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See detailThe 1.5-angstrom structure of Chryseobacterium meningosepticum zinc beta-lactamase in complex with the inhibitor, D-captopril
Garcia-Saez, I.; Hopkins, J.; Papamicael, C. et al

in Journal of Biological Chemistry (2003), 278(26), 23868-23873

The crystal structure of the class-B beta-lactamase, BlaB, from the pathogenic bacterium, Chryseobacterium meningosepticum, in complex with the inhibitor, D-captopril, has been solved at 1.5-Angstrom ... [more ▼]

The crystal structure of the class-B beta-lactamase, BlaB, from the pathogenic bacterium, Chryseobacterium meningosepticum, in complex with the inhibitor, D-captopril, has been solved at 1.5-Angstrom resolution. The enzyme has the typical alphabeta/betaalpha metallo-beta-lactamase fold and the characteristic two metal binding sites of members of the subclass B1, in which two Zn2+ ions were identified. D-Captopril, a diastereoisomer of the commercial drug, captopril, acts as an inhibitor by displacing the catalytic hydroxyl ion required for antibiotic hydrolysis and intercalating its sulfhydryl group between the two Zn2+ ions. Interestingly, D-captopril is located on one side of the active site cleft. The x-ray structure of the complex of the closely related enzyme, IMP-1, with a mercaptocarboxylate inhibitor, which also contains a sulfhydryl group bound to the two Zn2+ ions, shows the ligand to be located on the opposite side of the active site cleft. A molecule generated by fusion of these two inhibitors would cover the entire cleft, suggesting an interesting approach to the design of highly specific inhibitors. [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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See detailThe HBZ factor of human T-cell leukemia virus type I dimerizes with transcription factors JunB and c-Jun and modulates their transcriptional activity.
Basbous, Jihane; Arpin, Charlotte; Gaudray, Gilles ULg et al

in Journal of Biological Chemistry (2003), 278(44), 43620-7

The human T-cell leukemia virus type I (HTLV-I)-encoded Tax protein activates transcription from the viral promoter via association with the cellular basic leucine zipper factor cAMP-response element ... [more ▼]

The human T-cell leukemia virus type I (HTLV-I)-encoded Tax protein activates transcription from the viral promoter via association with the cellular basic leucine zipper factor cAMP-response element-binding protein-2. Tax is also able to induce cellular transformation of T lymphocytes probably by modulating transcriptional activity of cellular factors, including nuclear factor-kappaB, E2F, activator protein-1 (AP-1), and p53. Recently, we characterized in HTLV-I-infected cells the presence of a novel viral protein, HBZ, encoded by the complementary strand of the HTLV-I RNA genome (Gaudray, G., Gachon, F., Basbous, J., Biard-Piechaczyk, M., Devaux, C., and Mesnard, J.-M. (2002) J. Virol. 76, 12813-12822). HBZ is a nuclear basic leucine zipper protein that down-regulates Tax-dependent viral transcription by inhibiting the binding of cAMP-response element-binding protein-2 to the HTLV-I promoter. In searching for other cellular targets of HBZ, we identified two members of the Jun family, JunB and c-Jun. Co-immunoprecipitation and cellular colocalization confirmed that HBZ interacts in vivo with JunB and c-Jun. When transiently introduced into CEM cells with a reporter gene containing the AP-1 site from the collagenase promoter, HBZ suppressed transactivation by c-Jun. On the other hand, the combination of HBZ with Jun-B had higher transcriptional activity than JunB alone. Consistent with the structure of its basic domain, we demonstrate that HBZ decreases the DNA-binding activity of c-Jun and JunB. Last, we show that c-Jun is no longer capable of activating the basal expression of the HTLV-I promoter in the presence of HBZ in vivo. Our results support the hypothesis that HBZ could be a negative modulator of the Tax effect by controlling Tax expression at the transcriptional level and by attenuating activation of AP-1 by Tax. [less ▲]

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See detailP2X1-mediated ERK2 activation amplifies the collagen-induced platelet secretion by enhancing myosin light chain kinase activation.
Oury, Cécile ULg; Toth-Zsamboki, Emese; Cornelissen, Heidi et al

in Journal of Biological Chemistry (2003)

This study shows that, at low doses of collagen, glycoprotein VI activation leads to early protein kinase C- and MLC kinase-dependent platelet degranulation. Rapidly released ATP triggers P2X1 -mediated ... [more ▼]

