RESUMO
Leukotriene (LT) C(4) and its metabolites, LTD(4) and LTE(4), are involved in the pathobiology of bronchial asthma. LTC(4) synthase is the nuclear membrane-embedded enzyme responsible for LTC(4) biosynthesis, catalyzing the conjugation of two substrates that have considerably different water solubility; that amphipathic LTA(4) as a derivative of arachidonic acid and a water-soluble glutathione (GSH). A previous crystal structure revealed important details of GSH binding and implied a GSH activating function for Arg-104. In addition, Arg-31 was also proposed to participate in the catalysis based on the putative LTA(4) binding model. In this study enzymatic assay with mutant enzymes demonstrates that Arg-104 is required for the binding and activation of GSH and that Arg-31 is needed for catalysis probably by activating the epoxide group of LTA(4).
Assuntos
Arginina/química , Glutationa Transferase/química , Glutationa/química , Leucotrieno C4/química , Arginina/genética , Arginina/metabolismo , Asma/enzimologia , Asma/genética , Sítios de Ligação , Cristalografia por Raios X , Glutationa/genética , Glutationa/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Leucotrieno C4/biossíntese , Leucotrieno C4/genética , Mutação , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
Dodecyl-ß-D-selenomaltoside (SeDDM) is a seleno-detergent with a ß-glycosidic seleno-ether in place of the ether moiety in dodecyl-ß-D-maltoside. Seleno-detergents are candidates for heavy-atom agents in experimental phasing of membrane proteins in protein crystallography. Crystals of a nuclear membrane-embedded enzyme, leukotriene C(4) synthase (LTC(4)S), in complex with SeDDM were prepared and a multiwavelength anomalous diffraction (MAD) experiment was performed. The SeDDM in the LTC(4)S crystal exhibited sufficient anomalous diffraction for determination of the structure using MAD phasing.
Assuntos
Cristalografia por Raios X/métodos , Dissacarídeos/química , Glutationa Transferase/análise , Compostos de Organossilício/química , Animais , Glutationa Transferase/química , Humanos , Modelos Moleculares , Ligação ProteicaRESUMO
The beta-ketoacyl-(acyl carrier protein) synthases (beta-keto-ACP synthases; KAS) catalyse the addition of two-carbon units to the growing acyl chain during the elongation phase of fatty-acid synthesis. As key regulators of bacterial fatty-acid synthesis, they are promising targets for the development of new antibacterial agents. The crystal structure of 3-oxoacyl-ACP synthase II from Thermus thermophilus HB8 (TtKAS II) has been solved by molecular replacement and refined at 2.0 A resolution. The crystal is orthorhombic, space group P2(1)2(1)2, with unit-cell parameters a = 72.07, b = 185.57, c = 62.52 A, and contains one homodimer in the asymmetric unit. The subunits adopt the well known alpha-beta-alpha-beta-alpha thiolase fold that is common to ACP synthases. The structural and sequence similarities of TtKAS II to KAS I and KAS II enzymes of known structure from other sources support the hypothesis of comparable enzymatic activity. The dimeric state of TtKAS II is important to create each fatty-acid-binding pocket. Closer examination of KAS structures reveals that compared with other KAS structures in the apo form, the active site of TtKAS II is more accessible because of the ;open' conformation of the Phe396 side chain.
Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase/química , Thermus thermophilus/enzimologia , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/genética , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Dimerização , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Homologia de Sequência de AminoácidosRESUMO
Rabbit L-gulonate 3-dehydrogenase was crystallized using the oil-microbatch method at 295 K. X-ray diffraction data were collected to 1.70 A resolution from a crystal at 100 K using synchrotron radiation. The crystal belongs to the C-centred monoclinic space group C2, with unit-cell parameters a = 71.81, b = 69.08, c = 65.64 A, beta = 102.7 degrees. Assuming the presence of a monomeric protomer in the asymmetric unit gives a V(M) value of 2.21 A(3) Da(-1) and a solvent content of 44.4%. A cocrystal with NADH, which was isomorphous to the apo form, was also prepared and diffraction data were collected to 1.85 A resolution using Cu Kalpha radiation at 100 K.
