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See detailAllosteric inhibition of VIM metallo-beta-lactamase by a camelid nanobody
Sohier, Jean ULg; Laurent, Clémentine ULg; Chevigné, Andy et al

in Biochemical Journal (2013), 450(3), 477-486

Metallo-β-lactamase (MβL) enzymes are usually produced by multiresistant Gram-negative bacterial strains and have spread worldwide. An approach based on phage display was employed to select single-domain ... [more ▼]

Metallo-β-lactamase (MβL) enzymes are usually produced by multiresistant Gram-negative bacterial strains and have spread worldwide. An approach based on phage display was employed to select single-domain antibody fragments (VHHs also called Nanobodies) that would inhibit the clinically relevant VIM-4 MβL. Out of more than 50 selected nanobodies, only the NbVIM_38 nanobody inhibited VIM-4. The paratope, inhibition mechanism and epitope of NbVIM_38 nanobody were then characterised. An alanine scan of the NbVIM_38 paratope showed that its binding was driven by hydrophobic amino acids. The inhibitory concentration was in the µM range for all tested β-lactams. In addition, the inhibition was found to follow a mixed hyperbolic profile with a predominantly uncompetitive component. Moreover, substrate inhibition was recorded only after nanobody binding. These kinetic data are indicative of a binding site that is distant from the active site. This finding was confirmed by epitope mapping analysis that was performed using peptides, and which identified two stretches of amino acids in the L6 loop and at the end of the alpha2 helix. Because this binding site is distant from the active site and alters both the substrate binding and catalytic properties of VIM-4, this nanobody can be considered as an allosteric inhibitor. [less ▲]

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See detailThermodynamics and structural analysis of positive allosteric modulation of the ionotropic glutamate receptor GluA2
Krintel, Christian; Frydenvang, Karla; Olsen, Lars et al

in Biochemical Journal (2012), 441

Positive allosteric modulators of the ionotropic glutamate receptor-2 (GluA2) are promising compounds for the treatment of cognitive disorders, e.g. Alzheimer’s disease. These modulators bind within the ... [more ▼]

Positive allosteric modulators of the ionotropic glutamate receptor-2 (GluA2) are promising compounds for the treatment of cognitive disorders, e.g. Alzheimer’s disease. These modulators bind within the dimer interface of the LBD (ligand-binding domain) and stabilize the agonist-bound conformation slowing receptor desensitization and/or deactivation. In the present study, we employ isothermal titration calorimetry to determine binding affinities and thermodynamic details of binding of modulators of GluA2. A mutant of the LBD of GluA2 (LBD-L483Y-N754S) that forms a stable dimer in solution was used. The potent GluA2 modulator BPAM-97 was used as a reference compound. Evidence that BPAM-97 binds in the same pocket as the well-known GluA2 modulator cyclothiazide was obtained from X-ray structures. The LBD-L483Y-N754S:BPAM-97 complex has aKd of 5.6 μM (Δ H = − 4.9 kcal/mol, − T Δ S = − 2.3 kcal/mol; where 1 kcal ≈4.187 kJ). BPAM-97 was used in a displacement assay to determine a Kd of 0.46 mM (Δ H = − 1.2 kcal/mol, − T Δ S = − 3.3 kcal/mol) for the LBD-L483Y-N754S:IDRA-21 complex. The major structural factors increasing the potency of BPAM-97 over IDRA-21 are the increased van der Waals contacts to, primarily, Met496 in GluA2 imposed by the ethyl substituent of BPAM-97. These results add important information on binding affinities and thermodynamic details, and provide a new tool in the development of drugs against cognitive disorders. Key words: binding affinity, crystal structure, ionotropic glutamate receptor, isothermal titration calorimetry, positive allosteric modulator [less ▲]

