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921.
922.
Morand S Agnandji D Noel-Hudson MS Nicolas V Buisson S Macon-Lemaitre L Gnidehou S Kaniewski J Ohayon R Virion A Dupuy C 《The Journal of biological chemistry》2004,279(29):30244-30251
Dual oxidase 2 (Duox2) is a cell surface glycoprotein that probably provides thyroperoxidase with the H2O2 required to catalyze thyroid hormone synthesis. No functional H2O2-generating system has yet been obtained after transfecting Duox2 into non-thyroid cell lines, because it is retained in the endoplasmic reticulum (ER). We investigated the level of maturation of various Duox2 truncated proteins in an attempt to identify the region of Duox2 responsible for its remaining in the ER. Duox2-Q686X mutant, corresponding to the N-terminal ectodomain including the first putative transmembrane domain, was expressed in different cell lines. Carbohydrate content analysis revealed that complex type-specific Golgi apparatus (GA) oligosaccharides were present on pig Duox2-Q686X, whereas human truncated Duox2 carried only high mannose-type sugar chains characteristic of the ER. Further characterization using surface biotinylation and flow cytometry assays indicated that pig Duox2-Q686X was present at the plasma membrane, whereas human Duox2-Q686X remained inside the cell. The replacement of the last 90 residues of the human Duox2-Q686X with the pig equivalent region allowed the chimerical peptide to reach the Golgi apparatus. Pig mutants containing the complete first intracellular loop with or without the second transmembrane domain accumulated in the ER. These findings show that 1) the human Duox2-Q686X region encompassing residues 596-685 prevents mutant exportation from the ER and 2) there is a pig Duox2 retention domain in the first intracellular loop. In addition, missense mutations of four cysteines (Cys-351, -370, -568, or -582) completely inhibited the emergence of pig Duox2-Q686X from the ER compartment, indicating their importance in Duox2 maturation. 相似文献
923.
924.
Volpon L Lamthanh H Barbier J Gilles N Molgó J Ménez A Lancelin JM 《The Journal of biological chemistry》2004,279(20):21356-21366
Delta-conotoxin EVIA, from Conus ermineus, is a 32-residue polypeptide cross-linked by three disulfide bonds forming a four-loop framework. delta-Conotoxin EVIA is the first conotoxin known to inhibit sodium channel inactivation in neuronal membranes from amphibians and mammals (subtypes rNa(v)1.2a, rNa(v)1.3, and rNa(v)1.6), without affecting rat skeletal muscle (subtype rNa(v)1.4) and human cardiac muscle (subtype hNa(v)1.5) sodium channel (Barbier, J., Lamthanh, H., Le Gall, F., Favreau, P., Benoit, E., Chen, H., Gilles, N., Ilan, N., Heinemann, S. F., Gordon, D., Ménez, A., and Molgó, J. (2004) J. Biol. Chem. 279, 4680-4685). Its structure was solved by NMR and is characterized by a 1:1 cis/trans isomerism of the Leu(12)-Pro(13) peptide bond in slow exchange on the NMR time scale. The structure of both cis and trans isomers could be calculated separately. The isomerism occurs within a specific long disordered loop 2, including residues 11-19. These contribute to an important hydrophobic patch on the surface of the toxin. The rest of the structure matches the "inhibitor cystine-knot motif" of conotoxins from the "O superfamily" with a high structural order. To probe a possible functional role of the Leu(12)-Pro(13) cis/trans isomerism, a Pro(13) --> Ala delta-conotoxin EVIA was synthesized and shown to exist only as a trans isomer. P13A delta-conotoxin EVIA was estimated only two times less active than the wild-type EVIA in binding competition to rat brain synaptosomes and when injected intracerebroventricularly into mice. 相似文献
925.