Assuntos
Desidrogenases de Carboidrato/química , Animais , Desidrogenases de Carboidrato/genética , Desidrogenases de Carboidrato/metabolismo , Cristalização , Cristalografia por Raios X , CoelhosRESUMO
The purine nucleoside phosphorylase from Thermus thermophilus crystallized in space group P4(3)2(1)2 with the unit cell dimensions a = 131.9 A and c = 169.9 A and one biologically active hexamer in the asymmetric unit. The structure was solved by the molecular replacement method and refined at a 1.9A resolution to an r(free) value of 20.8%. The crystals of the binary complex with sulfate ion and ternary complexes with sulfate and adenosine or guanosine were also prepared and their crystal structures were refined at 2.1A, 2.4A and 2.4A, respectively. The overall structure of the T.thermophilus enzyme is similar to the structures of hexameric enzymes from Escherichia coli and Sulfolobus solfataricus, but significant differences are observed in the purine base recognition site. A base recognizing aspartic acid, which is conserved among the hexameric purine nucleoside phosphorylases, is Asn204 in the T.thermophilus enzyme, which is reminiscent of the base recognizing asparagine in trimeric purine nucleoside phosphorylases. Isothermal titration calorimetry measurements indicate that both adenosine and guanosine bind the enzyme with nearly similar affinity. However, the functional assays show that as in trimeric PNPs, only the guanosine is a true substrate of the T.thermophilus enzyme. In the case of adenosine recognition, the Asn204 forms hydrogen bonds with N6 and N7 of the base. While in the case of guanosine recognition, the Asn204 is slightly shifted together with the beta(9)alpha(7) loop and predisposed to hydrogen bond formation with O6 of the base in the transition state. The obtained experimental data suggest that the catalytic properties of the T.thermophilus enzyme are reminiscent of the trimeric rather than hexameric purine nucleoside phosphorylases.
Assuntos
Purina-Núcleosídeo Fosforilase/química , Thermus thermophilus/enzimologia , Adenosina/química , Sítios de Ligação , Calorimetria , Cristalização , Cristalografia por Raios X , Guanosina/química , Ligação de Hidrogênio , Estrutura Molecular , Conformação Proteica , Estrutura Quaternária de Proteína , Alinhamento de Sequência , Sulfatos/químicaRESUMO
The vacuole-type ATPases in eukaryotic cells translocate protons across various biological membranes including the vacuolar membrane by consuming ATP molecules. The E subunit of the multisubunit complex V-ATPase from Pyrococcus horikoshii OT3, which has a molecular weight of 22.88 kDa, has been cloned, overexpressed in Escherichia coli, purified and crystallized by the microbatch method using PEG 4000 as a precipitant at 296 K. A data set to 1.85 A resolution with 98.8% completeness and an Rmerge of 6.5% was collected from a single flash-cooled crystal using synchrotron radiation. The crystal belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 52.196, b = 55.317, c = 77.481 A, and is most likely to contain one molecule per asymmetric unit.
Assuntos
Pyrococcus horikoshii/enzimologia , ATPases Vacuolares Próton-Translocadoras/química , Proteínas Arqueais/química , Proteínas Arqueais/isolamento & purificação , Cristalização , Subunidades Proteicas/química , Subunidades Proteicas/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , ATPases Vacuolares Próton-Translocadoras/isolamento & purificação , Difração de Raios XRESUMO
Niemann-Pick disease type C (NP-C) is a progressive neurological disorder of lipid metabolism. The Balb/C npc1 mutant strain is a genetically authentic murine model of NPC, which reproduce the clinical and histologic features of human NP-C. In the present study, we show that cholecystokinin (CCK)-immunoreactive fibers in the thalamic VPL nuclei, which are densely distributed in controls, degenerate in NPC mice. This degeneration is associated with the appearance of CCK-immunoreactive axonal spheroids containing characteristic intracellular inclusions of NP-C. These observations provide supportive evidence of the occurrence of dying-back axonopathy of neurons in the dorsal column nuclei in this mouse model.
