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See detailCrystal structure of the Actinomadura R39 DD-peptidase reveals new domains in penicillin-binding proteins.
Sauvage, Eric ULg; Herman, Raphaël ULg; Petrella, Stephanie et al

in Journal of Biological Chemistry (2005), 280(35), 31249-56

Actinomadura sp. R39 produces an exocellular DD-peptidase/penicillin-binding protein (PBP) whose primary structure is similar to that of Escherichia coli PBP4. It is characterized by a high beta-lactam ... [more ▼]

Actinomadura sp. R39 produces an exocellular DD-peptidase/penicillin-binding protein (PBP) whose primary structure is similar to that of Escherichia coli PBP4. It is characterized by a high beta-lactam-binding activity (second order rate constant for the acylation of the active site serine by benzylpenicillin: k2/K = 300 mm(-1) s(-1)). The crystal structure of the DD-peptidase from Actinomadura R39 was solved at a resolution of 1.8 angstroms by single anomalous dispersion at the cobalt resonance wavelength. The structure is composed of three domains: a penicillin-binding domain similar to the penicillin-binding domain of E. coli PBP5 and two domains of unknown function. In most multimodular PBPs, additional domains are generally located at the C or N termini of the penicillin-binding domain. In R39, the other two domains are inserted in the penicillin-binding domain, between the SXXK and SXN motifs, in a manner similar to "Matryoshka dolls." One of these domains is composed of a five-stranded beta-sheet with two helices on one side, and the other domain is a double three-stranded beta-sheet inserted in the previous domain. Additionally, the 2.4-angstroms structure of the acyl-enzyme complex of R39 with nitrocefin reveals the absence of active site conformational change upon binding the beta-lactams. [less ▲]

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See detailRole Of The Lid Hydrophobicity Pattern In Pancreatic Lipase Activity
Thomas, Annick ULg; Allouche, M.; Basyn, F. et al

in Journal of Biological Chemistry (2005), 280(48), 40074-83

Pancreatic lipase is a soluble globular protein that must undergo structural modifications before it can hydrolyze oil droplets coated with bile salts. The binding of colipase and movement of the lipase ... [more ▼]

Pancreatic lipase is a soluble globular protein that must undergo structural modifications before it can hydrolyze oil droplets coated with bile salts. The binding of colipase and movement of the lipase lid open access to the active site. Mechanisms triggering lid mobility are unclear. The *KNILSQIVDIDGI* fragment of the lid of the human pancreatic lipase is predicted by molecular modeling to be a tilted peptide. Tilted peptides are hydrophobicity motifs involved in membrane fusion and more globally in perturbations of hydrophobic/hydrophilic interfaces. Analysis of this lid fragment predicts no clear consensus of secondary structure that suggests that its structure is not strongly sequence determined and could vary with environment. Point mutations were designed to modify the hydrophobicity profile of the [240-252] fragment and their consequences on the lipase-mediated catalysis were tested. Two mutants, in which the tilted peptide motif was lost, also have poor activity on bile salt-coated oil droplets and cannot be reactivated by colipase. Conversely, one mutant in which a different tilted peptide is created retains colipase dependence. These results suggest that the tilted hydrophobicity pattern of the [240-252] fragment is neither important for colipase binding to lipase, nor for interfacial binding but is important to trigger the maximal catalytic efficiency of lipase in the presence of bile salt. [less ▲]

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See detailDetermination Of The Functionality Of Common Apoa5 Polymorphisms
Talmud, Pj.; Palmen, J.; Putt, W. et al

in Journal of Biological Chemistry (2005), 280(31), 28215-20

Common variants of APOA5 have consistently shown association with differences in plasma triglyceride (TG) levels. These single nucleotide polymorphisms (SNPs) fall into three common haplotypes: APOA5*1 ... [more ▼]