This study shows that, at low doses of collagen, glycoprotein VI activation leads to early protein kinase C- and MLC kinase-dependent platelet degranulation. Rapidly released ATP triggers P2X1 -mediated Ca2+ influx, activating ERK2, in turn amplifying platelet secretion by reinforcing the early MLC kinase phosphorylation. Hence, the P2X1-ERK2-MLC axis contributes to collagen-induced platelet activation by enhancing platelet degranulation. [less ▲]

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See detailType I collagen triplet duplication mutation in lethal osteogenesis imperfecta shifts register of alpha chains throughout the helix and disrupts incorporation of mutant helices into fibrils and extracellular matrix.
Cabral, Wayne A; Mertts, Marianna V; Makareeva, Elena et al

in Journal of Biological Chemistry (2003), 278(12), 10006-12

The majority of collagen mutations causing osteogenesis imperfecta (OI) are glycine substitutions that disrupt formation of the triple helix. A rare type of collagen mutation consists of a duplication or ... [more ▼]

The majority of collagen mutations causing osteogenesis imperfecta (OI) are glycine substitutions that disrupt formation of the triple helix. A rare type of collagen mutation consists of a duplication or deletion of one or two Gly-X-Y triplets. These mutations shift the register of collagen chains with respect to each other in the helix but do not interrupt the triplet sequence, yet they have severe clinical consequences. We investigated the effect of shifting the register of the collagen helix by a single Gly-X-Y triplet on collagen assembly, stability, and incorporation into fibrils and matrix. These studies utilized a triplet duplication in COL1A1 exon 44 that occurred in the cDNA and gDNA of two siblings with lethal OI. The normal allele encodes three identical Gly-Ala-Hyp triplets at aa 868-876, whereas the mutant allele encodes four. The register shift delays helix formation, causing overmodification. Differential scanning calorimetry yielded a decrease in T(m) of 2 degrees C for helices with one mutant chain and a 6 degrees C decrease in helices with two mutant chains. An in vitro binary co-processing assay of N-proteinase cleavage demonstrated that procollagen with the triplet duplication has slower N-propeptide cleavage than in normal controls or procollagen with proalpha1(I) G832S, G898S, or G997S substitutions, showing that the register shift persists through the entire helix. The register shift disrupts incorporation of mutant collagen into fibrils and matrix. Proband fibrils formed inefficiently in vitro and contained only normal helices and helices with a single mutant chain. Helices with two mutant chains and a significant portion of helices with one mutant chain did not form fibrils. In matrix deposited by proband fibroblasts, mutant chains were abundant in the immaturely cross-linked fraction but constituted a minor fraction of maturely cross-linked chains. The profound effects of shifting the collagen triplet register on chain interactions in the helix and on fibril formation correlate with the severe clinical consequences. [less ▲]

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See detailDevelopment of pure prolactin receptor antagonists
Bernichtein, Sophie; Kayser, Christine; Dillner, Karin et al

in Journal of Biological Chemistry (2003), 278(38), 35988-99

Prolactin (PRL) promotes tumor growth in various experimental models and leads to prostate hyperplasia and mammary neoplasia in PRL transgenic mice. Increasing experimental evidence argues for the ... [more ▼]

Prolactin (PRL) promotes tumor growth in various experimental models and leads to prostate hyperplasia and mammary neoplasia in PRL transgenic mice. Increasing experimental evidence argues for the involvement of autocrine PRL in this process. PRL receptor antagonists have been developed to counteract these undesired proliferative actions of PRL. However, all forms of PRL receptor antagonists obtained to date exhibit partial agonism, preventing their therapeutic use as full antagonists. In the present study, we describe the development of new human PRL antagonists devoid of agonistic properties and therefore able to act as pure antagonists. This was demonstrated using several in vitro bioassays, including highly sensitive assays able to detect extremely low levels of receptor activation. These new compounds also act as pure antagonists in vivo, as assessed by analyzing their ability to competitively inhibit PRL-triggered signaling cascades in various target tissues (liver, mammary gland, and prostate). Finally, by using transgenic mice expressing PRL specifically in the prostate, which exhibit constitutively activated signaling cascades paralleling hyperplasia, we show that these new PRL analogs are able to completely revert PRL-activated events. These second generation human PRL antagonists are good candidates to be used as inhibitors of growth-promoting actions of PRL. [less ▲]