Assuntos
Vias Aferentes/metabolismo , Colecistocinina/metabolismo , Degeneração Neural/metabolismo , Doenças de Niemann-Pick/metabolismo , Doenças de Niemann-Pick/fisiopatologia , Núcleos Ventrais do Tálamo/metabolismo , Animais , Axônios/metabolismo , Axônios/ultraestrutura , Modelos Animais de Doenças , Imuno-Histoquímica/métodos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Mutantes , Microscopia Imunoeletrônica/métodos , Degeneração Neural/fisiopatologia , Neurônios/metabolismo , Neurônios/patologia , Neurônios/ultraestrutura , Proteína C1 de Niemann-Pick , Proteínas/genética , Núcleos Ventrais do Tálamo/patologia , Núcleos Ventrais do Tálamo/ultraestruturaRESUMO
The cysteinyl leukotrienes (cys-LTs), leukotriene C4 (LTC4) and its metabolites, LTD4 and LTE4, are proinflammatory lipid mediators in asthma and other inflammatory diseases. They are generated through the 5-lipoxygenase/LTC4 synthase (LTC4S) pathway and act via at least two distinct G protein-coupled receptors. The inhibition of human LTC4S will make a simple way to treat the cys-LT relevant inflammatory diseases. Here, we show that compounds having 5-(5-methylene-4-oxo-4,5-dihydrothiazol-2-ylamino) isophthalic acid moiety suppress LTC4 synthesis, glutathione conjugation to the precursor LTA4, in both an enzyme assay and a whole-cell assay. Hierarchical in silico screenings of 6 million compounds provided 300,000 dataset for docking, and after energy minimization based on the crystal structure of LTC4S, 111 compounds were selected as candidates for a competitive inhibitor to glutathione. One of those compounds showed significant inhibitory activity, and subsequently, its derivative 5-((Z)-5-((E)-2-methyl-3-phenylallylidene)-4-oxo-4,5-dihydrothiazol-2-ylamino) isophthalic acid (compound 1) was found to be the most potent inhibitor. The enzyme assay showed the IC50 was 1.9 µM and the corresponding 95% confidence interval was from 1.7 to 2.2 µM. The whole-cell assay showed that compound 1 was cell permeable and inhibited LTC4 synthesis in a concentration dependent manner.
Assuntos
Inibidores Enzimáticos/farmacologia , Glutationa Transferase/antagonistas & inibidores , Ácidos Ftálicos/farmacologia , Inibidores Enzimáticos/química , Estrutura Molecular , Ácidos Ftálicos/químicaRESUMO
l-Gulonate 3-dehydrogenase (GDH) is a bifunctional dimeric protein that functions not only as an NAD(+)-dependent enzyme in the uronate cycle but also as a taxon-specific lambda-crystallin in rabbit lens. Here we report the first crystal structure of GDH in both apo form and NADH-bound holo form. The GDH protomer consists of two structural domains: the N-terminal domain with a Rossmann fold and the C-terminal domain with a novel helical fold. In the N-terminal domain of the NADH-bound structure, we identified 11 coenzyme-binding residues and found 2 distinct side-chain conformers of Ser124, which is a putative coenzyme/substrate-binding residue. A structural comparison between apo form and holo form and a mutagenesis study with E97Q mutant suggest an induced-fit mechanism upon coenzyme binding; coenzyme binding induces a conformational change in the coenzyme-binding residues Glu97 and Ser124 to switch their activation state from resting to active, which is required for the subsequent substrate recruitment. Subunit dimerization is mediated by numerous intersubunit interactions, including 22 hydrogen bonds and 104 residue pairs of van der Waals interactions, of which those between two cognate C-terminal domains are predominant. From a structure/sequence comparison within GDH homologues, a much greater degree of interprotomer interactions (both polar and hydrophobic) in the rabbit GDH would contribute to its higher thermostability, which may be relevant to the other function of this enzyme as lambda-crystallin, a constitutive structural protein in rabbit lens. The present crystal structures and amino acid mutagenesis studies assigned the role of active-site residues: catalytic base for His145 and substrate binding for Ser124, Cys125, Asn196, and Arg231. Notably, Arg231 participates in substrate binding from the other subunit of the GDH dimer, indicating the functional significance of the dimeric state. Proper orientation of the substrate-binding residues for catalysis is likely to be maintained by an interprotomer hydrogen-bonding network of residues Asn196, Gln199, and Arg231, suggesting a network-based substrate recognition of GDH.