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See detailThree factors that modulate the activity of class D β-lactamases and interfere with the post-translational carboxylation of Lys 70
Vercheval, Lionel ULg; Di Paolo, Alexandre ULg; Borel, Franck et al

in Biochemical Journal (2010), 432(3), 495-504

Lys-70 carboxylation in the active site of class D β lactamases is essential for their activity. Structural, kinetic and affinity studies show that this post-translational modification can be affected by ... [more ▼]

Lys-70 carboxylation in the active site of class D β lactamases is essential for their activity. Structural, kinetic and affinity studies show that this post-translational modification can be affected by the presence of a poor substrate such as moxalactam but also by the V117T substitution. Val-117 is a strictly conserved hydrophobic residue located in the active site. In addition, inhibition of class D β lactamases by chloride ions is due to a competition between the side chain carboxylate of the modified Lys 70 and chloride ions. Determination of the individual kinetic constants shows that the deacylation of the acyl-enzyme is the rate limiting step for the wild type OXA 10 β lactamase. [less ▲]

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See detailHeart 6-phosphofructo-2-kinase activation by insulin requires PKB (protein kinase B), but not SGK3 (serum- and glucocorticoid-induced protein kinase 3).
Mouton, Veronique; Toussaint, Louise ULg; Vertommen, Didier et al

in Biochemical Journal (2010), 431(2), 267-75

On the basis of transfection experiments using a dominant-negative approach, our previous studies suggested that PKB (protein kinase B) was not involved in heart PFK-2 (6-phosphofructo2-kinase) activation ... [more ▼]

On the basis of transfection experiments using a dominant-negative approach, our previous studies suggested that PKB (protein kinase B) was not involved in heart PFK-2 (6-phosphofructo2-kinase) activation by insulin. Therefore we first tested whether SGK3 (serum- and glucocorticoid-induced protein kinase 3) might be involved in this effect. Treatment of recombinant heart PFK-2 with [gamma-32P]ATP and SGK3 in vitro led to PFK-2 activation and phosphorylation at Ser466 and Ser483. However, in HEK-293T cells [HEK (human embryonic kidney)-293 cells expressing the large T-antigen of SV40 (simian virus 40)] co-transfected with SGK3 siRNA (small interfering RNA) and heart PFK-2, insulin-induced heart PFK-2 activation was unaffected. The involvement of PKB in heart PFK-2 activation by insulin was re-evaluated using different models: (i) hearts from transgenic mice with a muscle/heart-specific mutation in the PDK1 (phosphoinositide-dependent protein kinase 1)-substrate-docking site injected with insulin; (ii) hearts from PKBbeta-deficient mice injected with insulin; (iii) freshly isolated rat cardiomyocytes and perfused hearts treated with the selective Akti-1/2 PKB inhibitor prior to insulin treatment; and (iv) HEK-293T cells co-transfected with heart PFK-2, and PKBalpha/beta siRNA or PKBalpha siRNA, incubated with insulin. Together, the results indicated that SGK3 is not required for insulin-induced PFK-2 activation and that this effect is likely mediated by PKBalpha. [less ▲]

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See detailBiochemical evidence of the interactions of membrane type-1 matrix metalloproteinase (MT1-MMP) with adenine nucleotide translocator (ANT): potential implications linking proteolysis with energy metabolism in cancer cells.
Radichev, I. A.; Remacle, A. G.; Sounni, Nor Eddine ULg et al

in Biochemical Journal (2009), 420(1), 37-47

Invasion-promoting MT1-MMP (membrane type-1 matrix metalloproteinase) is a key element in cell migration processes. To identify the proteins that interact and therefore co-precipitate with this proteinase ... [more ▼]

