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See detailEts-dependent regulation of target gene expression during megakaryopoiesis
Jackers, Pascale ULg; Szalai, Gabor; Moussa, Omar et al

in Journal of Biological Chemistry (2004), 279(50), 52183-52190

Megakaryopoiesis is the process by which hematopoietic stem cells in the bone marrow differentiate into mature megakaryocytes. The expression of megakaryocytic genes during megakaryopoiesis is controlled ... [more ▼]

Megakaryopoiesis is the process by which hematopoietic stem cells in the bone marrow differentiate into mature megakaryocytes. The expression of megakaryocytic genes during megakaryopoiesis is controlled by specific transcription factors. Fli-1 and GATA-1 transcription factors are required for development of megakaryocytes and promoter analysis has defined in vitro functional binding sites for these factors in several megakaryocytic genes, including GPIIb, GPIX, and C-MPL. Herein, we utilize chromatin immunoprecipitation to examine the presence of Ets-1, Fli-1, and GATA-1 on these promoters in vivo. Fli-1 and Ets-1 occupy the promoters of GPIIb, GPIX, and C-MPL genes in both Meg-01 and CMK11-5 cells. Whereas GPIIb is expressed in both Meg-01 and CMK11-5 cells, GPIX and C-MPL are only expressed in the more differentiated CMK11-5 cells. Thus, in vivo occupancy by an Ets factor is not sufficient to promote transcription of some megakaryocytic genes. GATA-1 and Fli-1 are both expressed in CMK11-5 cells and co-occupy the GPIX and C-MPL promoters. Transcription of all three megakaryocytic genes is correlated with the presence of acetylated histone H3 and phosphorylated RNA polymerase II on their promoters. We also show that exogenous expression of GATA-1 in Meg-01 cells leads to the expression of endogenous c-mpl and gpIX mRNA. Whereas GPIIb, GPIX, and C-MPL are direct target genes for Fli-1, both Fli-1 and GATA-1 are required for formation of an active transcriptional complex on the C-MPL and GPIX promoters in vivo. In contrast, GPIIb expression appears to be independent of GATA-1 in Meg-01 cells. [less ▲]

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See detailNOX3, a superoxide-generating NADPH oxidase of the inner ear
Banfi, Botond; Malgrange, Brigitte ULg; Knisz, Judit et al

in Journal of Biological Chemistry (2004), 279(44), 46065-46072

Reactive oxygen species (ROS) play a major role in drug-, noise-, and age-dependent hearing loss, but the source of ROS in the inner ear remains largely unknown. Herein, we demonstrate that NADPH oxidase ... [more ▼]

Reactive oxygen species (ROS) play a major role in drug-, noise-, and age-dependent hearing loss, but the source of ROS in the inner ear remains largely unknown. Herein, we demonstrate that NADPH oxidase (NOX) 3, a member of the NOX/dual domain oxidase family of NADPH oxidases, is highly expressed in specific portions of the inner ear. As assessed by real-time PCR, NOX3 mRNA expression in the inner ear is at least 50-fold higher than in any other tissues where its expression has been observed ( e. g. fetal kidney, brain, skull). Microdissection and in situ hybridization studies demonstrated that NOX3 is localized to the vestibular and cochlear sensory epithelia and to the spiral ganglions. Transfection of human embryonic kidney 293 cells with NOX3 revealed that it generates low levels of ROS on its own but produces high levels of ROS upon co-expression with cytoplasmic NOX subunits. NOX3-dependent superoxide production required a stimulus in the absence of subunits and upon co-expression with phagocyte NADPH oxidase subunits p47(phox) and p67(phox), but it was stimulus-independent upon co-expression with colon NADPH oxidase subunits NOX organizer 1 and NOX activator 1. Pre-incubation of NOX3-transfected human embryonic kidney 293 cells with the ototoxic drug cisplatin markedly enhanced superoxide production, in both the presence and the absence of subunits. Our data suggest that NOX3 is a relevant source of ROS generation in the cochlear and vestibular systems and that NOX3-dependent ROS generation might contribute to hearing loss and balance problems in response to ototoxic drugs. [less ▲]