Assuntos
Desidrogenases de Carboidrato/química , Cristalinas/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Biocatálise , Desidrogenases de Carboidrato/genética , Desidrogenases de Carboidrato/metabolismo , Cristalinas/genética , Cristalinas/metabolismo , Cisteína/metabolismo , Dimerização , Estabilidade Enzimática , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Coelhos , Homologia de Sequência de Aminoácidos , Serina/metabolismoRESUMO
In order to understand the induced fit and the thermostabilization mechanisms of ATP-dependent phosphoenolpyruvate carboxykinase, the crystal structure of the enzyme from the extreme thermophile Thermus thermophilus HB8 (TtPEPCK) was determined and compared with those of orthologues of known structure from two mesophilic organisms. The protomer structures in these orthologues, which exhibit open/closed interdomain conformations, are similar. Isomorphous crystals of unliganded and ATP-bound TtPEPCK were obtained. The asymmetric units of both crystal forms contain two protomers A and B with closed and open conformations, respectively. ATP was only observed in the interdomain cleft of the closed protomer, suggesting that the induced fit of TtPEPCK agrees with the so-called ;conformational selection' mechanism where ligand binding is not essential for domain closure although its binding leads to the stabilization of the closed state. A bound calcium observed in the N-terminal domain of TtPEPCK probably contributes to the thermal stability. A combination of hydrophobic effects, ion pairs and entropic effects might also contribute to the thermostability of TtPEPCK.
Assuntos
Proteínas de Bactérias/química , Fosfoenolpiruvato Carboxiquinase (ATP)/química , Thermus thermophilus/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cálcio/metabolismo , Varredura Diferencial de Calorimetria , Cristalografia por Raios X , Entropia , Estabilidade Enzimática , Modelos Moleculares , Dados de Sequência Molecular , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Conformação Proteica , Alinhamento de Sequência , Relação Estrutura-AtividadeRESUMO
BACKGROUND/AIMS: The human proto-oncogene c-erbB-2 (also called HER-2/neu) is located on chromosome 17q21-22. There have been no studies on gene amplification or mRNA expression of c-erbB-2 in human intrahepatic cholangiocarcinoma (CC) hitherto. METHODS: We investigated c-erbB-2 gene amplification by fluorescence in situ hybridization (FISH), c-erbB2 mRNA expression by ISH, and c-erbB-2 protein expression by immunohistochemistry in 22 archival cases of CC. RESULTS: FISH revealed that c-erbB-2 gene signals were increased in CC. ISH showed that c-erbB-2 mRNA signals were located in the nuclei and cytoplasms of cancer cells and were increased in cancer cells compared with non-cancerous bile ducts where no signals were present. Immunohistochemistry showed that the c-erbB-2 protein was expressed in the cell membrane of cancer cells, and was increased compared with non-cancerous bile ducts where no expression was found. There was a positive significant correlation between c-erbB-2 mRNA and protein expression. Clinicopathologically, there were no correlations between the c-erbB-2 expression and various pathological features. CONCLUSIONS: These data suggest that c-erbB-2 gene amplification does occur in CC, and that there is an overexpressed c-erbB-2 protein through the enhanced mRNA expression. The c-erbB-2 gene amplification may be related to the oncogenesis or tumor progression of CC.