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See detailDual effects of an extra disulfide bond on the activity and stability of a cold-adapted alpha-amylase
D'Amico, Salvino ULg; Gerday, Charles ULg; Feller, Georges ULg

in Journal of Biological Chemistry (2002), 277(48), 46110-46115

Chloride-dependent alpha-amylases constitute a well conserved family of enzymes thereby allowing investigation of the characteristics of each member to understand, for example, relevant properties ... [more ▼]

Chloride-dependent alpha-amylases constitute a well conserved family of enzymes thereby allowing investigation of the characteristics of each member to understand, for example, relevant properties required for environmental adaptation. In this context, we have constructed a double mutant (Q58C/A99C) of the cold-active and heat-labile alpha-amylase from the Antarctic bacterium Pseudoalteromonas haloplanktis, defined on the basis of its strong similarity with the mesophilic enzyme from pig pancreas. This mutant was characterized to understand the role of an extra disulfide bond specific to warm-blooded animals and located near the entrance of the catalytic cleft. We show that the catalytic parameters of the mutant are drastically modified and similar to those of the mesophilic enzyme. Calorimetric studies demonstrated that the mutant is globally stabilized (DeltaDeltaG = 1.87 kcal/mol at 20 degrees C) when compared with the wild-type enzyme, although the melting point (T-m) was not increased. Moreover, fluorescence quenching experiments indicate a more compact structure for the mutated a-amylase. However, the strain imposed on the active site architecture induces a 2-fold higher thermal inactivation rate at 45 degreesC as well as the appearance of a less stable calorimetric domain. It is concluded that stabilization by the extra disulfide bond arises from an enthalpy-entropy compensation effect favoring the enthalpic contribution. [less ▲]

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See detailPitx factors are involved in basal and hormone-regulated activity of the human prolactin promoter
Quentien, M. H.; Manfroid, Isabelle ULg; Moncet, D. et al

in Journal of Biological Chemistry (2002), 277(46), 44408-44416

The pituitary-specific POU homeodomain factor Pit-1 likely interacts with other factors for cell-specific expression of prolactin. Here we identify the paired-like homeobox transcription factors Pitx1 and ... [more ▼]

The pituitary-specific POU homeodomain factor Pit-1 likely interacts with other factors for cell-specific expression of prolactin. Here we identify the paired-like homeobox transcription factors Pitx1 and Pitx2 as factors functionally activating the proximal human prolactin promoter (hPRL-164luc). Using in vitro binding assays and a series of site-specific mutations of the proximal hPRL promoter, we mapped the 131 and B2 bicoid sites involved in Pitx-mediated transactivation of the hPRL-164luc construct. In somatolactotroph GH4C1 cells, basal proximal hPRL promoter activity was inhibited by a Pitx2 dominant-negative form in a dose-dependent manner, whereas binding disruptive mutations in the Pitx sites significantly reduced basal activity of the promoter. We also show that synergistic activation of hPRL-164luc by Pitx2 and Pit-1 requires the integrity of the B2 Pitx binding site, and at least one of the P1 and P2 Pit-1 response elements. In addition, mutation in the B2 Pitx site results in attenuation of the promoter's responsiveness to forskolin, thyrotropin-releasing hormone, and epidermal growth factor. Conversely, Pitx1 or Pitx2 overexpression in GH4C1 cells leads to an enhancement of the drugs stimulatory effects. Altogether, these results suggest that full responsiveness to several signaling pathways regulating the hPRL promoter requires the B2 Pitx binding site and that Pitx factors may be part of the proteic complex involved in these regulations. Finally, in situ hybridization analysis showing coexpression of the PRL and Pitx2 genes in rat and human lactotroph cells corroborates the physiological relevance of these results. [less ▲]

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See detailAssociation of the adaptor TANK with the IκB kinase (IKK) regulator NEMO connects IKK complexes with IKKε and TBK1 kinases
Chariot, Alain ULg; Leonardi, Antonio; Muller, Jean-Noel ULg et al

in Journal of Biological Chemistry (2002), 277(40), 37029-37036

Canonical activation of NF-kappaB is mediated via phosphorylation of the inhibitory IkappaB proteins by the IkappaB kinase complex (IKK). IKK is composed of a heterodimer of the catalytic IKKalpha and ... [more ▼]