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See detailUp-regulation of vascular endothelial growth factor-A by active membrane-type 1 matrix metalloproteinase through activation of Src-tyrosine kinases
Sounni, Nor Eddine ULg; Roghi, C.; Chabottaux, Vincent et al

in Journal of Biological Chemistry (2004), 279(14), 13564-13574

Membrane-type 1 matrix metalloproteinase (MT1-MMP) and vascular endothelial growth factor ( VEGF) are two key molecules involved in pericellular proteolysis and cell proliferation during tumor growth and ... [more ▼]

Membrane-type 1 matrix metalloproteinase (MT1-MMP) and vascular endothelial growth factor ( VEGF) are two key molecules involved in pericellular proteolysis and cell proliferation during tumor growth and angiogenesis. Our previous data showed that MT1-MMP overexpression in human breast carcinoma MCF7 cells induced an up-regulation of VEGF expression. This effect was associated in vivo with accelerated tumor growth and angiogenesis. We now provide evidence that MT1-MMP overexpression specifically affected VEGF-A production and failed to influence that of other VEGF family members ( VEGF, B, C, D, or PlGF) or their receptors. The up-regulation of VEGF-A by MT1-MMP was related to an increased transcriptional activation rather than to a modification of mRNA stability. It was blocked by synthetic MMP inhibitors, TIMP2, but not TIMP-1 and abolished by a partial deletion of the catalytic domain or the cytoplasmic tail of MT1-MMP. Analysis of the signal transduction mechanisms demonstrated that MT1-MMP acts through a signaling pathway involving Src tyrosine kinases. Thus, our results provide new insight into the mechanisms of action of MT1-MMP during angiogenesis and suggest that the full enzymatic activity of MT1-MMP is required for a specific up-regulation of VEGF-A through an activation of Src tyrosine kinase pathways. [less ▲]

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See detailEvidence of an intramolecular interaction between the two domains of the BlaR1 penicillin receptor during the signal transduction
Hanique, Sophie; Colombo, Maria-Luigi; Goormaghtigh, Erik et al

in Journal of Biological Chemistry (2004), 279(14), 14264-14272

The BlaR1 protein is a penicillin-sensory transducer involved in the induction of the Bacillus licheniformis beta-lactamase. The amino-terminal domain of the protein exhibits four transmembrane segments ... [more ▼]

The BlaR1 protein is a penicillin-sensory transducer involved in the induction of the Bacillus licheniformis beta-lactamase. The amino-terminal domain of the protein exhibits four transmembrane segments (TM1-TM4) that form a four-alpha-helix bundle embedded in the plasma bilayer. The carboxyl-terminal domain of 250 amino acids (BlaR-CTD) fused at the carboxyl end of TM4 possesses the amino acid sequence signature of penicillin-binding proteins. This membrane topology suggests that BlaR-CTD and the BlaR-amino-terminal domain are responsible for signal reception and signal transduction, respectively. With the use of phage display experiments, we highlight herein an interaction between BlaR-CTD and the extracellular, 63-amino acid L2 loop connecting TM2 and TM3. This interaction does not occur in the presence of penicillin. This result suggests that binding of the antibiotic to BlaR1 might entail the release of the interaction between L2 and BlaR-CTD, causing a motion of the alpha-helix bundle and transfer of the information to the cytoplasm of the cell. In addition, fluorescence spectroscopy, CD, and Fourier transform IR spectroscopy experiments indicate that in contrast to the behavior of the corresponding Staphylococcus aureus protein, the beta-lactam antibiotic does not induce a drastic conformational change in B. licheniformis BlaR-CTD. [less ▲]