Invasion-promoting MT1-MMP (membrane type-1 matrix metalloproteinase) is a key element in cell migration processes. To identify the proteins that interact and therefore co-precipitate with this proteinase from cancer cells, we used the proteolytically active WT (wild-type), the catalytically inert E240A and the C-end truncated (tailless; ΔCT) MT1-MMP–FLAG constructs as baits. The identity of the pulled-down proteins was determined by LC-MS/MS (liquid chromatography tandem MS) and then confirmed by Western blotting using specific antibodies. We determined that, in breast carcinoma MCF cells (MCF-7 cells), ANT (adenine nucleotide translocator) efficiently interacted with the WT, E240A and ΔCT constructs. The WT and E240A constructs also interacted with α-tubulin, an essential component of clathrin-mediated endocytosis. In turn, tubulin did not co-precipitate with the ΔCT construct because of the inefficient endocytosis of the latter, thus suggesting a high level of selectivity of our test system. To corroborate these results, we then successfully used the ANT2–FLAG construct as a bait to pull-down MT1-MMP, which was naturally produced by fibrosarcoma HT1080 cells. We determined that the presence of the functionally inert catalytic domain alone was sufficient to cause the proteinase to interact with ANT2, thus indicating that there is a non-proteolytic mode of these interactions. Overall, it is tempting to hypothesize that by interacting with pro-invasive MT1-MMP, ANT plays a yet to be identified role in a coupling mechanism between energy metabolism and pericellular proteolysis in migrating cancer cells. [less ▲]

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See detailUncoupling protein 1 inhibition by purine nucleotides is under the control of the endogenous ubiquinone redox state.
Swida-Barteczka, A.; Woyda-Ploszczyca, A.; Sluse, Francis ULg et al

in Biochemical Journal (2009), 424

We studied non-esterified fatty acid-induced uncoupling of heterologously expressed rat UCP1 (uncoupling protein 1) in yeast mitochondria, as well as UCP1 in rat BAT (brown adipose tissue) mitochondria ... [more ▼]

We studied non-esterified fatty acid-induced uncoupling of heterologously expressed rat UCP1 (uncoupling protein 1) in yeast mitochondria, as well as UCP1 in rat BAT (brown adipose tissue) mitochondria. The proton-conductance curves and the relationship between the ubiquinone reduction level and membrane potential were determined in non-phosphorylating BAT and yeast mitochondria. The ADP/O method was applied to determine the ADP phosphorylation rate and the relationship between the ubiquinone reduction level and respiration rate in yeast mitochondria. Our studies of the membranous ubiquinone reduction level in mitochondria demonstrate that activation of UCP1 leads to a purine nucleotide-sensitive decrease in the ubiquinone redox state. Results obtained for non-phosphorylating and phosphorylating mitochondria, as the endogenous ubiquinone redox state was gradually varied by a lowering rate of the ubiquinone-reducing or ubiquinol-oxidizing pathways, indicate that the endogenous ubiquinone redox state has no effect on non-esterified fatty acid-induced UCP1 activity in the absence of GTP, and can only regulate this activity through sensitivity to inhibition by the purine nucleotide. At a given oleic acid concentration, inhibition by GTP diminishes when ubiquinone is reduced sufficiently. The ubiquinone redox state-dependent alleviation of UCP1 inhibition by the purine nucleotide was observed at a high ubiquinone reduction level, when it exceeded 85-88%. [less ▲]

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See detailMutational analysis of the zinc- and substrate-binding sites in the CphA metallo-beta-lactamase from Aeromonas hydrophila.
Bebrone, Carine ULg; Anne, Christine; Kerff, Frédéric ULg et al

in Biochemical Journal (2008), 414(1), 151-9

The subclass B2 CphA (Carbapenemase hydrolysing Aeromonas) beta-lactamase from Aeromonas hydrophila is a Zn(2+)-containing enzyme that specifically hydrolyses carbapenems. In an effort to evaluate ... [more ▼]