Common variants of APOA5 have consistently shown association with differences in plasma triglyceride (TG) levels. These single nucleotide polymorphisms (SNPs) fall into three common haplotypes: APOA5*1, with common alleles at all sites; APOA5*2, with rare alleles of -1131T--> C, -3A--> G, 751G--> T, and 1891T--> C; and APOA5*3, distinguished by the c56C--> G (S19W). Molecular modeling of the apoAV signal peptide (SP) showed an increased angle of insertion (65 degrees ) at the lipid/water interface of Trp-19 SP compared with Ser-19 SP (40 degrees ), predicting reduced translocation. This was confirmed by 50% reduction of Trp-19-encoded SP.secretory alkaline phosphatase (SEAP) fusion protein secreted into the medium from HepG2 cells compared with the Ser-19.SEAP fusion protein (p < 0.002). Considering APOA5*2 SNPs, there was no significant difference in the relative luciferase expression in Huh7 cells transiently transfected with a -1131T construct compared with the -1131C (fragments -1177 to -516 or -1177 to -3). Similarly, for the -3A--> G in the Kozak sequence, in vitro transcription/translation assays and primer extension inhibition assays showed no alternate AUG initiation codon usage, demonstrating that -3A--> G did not influence translation efficiency. Although 1891T--> C in the 3'-untranslated region disrupts a putative Oct-1 transcription factor binding site, when inserted 3' of the luciferase gene the T--> C change demonstrated no significant difference in luciferase expression. Thus, association of APOA5*2 SNPs with TG levels is not due to the individual effects of any of these SNPs, although cooperativity between the SNPs cannot be excluded. Alternatively, the effect on TG levels may reflect the strong linkage disequilibrium with the functional APOC3 SNPs. [less ▲]

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See detailVaricella-zoster virus IE63 protein phosphorylation by roscovitine-sensitive cyclin-dependent kinases modulates its cellular localization and activity.
Habran, Lionel ULg; Bontems, Sébastien ULg; Di Valentin, Emmanuel ULg et al

in Journal of Biological Chemistry (2005), 280(32), 29135-43

During the first stage of Varicella-Zoster virus (VZV) infection, IE63 (immediate early 63 protein) is mostly expressed in the nucleus and also slightly in the cytoplasm, and during latency, IE63 ... [more ▼]

During the first stage of Varicella-Zoster virus (VZV) infection, IE63 (immediate early 63 protein) is mostly expressed in the nucleus and also slightly in the cytoplasm, and during latency, IE63 localizes in the cytoplasm quite exclusively. Because phosphorylation is known to regulate various cellular mechanisms, we investigated the impact of phosphorylation by roscovitine-sensitive cyclin-dependent kinase (RSC) on the localization and functional properties of IE63. We demonstrated first that IE63 was phosphorylated on Ser-224 in vitro by CDK1 and CDK5 but not by CDK2, CDK7, or CDK9. Furthermore, by using roscovitine and CDK1 inhibitor III (CiIII), we showed that CDK1 phosphorylated IE63 on Ser-224 in vivo. By mutagenesis and the use of inhibitors, we demonstrated that phosphorylation on Ser-224 was important for the correct localization of the protein. Indeed, the substitution of these residues by alanine led to an exclusive nuclear localization of the protein, whereas mutations into glutamic acid did not modify its subcellular distribution. When transfected or VZV-infected cells were treated with roscovitine or CiIII, an exclusive nuclear localization of IE63 was also observed. By using a transfection assay, we also showed that phosphorylation on Ser-224 and Thr-222 was essential for the down-regulation of the basal activity of the VZV DNA polymerase gene promoter. Similarly, roscovitine and CiIII impaired these properties of the wild-type form of IE63. These observations clearly demonstrated the importance of CDK1-mediated IE63 phosphorylation for a correct distribution of IE63 between both cellular compartments and for its repressive activity toward the promoter tested. [less ▲]

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See detailYersinia phosphatase induces mitochondrially dependent apoptosis of T cells.
Bruckner, Shane; Rahmouni, Souad ULg; Tautz, Lutz et al

in Journal of Biological Chemistry (2005), 280(11), 10388-94

To evade the immune system, the etiologic agent of plague, Yersinia pestis, injects an exceptionally active tyrosine phosphatase called YopH into host cells using a type III secretion system. We recently ... [more ▼]