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See detailMolecular basis of the amylose-like polymer formation catalyzed by Neisseria polysaccharea amylosucrase
Albenne, C.; Skov, L. K.; Mirza, O. et al

in Journal of Biological Chemistry (2004), 279(1), 726-734

Amylosucrase from Neisseria polysaccharea is a remarkable transglucosidase from family 13 of the glycosidehydrolases that synthesizes an insoluble amylose-like polymer from sucrose in the absence of any ... [more ▼]

Amylosucrase from Neisseria polysaccharea is a remarkable transglucosidase from family 13 of the glycosidehydrolases that synthesizes an insoluble amylose-like polymer from sucrose in the absence of any primer. Amylosucrase shares strong structural similarities with alpha-amylases. Exactly how this enzyme catalyzes the formation of alpha-1,4-glucan and which structural features are involved in this unique functionality existing in family 13 are important questions still not fully answered. Here, we provide evidence that amylosucrase initializes polymer formation by releasing, through sucrose hydrolysis, a glucose molecule that is subsequently used as the first acceptor molecule. Maltooligosaccharides of increasing size were produced and successively elongated at their nonreducing ends until they reached a critical size and concentration, causing precipitation. The ability of amylosucrase to bind and to elongate maltooligosaccharides is notably due to the presence of key residues at the OB1 acceptor binding site that contribute strongly to the guidance ( Arg(415), subsite +4) and the correct positioning (Asp(394) and Arg(446), subsite +1) of acceptor molecules. On the other hand, Arg(226) (subsites +2/+3) limits the binding of maltooligosaccharides, resulting in the accumulation of small products (G to G3) in the medium. A remarkable mutant (R226A), activated by the products it forms, was generated. It yields twice as much insoluble glucan as the wild-type enzyme and leads to the production of lower quantities of by-products. [less ▲]

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See detailLck dephosphorylation at Tyr-394 and inhibition of T cell antigen receptor signaling by Yersinia phosphatase YopH.
Alonso, Andres; Bottini, Nunzio; Bruckner, Shane et al

in Journal of Biological Chemistry (2004), 279(6), 4922-8

A key virulence factor for Yersinia pestis, the etiologic agent of plague, is the tyrosine phosphatase YopH, which the bacterium injects into host cells. We report that treatment of human T lymphocytes ... [more ▼]

A key virulence factor for Yersinia pestis, the etiologic agent of plague, is the tyrosine phosphatase YopH, which the bacterium injects into host cells. We report that treatment of human T lymphocytes with a recombinant membrane-permeable YopH resulted in severe reduction in intracellular tyrosine phosphorylation and inhibition of T cell activation. The primary signal transducer for the T cell antigen receptor, the Lck tyrosine kinase, was specifically precipitated by a substrate-trapping YopH mutant, and Lck was dephosphorylated at its positive regulatory site, Tyr-394, in cells containing active YopH. By turning off Lck, YopH blocks T cell antigen receptor signaling at its very first step, effectively preventing the development of a protective immune response against this lethal bacterium. [less ▲]

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See detailATP Augments von Willebrand Factor-dependent Shear-induced Platelet Aggregation through Ca2+-Calmodulin and Myosin Light Chain Kinase Activation
Oury, Cécile ULg; Sticker, Elsie; Cornelissen, Heidi et al

in Journal of Biological Chemistry (2004)

Shear stress triggers von Willebrand factor (VWF) binding to platelet glycoprotein Ibalpha and subsequent integrin alpha(IIb)beta(3)-dependent platelet aggregation. Concomitantly, nucleotides are released ... [more ▼]

Shear stress triggers von Willebrand factor (VWF) binding to platelet glycoprotein Ibalpha and subsequent integrin alpha(IIb)beta(3)-dependent platelet aggregation. Concomitantly, nucleotides are released from plateletdense granules, and ADP is known to contribute to shear-induced platelet aggregation (SIPA). This study shows that ATP also contributes to SIPA. The ATP-gated P2X(1) ion channel induces MLC-mediated cytoskeletal rearrangements that increases platelet degranulation during VWF-triggered platelet activation. [less ▲]