Leulliot N Quevillon-Cheruel S Sorel I Graille M Meyer P Liger D Blondeau K Janin J van Tilbeurgh H 《The Journal of biological chemistry》2004,279(22):23447-23452
Allantoicase (EC 3.5.3.4) catalyzes the conversion of allantoate into ureidoglycolate and urea, one of the final steps in the degradation of purines to urea. The mechanism of most enzymes involved in this pathway, which has been known for a long time, is unknown. In this paper we describe the three-dimensional crystal structure of the yeast allantoicase determined at a resolution of 2.6 A by single anomalous diffraction. This constitutes the first structure for an enzyme of this pathway. The structure reveals a repeated jelly roll beta-sheet motif, also present in proteins of unrelated biochemical function. Allantoicase has a hexameric arrangement in the crystal (dimer of trimers). Analysis of the protein sequence against the structural data reveals the presence of two totally conserved surface patches, one on each jelly roll motif. The hexameric packing concentrates these patches into conserved pockets that probably constitute the active site. 相似文献
926.
Bordon-Pallier F Jullian N Ferrari P Girard AM Bocquel MT Biton J Bouquin N Haesslein JL 《Biochimica et biophysica acta》2004,1697(1-2):211-223
There is today a blatant need for new antifungal agents, because of the recent increase in life-threatening infections involving an ever-greater number of fungal strains. Fungi make extensive use of kinases in the regulation of essential processes, in particular the cell cycle. Most fungal kinases, however, are shared with higher eukaryotes. Only the kinases which have no human homologs, such as the histidine kinases, can be used as targets for antifungal drugs design. This review describes efforts directed towards the discovery of drugs active against a novel target, the atypical cell cycle kinase, Civ1. 相似文献
927.
Gap junctions, specialised membrane structures that mediate cell-to-cell communication in almost all tissues, are composed of channel-forming integral membrane proteins termed connexins. The activity of these intercellular channels is closely regulated, particularly by intramolecular modifications as phosphorylations of proteins by protein kinases, which appear to regulate the gap junction at several levels, including assembly of channels in the plasma membrane, connexin turnover as well as directly affecting the opening and closure ("gating") of channels. The regulation of membrane channels by protein phosphorylation/dephosphorylation processes commonly requires the formation of a multiprotein complex, where pore-forming subunits bind to auxiliary proteins (e.g. scaffolding proteins, catalytic and regulatory subunits), that play essential roles in channel localisation and activity, linking signalling enzymes, substrates and effectors into a structure frequently anchored to the cytoskeleton. The present review summarises the up-to-date progress regarding the proteins capable of interacting or at least of co-localising with connexins and their functional importance. 相似文献
928.
Dihydropteridine reductase as an alternative to dihydrofolate reductase for synthesis of tetrahydrofolate in Thermus thermophilus
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Wilquet V Van de Casteele M Gigot D Legrain C Glansdorff N 《Journal of bacteriology》2004,186(2):351-355
A strategy devised to isolate a gene coding for a dihydrofolate reductase from Thermus thermophilus DNA delivered only clones harboring instead a gene (the T. thermophilus dehydrogenase [DH(Tt)] gene) coding for a dihydropteridine reductase which displays considerable dihydrofolate reductase activity (about 20% of the activity detected with 6,7-dimethyl-7,8-dihydropterine in the quinonoid form as a substrate). DH(Tt) appears to account for the synthesis of tetrahydrofolate in this bacterium, since a classical dihydrofolate reductase gene could not be found in the recently determined genome nucleotide sequence (A. Henne, personal communication). The derived amino acid sequence displays most of the highly conserved cofactor and active-site residues present in enzymes of the short-chain dehydrogenase/reductase family. The enzyme has no pteridine-independent oxidoreductase activity, in contrast to Escherichia coli dihydropteridine reductase, and thus appears more similar to mammalian dihydropteridine reductases, which do not contain a flavin prosthetic group. We suggest that bifunctional dihydropteridine reductases may be responsible for the synthesis of tetrahydrofolate in other bacteria, as well as archaea, that have been reported to lack a classical dihydrofolate reductase but for which possible substitutes have not yet been identified. 相似文献
929.