The subclass B2 CphA (Carbapenemase hydrolysing Aeromonas) beta-lactamase from Aeromonas hydrophila is a Zn(2+)-containing enzyme that specifically hydrolyses carbapenems. In an effort to evaluate residues potentially involved in metal binding and/or catalysis (His(118), Asp(120), His(196) and His(263)) and in substrate specificity (Val(67), Thr(157), Lys(224) and Lys(226)), site-directed mutants of CphA were generated and characterized. Our results confirm that the first zinc ion is in interaction with Asp(120) and His(263), and thus is located in the 'cysteine' zinc-binding site. His(118) and His(196) residues seem to be interacting with the second zinc ion, as their replacement by alanine residues has a negative effect on the affinity for this second metal ion. Val(67) plays a significant role in the binding of biapenem and benzylpenicillin. The properties of a mutant with a five residue (LFKHV) insertion just after Val(67) also reveals the importance of this region for substrate binding. This latter mutant has a higher affinity for the second zinc ion than wild-type CphA. The T157A mutant exhibits a significantly modified activity spectrum. Analysis of the K224Q and N116H/N220G/K224Q mutants suggests a significant role for Lys(224) in the binding of substrate. Lys(226) is not essential for the binding and hydrolysis of substrates. Thus the present paper helps to elucidate the position of the second zinc ion, which was controversial, and to identify residues important for substrate binding. [less ▲]

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See detailThe linker region plays a key role in the adaptation to cold of the cellulase from an Antarctic bacterium
Sonan, Guillaume K; Receveur-Brechot, Véronique; Duez, Colette ULg et al

in Biochemical Journal (2007), 407(Part 2), 293-302

The psychrophilic cellulase, Cel5G, from the Antarctic bacterium Pseudoalteromonas haloplanktis is composed of a catalytic module (CM) joined to a carbohydrate-binding module (CBM) by an unusually long ... [more ▼]

The psychrophilic cellulase, Cel5G, from the Antarctic bacterium Pseudoalteromonas haloplanktis is composed of a catalytic module (CM) joined to a carbohydrate-binding module (CBM) by an unusually long, extended and flexible linker region (LR) containing three loops closed by three disulfide bridges. To evaluate the possible role of this region in cold adaptation, the LR was sequentially shortened by protein engineering, successively deleting one and two loops of this module, whereas the last disulfide bridge was also suppressed by replacing the last two cysteine residue by two alanine residues. The kinetic and thermodynamic properties of the mutants were compared with those of the full-length enzyme, and also with those of the cold-adapted CM alone and with those of the homologous mesophilic enzyme, Cel5A, from Erwinia chrysanthemi. The thermostability of the mutated enzymes as well as their relative flexibility were evaluated by differential scanning calorimetry and fluorescence quenching respectively. The topology of the structure of the shortest mutant was determined by SAXS (small-angle X-ray scattering). The data indicate that the sequential shortening of the LR induces a regular decrease of the specific activity towards macromolecular substrates, reduces the relative flexibility and concomitantly increases the thermostability of the shortened enzymes. This demonstrates that the long LR of the full-length enzyme favours the catalytic efficiency at low and moderate temperatures by rendering the structure not only less compact, but also less stable, and plays a crucial role in the adaptation to cold of this cellulolytic enzyme. [less ▲]

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See detailThe activity of the dinuclear cobalt-beta-lactamase from Bacillus cereus in catalysing the hydrolysis of beta-lactams
Badarau, Adriana; Damblon, Christian ULg; Page, Michael I

in Biochemical Journal (2007), 401(Part 1), 197-203

Metallo-beta-lactamases are native zinc enzymes that catalyse the hydrolysis of beta-lactam antibiotics, but are also able to function with cobalt(II) and require one or two nnetal-ions for catalytic ... [more ▼]