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See detailIdentification of residues within human glycoprotein VI involved in the binding to collagen: evidence for the existence of distinct binding sites.
Lecut, Christelle ULg; Arocas, Veronique; Ulrichts, Hans et al

in Journal of Biological Chemistry (2004), 279(50), 52293-9

Glycoprotein VI (GPVI) has a crucial role in platelet responses to collagen. Still, little is known about its interaction with its ligands. In binding assays using soluble or cell-expressed human GPVI, we ... [more ▼]

Glycoprotein VI (GPVI) has a crucial role in platelet responses to collagen. Still, little is known about its interaction with its ligands. In binding assays using soluble or cell-expressed human GPVI, we observed that (i) collagen, and the GPVI-specific ligands collagen-related peptides (CRP) and convulxin, competed with one another for the binding to GPVI and (ii) monoclonal antibodies directed against the extracellular part of the human receptor displayed selective inhibitory properties on GPVI interaction with its ligands. Monoclonal antibody 9E18 strongly reduced the binding of GPVI to collagen/CRP, 3F8 inhibited its interaction with convulxin, whereas 9O12 prevented all three interactions. These observations suggest that ligand-binding sites are distinct, exhibiting specific features but at the same time also sharing some common residues participating in the recognition of these ligands. The epitope of 9O12 was mapped by phage display, along with molecular modeling of human GPVI, which allowed the identification of residues within GPVI potentially involved in ligand recognition. Site-directed mutagenesis revealed that valine 34 and leucine 36 are critical for GPVI interaction with collagen and CRP. The loop might thus be part of a collagen/CRP-binding site. [less ▲]

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See detail15-deoxy-delta12,14-prostaglandin J2 inhibits Bay 11-7085-induced sustained extracellular signal-regulated kinase phosphorylation and apoptosis in human articular chondrocytes and synovial fibroblasts
Relic, Biserka ULg; Benoit, Valerie; Franchimont, Nathalie et al

in Journal of Biological Chemistry (2004), 279(21), 399-403

We have previously shown that nuclear factor-kappaB inhibition by adenovirus expressing mutated IkappaB-alpha or by proteasome inhibitor increases human articular chondrocytes sensibility to apoptosis ... [more ▼]

We have previously shown that nuclear factor-kappaB inhibition by adenovirus expressing mutated IkappaB-alpha or by proteasome inhibitor increases human articular chondrocytes sensibility to apoptosis. Moreover, the nuclear factor-kappaB inhibitor BAY11-7085, a potent anti-inflammatory drug in rat adjuvant arthritis, is itself a proapoptotic agent for chondrocytes. In this work, we show that BAY 11-7085 but not the proteasome inhibitor MG-132 induced a rapid and sustained phosphorylation of extracellular signal-regulated kinases (ERK1/2) in human articular chondrocytes. The level of ERK1/2 phosphorylation correlated with BAY 11-7085 concentration and chondrocyte apoptosis. 15-Deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2) and its precursor prostaglandin (PG) D2 but not PGE2 and PGF2alpha rescued chondrocytes from BAY 11-7085-induced apoptosis. 15d-PGJ2 markedly inhibited BAY 11-7085-induced phosphorylation of ERK1/2. BAY 11-7085 also induced ERK1/2 phosphorylation and apoptosis in human synovial fibroblasts, and these reactions were down-regulated by 15d-PGJ2. Further analysis in synovial fibroblasts showed that only molecules that suppressed BAY 11-7085-induced phosphorylation of ERK1/2 (i.e. 15d-PGJ2, PGD2, and to a lesser extent, MEK1/2 inhibitor UO126, but not prostaglandins E2 and F2alpha or peroxisome proliferator-activated receptor-gamma agonist ciglitazone) were able protect cells from apoptosis. These results suggested that the antiapoptotic effect of 15d-PGJ2 on chondrocytes and synovial fibroblasts might involve inhibition of ERK1/2 phosphorylation. [less ▲]