Assuntos
Colangiocarcinoma/fisiopatologia , Neoplasias Hepáticas/fisiopatologia , Receptor ErbB-2/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Colangiocarcinoma/química , Feminino , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Hibridização In Situ , Hibridização in Situ Fluorescente , Neoplasias Hepáticas/química , Masculino , Pessoa de Meia-Idade , Proto-Oncogene Mas , RNA Mensageiro/análise , Receptor ErbB-2/análiseRESUMO
Niemann-Pick disease type C (NP-C) is a progressive and fatal neurological disorder characterized by intracellular accumulation of cholesterol and glycolipid. A Balb/c-npc1 mutant strain is a genetically authentic murine model of NP-C, and homozygous mice show progressive weight loss and tremor or ataxia until death at 12-14 weeks of age. Neuropathologically, this model is known to faithfully reproduce the cardinal histologic features of NP-C including neuronal storage, appearance of swollen axons (spheroids), and neuronal loss, although the cellular mechanisms of neural degeneration are largely unknown. To investigate the mode of neural degeneration of sensory neurons in NP-C, we studied the central processes of dorsal root ganglion (DRG) neurons at the level of the medullary dorsal column nuclei and the spinal dorsal horn with special attention to the ultrastructural changes of presynaptic axon terminals. The appearance of axonal spheroids in the dorsal column nuclei and the loss of axons in the spinal nerve roots were assessed quantitatively. We show that the gracile nuclei develop numerous axonal spheroids after only 3 weeks. At 6 and 9 weeks, dystrophic axons, which were separated from simple axonal spheroids by the ultrastructural presence of distinctive tubulo-vesicular elements, progressively increased in size and number. These neuropathological findings are identical to those of gracile axonal dystrophy (GAD) of the normal aging mouse. Presynaptic elements were exclusively involved in spheroid formation. The cuneate nuclei and the spinal dorsal horn revealed fewer axonal spheroids and only rare dystrophic changes. This was associated with a significant drop in the number of L4-5 dorsal root axons in NP-C mouse at 9 weeks of age compared with controls. These results support the existence of a length-dependent axonopathy in the central processes of DRG neurons and are consistent with the view that altered axonal transport, which is implicated in the pathogenesis of GAD in physiological aging, may be an underlying mechanism in neuronal degeneration in NP-C. Clinically, the premature development of GAD may be responsible for ataxia, one of the early manifestations of this disease.
Assuntos
Axônios/patologia , Gânglios Espinais/patologia , Neurônios Aferentes/patologia , Doenças de Niemann-Pick/patologia , Fatores Etários , Animais , Axônios/ultraestrutura , Contagem de Células , Modelos Animais de Doenças , Gânglios Espinais/ultraestrutura , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Mutantes Neurológicos , Neurônios Aferentes/ultraestrutura , Células do Corno Posterior/patologia , Células do Corno Posterior/ultraestrutura , Terminações Pré-Sinápticas/ultraestrutura , Sinapses/patologia , Sinapses/ultraestruturaRESUMO
2-Keto-3-deoxygluconate kinase (KDGK) catalyzes the phosphorylation of 2-keto-3-deoxygluconate (KDG) to 2-keto-3-deoxy-6-phosphogluconate. Two crystal forms of KDGK from Thermus thermophilus were obtained by vapour-diffusion and microbatch methods. Crystals in the form of triangular plates (TtKDGK-1) were obtained that belong to space group P3, with unit-cell parameters a = b = 145.83, c = 74.63 A, and diffract to 3.2 A. These crystals exhibited nearly perfect hemihedral twinning. Assigning six subunits of TtKDGK to the asymmetric unit of the crystal corresponds to a 46.2% solvent content. A single plate-like crystal (TtKDGK-2) belonged to space group P6(3), with unit-cell parameters a = b = 84.83, c = 168.49 A, and diffracts to 2.25 A. This crystal exhibits only partial hemihedral twinning, with a twin fraction of 24.4%. Diffraction-quality crystals of TtKDGK with bound ATP (TtKDGK-ATP), a = b = 84.72, c = 321.61 A and with bound KDG plus the ATP analogue AMP-PNP (TtKDGK-ATP-KDG), with unit-cell parameters a = b = 84.32, c = 168.7 A, were also prepared and characterized.