Canonical activation of NF-kappaB is mediated via phosphorylation of the inhibitory IkappaB proteins by the IkappaB kinase complex (IKK). IKK is composed of a heterodimer of the catalytic IKKalpha and IKKbeta subunits and a presumed regulatory protein termed NEMO (NF-kappaB essential modulator) or IKKgamma. NEMO/IKKgamma is indispensable for activation of the IKKs in response to many signals, but its mechanism of action remains unclear. Here we identify TANK (TRAF family member-associated NF-kappaB activator) as a NEMO/IKKgamma-interacting protein via yeast two-hybrid analyses. This interaction is confirmed in mammalian cells, and the domains required are mapped. TANK was previously shown to assist NF-kappaB activation in a complex with TANK-binding kinase 1 (TBK1) or IKKepsilon, two kinases distantly related to IKKalpha/beta, but the underlying mechanisms remained unknown. Here we show that TBK1 and IKKepsilon synergize with TANK to promote interaction with the IKKs. The TANK binding domain within NEMO/IKKgamma is required for proper functioning of this IKK subunit. These results indicate that TANK can synergize with IKKepsilon or TBK1 to link them to IKK complexes, where the two kinases may modulate aspects of NF-kappaB activation. [less ▲]

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See detailA novel family 8 xylanase, functional and physicochemical characterization
Collins, T.; Meuwis, Marie-Alice ULg; Stals, I. et al

in Journal of Biological Chemistry (2002), 277(38), 35133-35139

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See detailSubstrate-activated zinc binding of metallo-beta-lactamases - Physiological importance of the mononuclear enzymes
Wommer, S.; Rival, S.; Heinz, U. et al

in Journal of Biological Chemistry (2002), 277(27), 24142-24147

We have investigated the influence of substrate binding on the zinc ion affinity of representatives from the three metallo-g-lactamase subclasses, B1 (BcII from Bacillus cereus and BlaB from ... [more ▼]

We have investigated the influence of substrate binding on the zinc ion affinity of representatives from the three metallo-g-lactamase subclasses, B1 (BcII from Bacillus cereus and BlaB from Chryseobacterium meningosepticum), B2 (CphA from Aeromonas hydrophila), and B3 (L1 from Stenotrophomonas maltophilia). By competition experiments with metal-free apoenzymes and chromophoric zinc chelators or EDTA, we determined the dissociation constants in the absence and presence of substrates. For the formation of the monozine enzymes we determined constants of 1.8, 5.1, 0.007, and 2.6 nm in the absence and 13.6, 1.8, 1.2, and 5.7 pm in the presence of substrates for Bell, BlaB, CphA, and L1, respectively. A second zinc ion binds in the absence (presence) of substrates with considerably higher dissociation constants, namely 1.8 (0.8), 0.007 (0.025), 50 (1.9), and 0.006 (0.12) mum for BcII, BlaB, CphA, and L1, respectively. We have concluded that the apo form might be the prevailing state of most of the metallo-beta-lactamases under physiological conditions in the absence of substrates. Substrate availability induces a spontaneous self-activation due to a drastic decrease of the dissociation constants, resulting in the formation of active mononuclear enzymes already at picomolar free zinc ion concentrations. In the presence of substrates, the binuclear state of the enzymes only exists at unphysiologic high zinc concentrations and might be of no biological relevance. From the competition experiments with EDTA it is further concluded that the reactivation rate does not depend on the pool of free zinc ions but proceeds via the EDTA-Zn(II)-enzyme ternary complexes. [less ▲]

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See detailPhosphorylation of varicella-zoster virus IE63 protein by casein kinases influences its cellular localization and gene regulation activity
Bontems, Sébastien ULg; Di Valentin, Emmanuel ULg; Baudoux, Laurence et al

in Journal of Biological Chemistry (2002), 277(23), 21050-21060

During the early phase of varicella-zoster virus (VZV) infection, Immediate Early protein 63 (IE63) is expressed rapidly and abundantly in the nucleus, while during latency, this protein is confined ... [more ▼]