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See detailBistability analyses of a caspase activation model for receptor-induced apoptosis
Eissing, Thomas; Conzelmann, Holger; Gilles, Ernst Dieter et al

in Journal of Biological Chemistry (2004), 279(35), 36892-36897

Apoptosis is an important physiological process crucially involved in development and homeostasis of multicellular organisms. Although the major signaling pathways have been unraveled, a detailed ... [more ▼]

Apoptosis is an important physiological process crucially involved in development and homeostasis of multicellular organisms. Although the major signaling pathways have been unraveled, a detailed mechanistic understanding of the complex underlying network remains elusive. We have translated here the current knowledge of the molecular mechanisms of the death-receptor-activated caspase cascade into a mathematical model. A reduction down to the apoptotic core machinery enables the application of analytical mathematical methods to evaluate the system behavior within a wide range of parameters. Using parameter values from the literature, the model reveals an unstable status of survival indicating the need for further control. Based on recent publications we tested one additional regulatory mechanism at the level of initiator caspase activation and demonstrated that the resulting system displays desired characteristics such as bistability. In addition, the results from our model studies allowed us to reconcile the fast kinetics of caspase 3 activation observed at the single cell level with the much slower kinetics found at the level of a cell population. [less ▲]

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See detailCofactor binding modulates the conformational stabilities and unfolding patterns of NAD(+)-dependent DNA ligases from Escherichia coli and Thermus scotoductus
Georlette, D.; Blaise, Vinciane ULg; Dohmen, C. et al

in Journal of Biological Chemistry (2003), 278(50), 49945-49953

DNA ligases are important enzymes required for cellular processes such as DNA replication, recombination, and repair. NAD(+)-dependent DNA ligases are essentially restricted to eubacteria, thus ... [more ▼]

DNA ligases are important enzymes required for cellular processes such as DNA replication, recombination, and repair. NAD(+)-dependent DNA ligases are essentially restricted to eubacteria, thus constituting an attractive target in the development of novel antibiotics. Although such a project might involve the systematic testing of a vast number of chemical compounds, it can essentially gain from the preliminary deciphering of the conformational stability and structural perturbations associated with the formation of the catalytically active adenylated enzyme. We have, therefore, investigated the adenylation-induced conformational changes in the mesophilic Escherichia coli and thermophilic Thermus scotoductus NAD(+)-DNA ligases, and the resistance of these enzymes to thermal and chemical (guanidine hydrochloride) denaturation. Our results clearly demonstrate that anchoring of the cofactor induces a conformational rearrangement within the active site of both mesophilic and thermophilic enzymes accompanied by their partial compaction. Furthermore, the adenylation of enzymes increases their resistance to thermal and chemical denaturation, establishing a thermodynamic link between cofactor binding and conformational stability enhancement. Finally, guanidine hydrochloride-induced unfolding of NAD(+)-dependent DNA ligases is shown to be a complex process that involves accumulation of at least two equilibrium intermediates, the molten globule and its precursor. [less ▲]

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See detailCytoplasmic I kappa B alpha increases NF-kappa B-independent transcription through binding to histone deacetylase (HDAC) 1 and HDAC3
Viatour, Patrick ULg; Legrand-Poels, Sylvie; van Lint, Carine et al

in Journal of Biological Chemistry (2003), 278(47), 46541-46548

IkappaBalpha is an inhibitory molecule that sequesters NF-kappaB dimers in the cytoplasm of unstimulated cells. Upon stimulation, NF-kappaB moves to the nucleus and induces the expression of a variety of ... [more ▼]