To evade the immune system, the etiologic agent of plague, Yersinia pestis, injects an exceptionally active tyrosine phosphatase called YopH into host cells using a type III secretion system. We recently reported that YopH acutely inhibits T cell antigen receptor signaling by dephosphorylating the Lck tyrosine kinase. Here, we show that prolonged presence of YopH in primary T cells or Jurkat T leukemia cells causes apoptosis, detected by annexin V binding, mitochondrial breakdown, caspase activation, and internucleosomal fragmentation. YopH also causes cell death when expressed in HeLa cells, and this cell death was inhibited by YopH-specific small molecule inhibitors. Cell death induced by YopH was also prevented by caspase inhibition or co-expression of Bcl-xL. We conclude that YopH not only paralyzes T cells acutely, but also ensures that the cells will not recover to induce a protective immune response but instead undergo mitochondrially regulated programmed cell death. [less ▲]

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See detailThe homeobox protein MSX2 interacts with tax oncoproteins and represses their transactivation activity.
Twizere, Jean-Claude ULg; Lefebvre, Laurent; Collete, Delphine et al

in Journal of Biological Chemistry (2005), 280(33), 29804-11

Bovine leukemia virus (BLV) tax is an essential gene involved in the transcriptional activation of viral expression. Tax is also believed to be implicated in leukemogenesis because of its ability to ... [more ▼]

Bovine leukemia virus (BLV) tax is an essential gene involved in the transcriptional activation of viral expression. Tax is also believed to be implicated in leukemogenesis because of its ability to immortalize primary cells in vitro. To gain insight into the molecular pathways mediating the activities of this important gene, we identified cellular proteins interacting with Tax. By means of a two-hybrid approach, we show that Tax specifically interacts with MSX2, a general repressor of gene expression. GST pull-down experiments and co-immunoprecipitation assays further confirmed binding specificity. Furthermore, the N-terminal residues 1-79 of MSX2 are required for binding, whereas the C-terminal residues 201-267 of MSX2 do not play a critical role. Whereas the oncogenic potential of Tax in primary cells was only slightly affected by overexpression of MSX2, the other function of Tax, namely LTR-dependent transcriptional activation, was inhibited by MSX2 in human HeLa and bovine B-lymphoblastoid (BL3) cell lines. This MSX2 repression function can be counteracted by overexpression of transcription factors CREB2 and RAP74. The Tax/MSX2 interplay thus results in repression of viral transcriptional activation possibly acting as a regulatory feedback loop. Importantly, this viral gene silencing is not strictly associated with a concomitant loss of Tax oncogenicity as measured by its ability to immortalize primary cells. And interestingly, MSX2 also interacts with and inhibits the transactivation function of the related Tax1 protein encoded by the Human T-cell leukemia virus type 1 (HTLV-1). [less ▲]

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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 detailProbing the specificity of the subclass B3 FEZ-1 metallo-beta-lactamase by site-directed mutagenesis
Mercuri, P. S.; Garcia-Saez, I.; De Vriendt, K. et al

in Journal of Biological Chemistry (2004), 279(32), 33630-33638

The subclass B3 FEZ-1 beta-lactamase produced by Fluoribacter (Legionella) gormanii is a Zn(II)-containing enzyme that hydrolyzes the beta-lactam bond in penicillins, cephalosporins, and carbapenems. FEZ ... [more ▼]

The subclass B3 FEZ-1 beta-lactamase produced by Fluoribacter (Legionella) gormanii is a Zn(II)-containing enzyme that hydrolyzes the beta-lactam bond in penicillins, cephalosporins, and carbapenems. FEZ-1 has been extensively studied using kinetic, computational modeling and x-ray crystallography. In an effort to probe residues potentially involved in substrate binding and zinc binding, five site-directed mutants of FEZ-1 (H121A, Y156A, S221A, N225A, and Y228A) were prepared and characterized using metal analyses and steady state kinetics. The activity of H121A is dependent on zinc ion concentration. The H121A monozinc form is less active than the dizinc form, which exhibits an activity similar to that of the wild type enzyme. Tyr156 is not essential for binding and hydrolysis of the substrate. Substitution of residues Ser221 and Asn225 modifies the substrate profile by selectively decreasing the activity against carbapenems. The Y228A mutant is inhibited by the product formed upon hydrolysis of cephalosporins. A covalent bond between the side chain of Cys200 and the hydrolyzed cephalosporins leads to the formation of an inactive and stable complex. [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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