Metallo-beta-lactamases are native zinc enzymes that catalyse the hydrolysis of beta-lactam antibiotics, but are also able to function with cobalt(II) and require one or two nnetal-ions for catalytic activity. The hydrolysis of cefoxitin, cephaloridine and benzylpenicillin catalysed by CoBcII (cobalt-substituted beta-lactamase from Bacillus cereus) has been studied at different pHs and metal-ion concentrations. An enzyme group of pK(a) 6.52 +/- 0.1 is found to be required in its deprotionated form for metal-ion binding and catalysis. The species that results from the loss of one cobalt ion from the enzyme has no significant catalytic activity and is thought to be the mononuclear CoBcII. It appears that dinuclear CoBcII is the active form of the enzyme necessary for turnover, while the mononuclear CoBcII is only involved in substrate binding. The cobalt-substituted enzyme is a more efficient catalyst than the native enzyme for the hydrolysis of some beta-lactam antibiotics suggesting that the role of the metal-ion is predominantly to provide the nucleophilic hydroxide, rather than to act as a Lewis acid to polarize the carbonyl group and stabilize the oxyanion tetrahedral intermediate. [less ▲]

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See detailTNFa and IKKb-mediated TANK/I-TRAF phosphorylation: implications for interaction with NEMO/IKKg and NF-kB activation
Bonif, Marianne; Meuwis, Marie-Alice ULg; Close, Pierre ULg et al

in Biochemical Journal (2006), 394

Pro-inflammatory cytokines trigger signalling cascades leading to NF-kappaB (nuclear factor-kappaB)-dependent gene expression through IKK [IkappaB (inhibitory kappaB) kinase]-dependent phosphorylation and ... [more ▼]

Pro-inflammatory cytokines trigger signalling cascades leading to NF-kappaB (nuclear factor-kappaB)-dependent gene expression through IKK [IkappaB (inhibitory kappaB) kinase]-dependent phosphorylation and subsequent degradation of the IkappaB proteins and via induced phosphorylation of p65. These signalling pathways rely on sequentially activated kinases which are assembled by essential and non-enzymatic scaffold proteins into functional complexes. Here, we show that the pro-inflammatory cytokine TNFalpha (tumour necrosis factor alpha) promotes TANK [TRAF (TNF receptor-associated factor) family member associated NF-kappaB activator] recruitment to the IKK complex via a newly characterized C-terminal zinc finger. Moreover, we show that TANK is phosphorylated by IKKbeta upon TNFalpha stimulation and that this modification negatively regulates TANK binding to NEMO (NF-kappaB essential modulator). Interestingly, reduced TANK expression by RNA interference attenuates TNFalpha-mediated induction of a subset of NF-kappaB target genes through decreased p65 transactivation potential. Therefore the scaffold protein TANK is required for the cellular response to TNFalpha by connecting upstream signalling molecules to the IKKs and p65, and its subsequent IKKbeta-mediated phosphorylation may be a mechanism to terminate the TANK-dependent wave of NF-kappaB activation. [less ▲]

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See detailPerturbation of actin dynamics induces NF-kappa B activation in myelomonocytic cells through an NADPH oxidase-dependent pathway
Kustermans, Gaëlle ULg; El Benna, Jamel; Piette, Jacques ULg et al

in Biochemical Journal (2005), 387(Pt 2), 531-540

Although several reports showed the effect of compounds disrupting microtubules on NF-kappa B (nuclear factor kappa B) activation, nothing is known about agents perturbing actin dynamics. In the present ... [more ▼]

Although several reports showed the effect of compounds disrupting microtubules on NF-kappa B (nuclear factor kappa B) activation, nothing is known about agents perturbing actin dynamics. In the present study, we have shown that actin cytoskeleton disruption induced by actin-depolymerizing agents such as cytochalasin D and latrunculin B and actin-polymerizing compounds such as jasplakinolide induced NF-kappa B activation in myelomonocytic cells. The transduction pathway involved the I kappa B (inhibitory kappa B) kinase complex and a degradation of I kappa B alpha. We have shown that NF-kappa B activation in response to the perturbation of actin dynamics required reactive oxygen species. as demonstrated by the effect of antioxidants. Actin cytoskeleton disruption by cytochalasin D induced O-2(-) release from human monocytes, through the activation of the NADPH oxidase, as confirmed by the phosphorylation and by the membrane translocation of p47(phox). NF-kappa B activation after actin cytoskeleton disruption could be physiologically relevant during monocyte activation and/or recruitment into injured tissues, where cellular attachment, migration and phagocytosis result in cyclic shifts in cytoskeletal organization and disorganization. [less ▲]