IkappaBalpha is an inhibitory molecule that sequesters NF-kappaB dimers in the cytoplasm of unstimulated cells. Upon stimulation, NF-kappaB moves to the nucleus and induces the expression of a variety of genes including IkappaBalpha. This newly synthesized IkappaBalpha also translocates to the nucleus, removes activated NF-kappaB from its target genes, and brings it back to the cytoplasm to terminate the phase of NF-kappaB activation. We show here that IkappaBalpha enhances the transactivation potential of several homeodomain-containing proteins such as HOXB7 and Pit-1 through a NF-kappaB-independent association with histone deacetylase (HDAC) 1 and HDAC3 but not with HDAC2, -4, -5, and -6. IkappaBalpha bound both HDAC proteins through its ankyrin repeats, and this interaction was disrupted by p65. Immunofluorescence experiments demonstrated further that IkappaBalpha acts by partially redirecting HDAC3 to the cytoplasm. At the same time, an IkappaBalpha mutant, which lacked a functional nuclear localization sequence, interacted very efficiently with HDAC1 and -3 and intensively enhanced the transactivation potential of Pit-1. Our results support the hypothesis that the NF-kappaB inhibitor IkappaBalpha regulates the transcriptional activity of homeodomain-containing proteins positively through cytoplasmic sequestration of HDAC1 and HDAC3, a mechanism that would assign a new and unexpected role to IkappaBalpha. [less ▲]

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See detailStructural and functional adaptations to extreme temperatures in psychrophilic, mesophilic, and thermophilic DNA ligases
Georlette, D.; Damien, B.; Blaise, Vinciane ULg et al

in Journal of Biological Chemistry (2003), 278(39), 37015-37023

Psychrophiles, host of permanently cold habitats, display metabolic fluxes comparable to those exhibited by mesophilic organisms at moderate temperatures. These organisms have evolved by producing, among ... [more ▼]

Psychrophiles, host of permanently cold habitats, display metabolic fluxes comparable to those exhibited by mesophilic organisms at moderate temperatures. These organisms have evolved by producing, among other peculiarities, cold-active enzymes that have the properties to cope with the reduction of chemical reaction rates induced by low temperatures. The emerging picture suggests that these enzymes display a high catalytic efficiency at low temperatures through an improved flexibility of the structural components involved in the catalytic cycle, whereas other protein regions, if not implicated in catalysis, may be even more rigid than their mesophilic counterparts. In return, the increased flexibility leads to a decreased stability of psychrophilic enzymes. In order to gain further advances in the analysis of the activity/flexibility/stability concept, psychrophilic, mesophilic, and thermophilic DNA ligases have been compared by three-dimensional-modeling studies, as well as regards their activity, surface hydrophobicity, structural permeability, conformational stabilities, and irreversible thermal unfolding. These data show that the cold-adapted DNA ligase is characterized by an increased activity at low and moderate temperatures, an overall destabilization of the molecular edifice, especially at the active site, and a high conformational flexibility. The opposite trend is observed in the mesophilic and thermophilic counterparts, the latter being characterized by a reduced low temperature activity, high stability and reduced flexibility. These results strongly suggest a complex relationship between activity, flexibility and stability. In addition, they also indicate that in cold-adapted enzymes, the driving force for denaturation is a large entropy change. [less ▲]

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See detailDimerization and DNA binding properties of the Bacillus licheniformis 749/I BlaI repressor
Filée, Patrice ULg; Vreuls, Christelle ULg; Herman, Raphaël ULg et al

in Journal of Biological Chemistry (2003), 278(19), 16482-16487

In the absence of penicillin, the beta-lactamase encoding gene blaP of Bacillus licheniformis 749/I is negatively regulated by the transcriptional repressor BlaI. Three palindromic operator regions are ... [more ▼]