Quevillon-Cheruel S Leulliot N Meyer P Graille M Bremang M Blondeau K Sorel I Poupon A Janin J van Tilbeurgh H 《The Journal of biological chemistry》2004,279(1):619-625
Chorismate synthase (EC 4.2.3.5), the seventh enzyme in the shikimate pathway, catalyzes the transformation of 5-enolpyruvylshikimate 3-phosphate (EPSP) to chorismate, which is the last common precursor in the biosynthesis of numerous aromatic compounds in bacteria, fungi, and plants. The chorismate synthase reaction involves a 1,4-trans-elimination of phosphoric acid from EPSP and has an absolute requirement for reduced FMN as a cofactor. We have determined the three-dimensional x-ray structure of the yeast chorismate synthase from selenomethionine-labeled crystals at 2.2-A resolution. The structure shows a novel betaalphabetaalpha fold consisting of an alternate tight packing of two alpha-helical and two beta-sheet layers, showing no resemblance to any documented protein structure. The molecule is arranged as a tight tetramer with D2 symmetry, in accordance with its quaternary structure in solution. Electron density is missing for 23% of the amino acids, spread over sequence regions that in the three-dimensional structure converge on the surface of the protein. Many totally conserved residues are contained within these regions, and they probably form a structured but mobile domain that closes over a cleft upon substrate binding and catalysis. This hypothesis is supported by previously published spectroscopic measurements implying that the enzyme undergoes considerable structural changes upon binding of both FMN and EPSP. 相似文献
930.
Barriault D Lépine F Mohammadi M Milot S Leberre N Sylvestre M 《The Journal of biological chemistry》2004,279(46):47489-47496
2,2'-Dichlorobiphenyl (CB) is transformed by the biphenyl dioxygenase of Burkholderia xenovorans LB400 (LB400 BPDO) into two metabolites (1 and 2). The most abundant metabolite, 1, was previously identified as 2,3-dihydroxy-2'-chlorobiphenyl and was presumed to originate from the initial attack by the oxygenase on the chlorine-bearing ortho carbon and on its adjacent meta carbon of one phenyl ring. 2,3,2',3'-Tetrachlorobiphenyl is transformed by LB400 BPDO into two metabolites that had never been fully characterized structurally. We determined the precise identity of the metabolites produced by LB400 BPDO from 2,2'-CB and 2,3,2',3'-CB, thus providing new insights on the mechanism by which 2,2'-CB is dehalogenated to generate 2,3-dihydroxy-2'-chlorobiphenyl. We reacted 2,2'-CB with the BPDO variant p4, which produces a larger proportion of metabolite 2. The structure of this compound was determined as cis-3,4-dihydro-3,4-dihydroxy-2,2'-dichlorobiphenyl by NMR. Metabolite 1 obtained from 2,2'-CB-d(8) was determined to be a dihydroxychlorobiphenyl-d(7) by gas chromatographic-mass spectrometric analysis, and the observed loss of only one deuterium clearly shows that the oxygenase attack occurs on carbons 2 and 3. An alternative attack at the 5 and 6 carbons followed by a rearrangement leading to the loss of the ortho chlorine would have caused the loss of more than one deuterium. The major metabolite produced from catalytic oxygenation of 2,3,2',3'-CB by LB400 BPDO was identified by NMR as cis-4,5-dihydro-4,5-dihydroxy-2,3,2',3'-tetrachlorobiphenyl. These findings show that LB400 BPDO oxygenates 2,2'-CB principally on carbons 2 and 3 and that BPDO regiospecificity toward 2,2'-CB and 2,3,2,',3'-CB disfavors the dioxygenation of the chlorine-free ortho-meta carbons 5 and 6 for both congeners. 相似文献