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See detailYB-1 represses AP1-dependent gene transactivation and interacts with an AP-1 DNA sequence.
Samuel, Shaija; Twizere, Jean-Claude ULg; Bernstein, Lori R

in Biochemical Journal (2005), 388(Pt 3), 921-8

Involvement of the AP-1 (activator protein-1) transcription factor has been demonstrated previously in the regulation of cell proliferation and cell-cycle progression, in the control of cell migration ... [more ▼]

Involvement of the AP-1 (activator protein-1) transcription factor has been demonstrated previously in the regulation of cell proliferation and cell-cycle progression, in the control of cell migration, invasion and metastasis, and in signal transduction, stress responsiveness, DNA replication and DNA repair. YB-1 (Y-box-binding protein-1) has also been implicated in many of these processes. However, the mechanism by which YB-1 mediates these processes is poorly understood. In the present study, we report that overexpression of a transfected gene encoding YB-1 in human HeLa cervical carcinoma cells significantly represses the transactivation of a minimal AP-1 reporter construct in response to the tumour promoter PMA. YB-1 also represses mRNA expression and PMA-induced promoter transactivation of the endogenous AP-1 target gene encoding matrix metalloproteinase-12 (metalloelastase). YB-1 transrepression of both the minimal and matrix metalloproteinase-12 promoter reporter constructs is dependent on the AP-1 sequence. To identify new nuclear proteins that bind specifically to the AP-1 DNA-binding site, we devised a DNA-affinity-chromatography-based assay termed NAPSTER (nucleotide-affinity preincubation specificity test of recognition) and discovered a 49 kDa protein from human cancer cells that binds in a sequence-specific manner to the AP-1 DNA sequence. By tandem MS fragmentation sequencing analyses we determined that p49 is a YB-1. Immunoblotting of the NAPSTER-purified p49 protein using anti-YB-1 antibodies confirmed YB-1 binding to the AP-1 DNA sequence, as did gel mobility-supershift assays using YB-1 antibodies. This is the first report of YB-1 transrepression and interaction at the AP-1 DNA-binding site. [less ▲]

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See detailZoledronic acid up-regulates bone sialoprotein expression in osteoblastic cells through Rho GTPase inhibition
Chaplet, Michaël; Deroanne, Christophe ULg; Fisher, Larry W. et al

in Biochemical Journal (2004), 384(Pt 3), 591-598

Clinical practice reveals that osteoporotic women treated with BPs (bisphosphonates) show an increased bone mass density and a reduced risk of fractures. However, the mechanisms leading to these ... [more ▼]

Clinical practice reveals that osteoporotic women treated with BPs (bisphosphonates) show an increased bone mass density and a reduced risk of fractures. However, the mechanisms leading to these beneficial effects of BPs are still poorly understood. We hypothesized that ZOL (zoledronic acid), a potent third-generation BP, may induce the expression of proteins associated with the bone-forming potential of osteoblastic cells such as BSP (bone sialoprotein). Expression of BSP gene is up-regulated by hormones that promote bone formation and has been associated with de novo bone mineralization. Using real-time reverse transcriptase-PCR and Western-blot analysis, we demonstrated that ZOL increased BSP expression in Saos-2 osteoblast-like cells. Nuclear run-on and mRNA decay assays showed no effect at the transcriptional level but a stabilization of BSP transcripts in ZOL-treated cells. ZOL effect on BSP expression occurred through an interference with the mevalonate pathway since it was reversed by either mevalonate pathway intermediates or a Rho GTPase activator. We showed that ZOL impaired membrane localization of RhoA in Saos-2 cells indicating reduced prenylation of this protein. By the use of small interfering RNAs directed to RhoA and Rac1, we identified both Rho GTPases as negative regulators of BSP expression in Saos-2 cells. Our study demonstrates that ZOL induces BSP expression in osteoblast-like cells through inactivation of Rho GTPases and provides a potential mechanism to explain the favourable effects of ZOL treatment on bone mass and integrity. [less ▲]