In the absence of penicillin, the beta-lactamase encoding gene blaP of Bacillus licheniformis 749/I is negatively regulated by the transcriptional repressor BlaI. Three palindromic operator regions are recognized by BlaI: two in the blaP promoter (OP1 and OP2) and one (OP3) in the promoter of the blaI-blaR1 operon. In this study, the dissociation constant of the purified BlaI dimer was estimated at 25 muM by equilibrium ultracentrifugation. Quantitative Western blot analysis indicates that the intracellular concentrations of BlaI in B. licheniformis 749/I and Bacillus subtilis transformed by a multicopy plasmid harboring the beta-lactamase locus (blaP-blaI-blaR1) were lower than (1.9 muM) or in the same range as (75 muM) the dissociation constant, respectively. This suggests that BlaI is partially dimeric in the cytoplasm of these strains and interacts in vivo with its operators as a preformed dimer. This hypothesis is supported by band shift assays on an operator containing a randomized half-operator sequence. The global dissociation constants of the operator-BlaI dimer complexes were measured by band shift assays and estimated as K-dOP1=1.7+/-0.5 10(-15) M-2, K-dOP2=3.3+/-0.9 10(-15) M-2, and K-dOP3=10.5+/-2.5 10(-15) M-2. The role of the DNA binding properties of BlaI on the beta-lactamase regulation is discussed. [less ▲]

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See detailActivity-stability relationships in extremophilic enzymes
D'Amico, Salvino ULg; Marx, J. C.; Gerday, Charles ULg et al

in Journal of Biological Chemistry (2003), 278(10), 7891-7896

Psychrophilic, mesophilic, and thermophilic alpha-amylases have been studied as regards their conformational stability, heat inactivation, irreversible unfolding, activation parameters of the reaction ... [more ▼]

Psychrophilic, mesophilic, and thermophilic alpha-amylases have been studied as regards their conformational stability, heat inactivation, irreversible unfolding, activation parameters of the reaction, properties of the enzyme in complex with a transition state analog, and structural permeability. These data allowed us to propose an energy landscape for a family of extremophilic enzymes based on the folding funnel model, integrating the main differences in conformational energy, cooperativity of protein unfolding, and temperature dependence of the activity. In particular, the shape of the funnel bottom, which depicts the stability of the native state ensemble, also accounts for the thermodynamic parameters of activation that characterize these extremophilic enzymes, therefore providing a rational basis for stability-activity relationships in protein adaptation to extreme temperatures. [less ▲]

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See detailThe structure of a cold-adapted family 8 xylanase at 1.3 angstrom resolution - Structural adaptations to cold and investigation of the active site
Van Petegem, F.; Collins, T.; Meuwis, Marie-Alice ULg et al

in Journal of Biological Chemistry (2003), 278(9), 7531-7539

Enzymes from psychrophilic organisms differ from their mesophilic counterparts in having a lower thermo-stability and a higher specific activity at low and moderate temperatures. The current consensus is ... [more ▼]

Enzymes from psychrophilic organisms differ from their mesophilic counterparts in having a lower thermo-stability and a higher specific activity at low and moderate temperatures. The current consensus is that they have an increased flexibility, enhancing accommodation and transformation of the substrates at low energy costs. Here we describe the structure of the xylanase from the Antarctic bacterium Pseudoalteromonas haloplanktis at 1.3 Angstrom resolution. Xylanases are usually grouped into glycosyl hydrolase families 10 and 11, but this enzyme belongs to family 8. The fold differs from that of other known xylanases and can be described as an (alpha/alpha)(6) barrel. Various parameters that may explain the cold-adapted properties were examined and indicated that the protein has a reduced number of salt bridges and an increased exposure of hydrophobic residues. The crystal structures of a complex with xylobiose and of mutant D144N were obtained at 1.2 and 1.5 A resolution, respectively. Analysis of the various substrate binding sites shows that the +3 and -3 subsites are rearranged as compared to those of a family 8 homolog, while the xylobiose complex suggests the existence of a +4 subsite. A decreased acidity of the substrate binding cleft and an increased flexibility of aromatic residues lining the subsites may enhance the rate at which substrate is bound. [less ▲]

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See detailThe inhibitor thiomandelic acid binds to both metal ions in metallo-beta-lactamase and induces positive cooperativity in metal binding.
Damblon, Christian ULg; Jensen, Mikael; Ababou, Abdessamad et al

in Journal of Biological Chemistry (2003), 278(31), 29240-51

Thiomandelic acid is a simple, broad spectrum, and reasonably potent inhibitor of metallo-beta-lactamases, enzymes that mediate resistance to beta-lactam antibiotics. We report studies by NMR and ... [more ▼]

