enzimología , Humanos , Inmunidad Innata , Lípido A/metabolismo , Espectroscopía de Resonancia Magnética , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Polimixinas/metabolismo , Receptor Toll-Like 4/metabolismoRESUMEN
Inositol pyrophosphates are a diverse group of high-energy signaling molecules whose cellular roles remain an active area of study. We report a previously uncharacterized class of inositol pyrophosphate synthase and find it is identical to yeast Vip1 and Asp1 proteins, regulators of actin-related protein-2/3 (ARP 2/3) complexes. Vip1 and Asp1 acted as enzymes that encode inositol hexakisphosphate (IP6) and inositol heptakisphosphate (IP7) kinase activities. Alterations in kinase activity led to defects in cell growth, morphology, and interactions with ARP complex members. The functionality of Asp1 and Vip1 may provide cells with increased signaling capacity through metabolism of IP6.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Ácido Fítico/metabolismo , Saccharomyces cerevisiae/enzimología , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimología , Alineación de Secuencia , Proteína 3 Relacionada con la Actina/metabolismo , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Cromatografía Líquida de Alta Presión , Secuencia Conservada , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/aislamiento & purificación , Humanos , Fosfatos de Inositol/metabolismo , Datos de Secuencia Molecular , Enzimas Multifuncionales , Fosforilación , Fosfotransferasas (Aceptor del Grupo Fosfato)/química , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Fosfotransferasas (Aceptor del Grupo Fosfato)/aislamiento & purificación , Estructura Terciaria de Proteína , Pirofosfatasas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/genética , Schizosaccharomyces/crecimiento & desarrollo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/aislamiento & purificación , Especificidad por Sustrato , TemperaturaRESUMEN
Francisella tularensis subsp. novicida U112 phospholipids, extracted without hydrolysis, consist mainly of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, and two lipid A species, designated A1 and A2. These lipid A species, present in a ratio of 7:1, comprise 15% of the total phospholipids, as judged by 32Pi labeling. Although lipopolysaccharide is detectable in F. tularensis subsp. novicida U112, less than 5% of the total lipid A is covalently linked to it. A1 and A2 were analyzed by electrospray ionization and matrix-assisted laser desorption ionization mass spectrometry, gas chromatography/mass spectrometry, and NMR spectroscopy. Both compounds are disaccharides of glucosamine, acylated with primary 3-hydroxystearoyl chains at positions 2, 3, and 2' and a secondary palmitoyl residue at position 2'. Minor isobaric species and some lipid A molecules containing a 3-hydroxypalmitoyl chain in place of 3-hydroxystearate are also present. The 4'- and 3'-positions of A1 and A2 are not derivatized, and 3-deoxy-d-manno-octulosonic acid (Kdo) is not detectable. The 1-phosphate groups of both A1 and A2 are modified with an alpha-linked galactosamine residue, as shown by NMR spectroscopy and gas chromatography/mass spectrometry. An alpha-linked glucose moiety is attached to the 6'-position of A2. The lipid A released by mild acid hydrolysis of F. tularensis subsp. novicida lipopolysaccharide consists solely of component A1. F. tularensis subsp. novicida mutants lacking the arnT gene do not contain a galactosamine residue on their lipid A. Formation of free lipid A in F. tularensis subsp. novicida might be initiated by an unusual Kdo hydrolase present in the membranes of this organism.
Asunto(s)
Francisella tularensis/química , Francisella tularensis/metabolismo , Lípido A/biosíntesis , Lípido A/química , Álcalis/química , Conformación de Carbohidratos , Galactosamina/química , Galactosamina/metabolismo , Hidrólisis , Iones/química , Lípido A/aislamiento & purificación , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Fosfolípidos/química , Fosfolípidos/metabolismoRESUMEN
The Salmonella and related bacteria modify the structure of the lipid A portion of their lipopolysaccharide in response to environmental stimuli. Some lipid A modifications are required for virulence and resistance to cationic antimicrobial peptides. We now demonstrate that membranes of Salmonella typhimurium contain a novel hydrolase that removes the 3'-acyloxyacyl residue of lipid A in the presence of 5 mM Ca2+. We have identified the gene encoding the S. typhimurium lipid A 3'-O-deacylase, designated lpxR, by screening an ordered S. typhimurium genomic DNA library, harbored in Escherichia coli K-12, for expression of Ca2+-dependent 3'-O-deacylase activity in membranes. LpxR is synthesized with an N-terminal type I signal peptide and is localized to the outer membrane. Mass spectrometry was used to confirm the position of lipid A deacylation in vitro and the release of the intact 3'-acyloxyacyl group. Heterologous expression of lpxR in the E. coli K-12 W3110, which lacks lpxR, resulted in production of significant amounts of 3'-O-deacylated lipid A in growing cultures. Orthologues of LpxR are present in the genomes of E. coli O157:H7, Yersinia enterocolitica, Helicobacter pylori, and Vibrio cholerae. The function of LpxR is unknown, but it could play a role in pathogenesis because it might modulate the cytokine response of an infected animal.