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See detailCyclo-oxygenase type 2-dependent prostaglandin E-2 secretion is involved in retrovirus-induced T-cell dysfunction in mice
Rahmouni, Souad ULg; Aandahl, Einar Martin; Nayjib, Btissam ULg et al

in Biochemical Journal (2004), 384(Pt 3), 469-476

MAIDS (murine AIDS) is caused by infection with the murine leukaemia retrovirus RadLV-Rs and is characterized by a severe immunodeficiency and T-cell anergy combined with a lymphoproliferative disease ... [more ▼]

MAIDS (murine AIDS) is caused by infection with the murine leukaemia retrovirus RadLV-Rs and is characterized by a severe immunodeficiency and T-cell anergy combined with a lymphoproliferative disease affecting both B- and T-cells. Hyperactivation of the cAMP-protein kinase A pathway is involved in the T-cell dysfunction of MAIDS and HIV by inhibiting T-cell activation through the T-cell receptor. In the present study, we show that MAIDS involves a strong and selective up-regulation of cyclo-oxygenase type 2 in the CD11b+ subpopulation of T- and B-cells of the lymph nodes, leading to increased levels of PGE2 (prostaglandin E2). PGE2 activates the cAMP pathway through G-protein-coupled receptors. Treatment with cyclo-oxygenase type 2 inhibitors reduces the level of PGE2 and thereby reverses the T-cell anergy, restores the T-cell immune function and ameliorates the lymphoproliferative disease. [less ▲]

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See detailKinetic and structural optimization to catalysis at low temperatures in a psychrophilic cellulase from the Antarctic bacterium Pseudoalteromonas haloplanktis
Garsoux, G.; Lamotte, J.; Gerday, Charles ULg et al

in Biochemical Journal (2004), 384(Pt 2), 247-253

The cold-adapted cellulase CelG has been purified from the culture supernatant of the Antarctic bacterium Pseudoalteromonas haloplanktis and the gene coding for this enzyme has been cloned, sequenced and ... [more ▼]

The cold-adapted cellulase CelG has been purified from the culture supernatant of the Antarctic bacterium Pseudoalteromonas haloplanktis and the gene coding for this enzyme has been cloned, sequenced and expressed in Escherichia coli. This cellulase is composed of three structurally and functionally distinct regions: an N-terminal catalytic domain belonging to glycosidase family 5 and a C-terminal cellulose-binding domain belonging to carbohydrate-binding module family 5. The linker of 107 residues connecting both domains is one of the longest found in cellulases, and optimizes substrate accessibility to the catalytic domain by drastically increasing the Surface of cellulose available to a bound enzyme molecule. The psychrophilic enzyme is closely related to the cellulase Cel5 from Erwinia chrysanthemi. Both k(cat) and k(cat)/K-m values at 4 degreesC for the psychrophilic cellulase are similar to the values for Cel5 at 30-35 degreesC, suggesting temperature adaptation of the kinetic parameters. The thermodynamic parameters of activation of CelG suggest a heat-labile, relatively disordered active site with low substrate affinity, in agreement with the experimental data. The structure of CelG has been constructed by homology modelling with a molecule of cellotetraose docked into the active site. No structural alteration related to cold-activity can be found in the catalytic cleft, whereas several structural factors in the overall structure can explain the weak thermal stability, suggesting that the loss of stability provides the required active-site mobility at low temperatures. [less ▲]