Thiomandelic acid is a simple, broad spectrum, and reasonably potent inhibitor of metallo-beta-lactamases, enzymes that mediate resistance to beta-lactam antibiotics. We report studies by NMR and perturbed angular correlation (PAC) spectroscopy of the mode of binding of the R and S enantiomers of thiomandelic acid, focusing on their interaction with the two metal ions in cadmium-substituted Bacillus cereus metallo-beta-lactamase. The 113Cd resonances are specifically assigned to the metals in the two individual sites on the protein by using 113Cd-edited 1H NMR spectra. Each enantiomer of thiomandelate produces large downfield shifts of both 113Cd resonances and changes in the PAC spectra, which indicate that they bind such that the thiol of the inhibitor bridges between the two metals. For R-thiomandelate, this is unambiguously confirmed by the observation of scalar coupling between Halpha of the inhibitor and both cadmium ions. The NMR and PAC spectra reveal that the two chiral forms of the inhibitor differ in the details of their coordination geometry. The complex with R-thiomandelate, but not that with the S-enantiomer, shows evidence in the PAC spectra of a dynamic process in the nanosecond time regime, the possible nature of which is discussed. The thiomandelate complex of the mononuclear enzyme can be detected only at low metal to enzyme stoichiometry; the relative populations of mononuclear and binuclear enzyme as a function of cadmium concentration provide clear evidence for positive cooperativity in metal ion binding in the presence of the inhibitor, in contrast to the negative cooperativity observed in the free enzyme. [less ▲]

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See detailRegulation of hypoxia-inducible factor-1alpha protein level during hypoxic conditions by the phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase 3beta pathway in HepG2 cells.
Mottet, Denis ULg; Dumont, Valery; Deccache, Yann et al

in Journal of Biological Chemistry (2003), 278(33), 31277-85

Hypoxia initiates an intracellular signaling pathway leading to the activation of the transcription factor hypoxia-inducible factor-1 (HIF-1). HIF-1 activity is regulated through different mechanisms ... [more ▼]

Hypoxia initiates an intracellular signaling pathway leading to the activation of the transcription factor hypoxia-inducible factor-1 (HIF-1). HIF-1 activity is regulated through different mechanisms involving stabilization of HIF-1alpha, phosphorylations, modifications of redox conditions, and interactions with coactivators. However, it appears that some of these steps can be cell type-specific. Among them, the involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in the regulation of HIF-1 by hypoxia remains controversial. Here, we investigated the activation state of PI3K/Akt/glycogen synthase kinase 3beta (GSK3beta) in HepG2 cells. Increasing incubation times in hypoxia dramatically decreased both the phosphorylation of Akt and the inhibiting phosphorylation of GSK3beta. The PI3K/Akt pathway was necessary for HIF-1alpha stabilization early during hypoxia. Indeed, its inhibition was sufficient to decrease HIF-1alpha protein level after 5-h incubation in hypoxia. However, longer exposure (16 h) in hypoxia resulted in a decreased HIF-1alpha protein level compared with early exposure (5 h). At that time, Akt was no longer present or active, which resulted in a decrease in the inhibiting phosphorylation of GSK3beta on Ser-9 and hence in an increased GSK3beta activity. GSK3 inhibition reverted the effect of prolonged hypoxia on HIF-1alpha protein level; more stabilized HIF-1alpha was observed as well as increased HIF-1 transcriptional activity. Thus, a prolonged hypoxia activates GSK3beta, which results in decreased HIF-1alpha accumulation. In conclusion, hypoxia induced a biphasic effect on HIF-1alpha stabilization with accumulation in early hypoxia, which depends on an active PI3K/Akt pathway and an inactive GSK3beta, whereas prolonged hypoxia results in the inactivation of Akt and activation of GSK3beta, which then down-regulates the HIF-1 activity through down-regulation of HIF-1alpha accumulation. [less ▲]

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