During the early phase of varicella-zoster virus (VZV) infection, Immediate Early protein 63 (IE63) is expressed rapidly and abundantly in the nucleus, while during latency, this protein is confined mostly to the cytoplasm. Because phosphorylation is known to regulate many cellular events, we investigated the importance of this modification on the cellular localization of IE63 and on its regulatory properties. We demonstrate here that cellular casein kinases I and II are implicated in the in vitro and in vivo phosphorylation of IE63. A mutational approach also indicated that phosphorylation of the protein is important for its correct cellular localization in a cell type-dependent fashion. Using an activity test, we demonstrated that IE63 was able to repress the gene expression driven by two VZV promoters and that phosphorylation of the protein was required for its full repressive properties. Finally, we showed that IE63 was capable of exerting its repressive activity in the cytoplasm, as well as in the nucleus, suggesting a regulation at the transcriptional and/or post-transcriptional level. [less ▲]

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See detailSubdivision of the helix-turn-helix GntR family of bacterial regulators in the FadR, HutC, MocR, and YtrA subfamilies
Rigali, Sébastien ULg; Derouaux, Adeline ULg; Giannotta, F. et al

in Journal of Biological Chemistry (2002), 277(15), 12507-12515

Haydon and Guest (Haydon, D. J, and Guest, J. R. (1991) FEMS Microbiol Lett. 63, 291-295) first described the helix-turn-helix GntR family of bacterial regulators. They presented them as transcription ... [more ▼]

Haydon and Guest (Haydon, D. J, and Guest, J. R. (1991) FEMS Microbiol Lett. 63, 291-295) first described the helix-turn-helix GntR family of bacterial regulators. They presented them as transcription factors sharing a similar N-terminal DNA-binding (D-b) domain, but they observed near-maximal divergence in the C-terminal effector-binding and oligomerization (E-b/O) domain. To elucidate this C-terminal heterogeneity, structural, phylogenetic, and functional analyses were performed on a family that now comprises about 270 members. Our comparative study first focused on the C-terminal E-b/O domains and next on DNA-binding domains and palindromic operator sequences, has classified the GntR members into four subfamilies that we called FadR, HutC, MocR, and YtrA. Among these subfamilies a degree of similarity of about 55% was observed throughout the entire sequence. Structure/function associations were highlighted although they were not absolutely stringent. The consensus sequences deduced for the DNA-binding domain were slightly different for each subfamily, suggesting that fusion between the D-b and E-b/O domains have occurred separately, with each subfamily having its own D-b domain ancestor. Moreover, the compilation of the known or predicted palindromic cis-acting elements has highlighted different operator sequences according to our subfamily subdivision. The observed C-terminal E-b/O domain heterogeneity was therefore reflected on the DNA-binding domain and on the cis-acting elements, suggesting the existence of a tight link between the three regions involved in the regulating process. [less ▲]

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See detailCloning and characterization of ADAMTS-14, a novel ADAMTS displaying high homology with ADAMTS-2 and ADAMTS-3.
Colige, Alain ULg; Vandenberghe, Isabel; Thiry, Marc ULg et al

in Journal of Biological Chemistry (2002), 277(8), 5756-66

The processing of amino- and carboxyl-propeptides of fibrillar collagens is required to generate collagen monomers that correctly assemble into fibrils. Mutations in the ADAMTS2 gene, the ... [more ▼]

The processing of amino- and carboxyl-propeptides of fibrillar collagens is required to generate collagen monomers that correctly assemble into fibrils. Mutations in the ADAMTS2 gene, the aminopropeptidase of procollagen I and II, result in the accumulation of non-fully processed type I procollagen, causing human Ehlers-Danlos syndrome type VIIC and animal dermatosparaxis. In this study, we show that the aminopropeptide of type I procollagen can be cleaved in vivo in absence of ADAMTS-2 activity and that this processing is performed at the cleavage site for ADAMTS-2. In an attempt to identify the enzyme responsible for this alternative aminoprocollagen peptidase activity, we have cloned the cDNA and determined the primary structure of human and mouse ADAMTS-14, a novel ADAMTS displaying striking homologies with ADAMTS-2 and -3. The structure of the human gene, which maps to 10q21.3, and the mechanisms of generation of the various transcripts are described. The existence of two sites of initiation of transcription, in two different promoter contexts, suggests that transcripts resulting from these two sites can be differently regulated. The tissue distribution of ADAMTS-14, the regulation of the gene expression by various cytokines and the activity of the recombinant enzyme are evaluated. The potential function of ADAMTS-14 as a physiological aminoprocollagen peptidase in vivo is discussed. [less ▲]

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