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See detailGuanidinium chloride denaturation of the dimeric Bacillus licheniformis Blal repressor highlights an independent domain unfolding pathway
Vreuls, Christelle ULg; Filée, Patrice ULg; Van Melckebeke, H. et al

in Biochemical Journal (2004), 384(Pt 1), 179-190

The Bacillus licheniformis 74911 BlaI repressor is a prokaryotic regulator that, in the absence of a P-lactam antibiotic, prevents the transcription of the blaP gene, which encodes the BlaP beta-lactamase ... [more ▼]

The Bacillus licheniformis 74911 BlaI repressor is a prokaryotic regulator that, in the absence of a P-lactam antibiotic, prevents the transcription of the blaP gene, which encodes the BlaP beta-lactamase. The BlaI repressor is composed of two structural domains. The 82-residue NTD (N-terminal domain) is a DNA-binding domain, and the CTD (C-terminal domain) containing the next 46 residues is a dimerization domain. Recent studies have shown the existence of the monomeric, dimeric and tetrameric forms of BlaI in solution. In the present study, we analyse the equilibrium unfolding of BlaI in the presence of GdmCl (guanidinium chloride) using different techniques: intrinsic and ANS (8-anilinonaphthalene-1-sulphonic acid) fluorescence, far- and near-UV CD spectroscopy, cross-linking, analytical ultracentrifugation, size exclusion chromatography and NMR spectroscopy. In addition, the intact NTD and CTD were purified after proteolysis of BlaI by papain, and their unfolding by GdmCl was also studied. GdmCl-induced equilibrium unfolding was shown to be fully reversible for BlaI and for the two isolated fragments. The results demonstrate that the NTD and CTD of BlaI fold/unfold independently in a four-step process, with no significant cooperative interactions between them. During the first step, the unfolding of the Blal CTD occurs, followed in the second step by the formation of an 'ANS-bound' intermediate state. Crosslinking and analytical ultracentrifugation experiments suggest that the dissociation of the dimer into two partially unfolded monomers takes place in the third step. Finally, the unfolding of the Blal NTD occurs at a GdmCI concentration of approx. 4 M. In summary, it is shown that the Blal CTD is structured, more flexible and less stable than the NTD upon GdmCI denaturation. These results contribute to the characterization of the Blal dimerization domain (i.e. CTD) involved in the induction process. [less ▲]

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See detailStructural organization of mitochondrial human complex I: role of the ND4 and ND5 mitochondria-encoded subunits and interaction with prohibitin
Bourges, I.; Ramus, C.; de Camaret, B. M. et al

in Biochemical Journal (2004), 383(Part 3), 491-499

Mitochondria-encoded ND (NADH dehydrogenase) subunits, as components of the hydrophobic part of complex I, are essential for NADH:ubiquinone oxidoreductase activity. Mutations or lack of expression of ... [more ▼]

Mitochondria-encoded ND (NADH dehydrogenase) subunits, as components of the hydrophobic part of complex I, are essential for NADH:ubiquinone oxidoreductase activity. Mutations or lack of expression of these subunits have significant pathogenic consequences in humans. However, the way these events affect complex I assembly is poorly documented. To understand the effects of particular mutations in ND subunits on complex I assembly, we studied four human cell lines: ND4 non-expressing cells, ND5 non-expressing cells, and rhodegrees cells that do not express any ND subunits, in comparison with normal complex I control cells. In control cells. all the seven analysed nuclear-encoded complex I subunits Were found to be attached to the mitochondrial inner membrane, except for the 24 kDa subunit, which was nearly equally partitioned between the membranes and the matrix. Absence of a single ND subunit, or even all the seven ND subunits, caused no major changes in the nuclear-encoded complex I subunit content of mitochondria. However, in cells lacking ND4 or ND5, very low amounts of 24 kDa subunit were found associated with the membranes, whereas most of the other nuclear-encoded subunits remained attached. In contrast, membrane association of most of the nuclear subunits was significantly reduced in the absence of all seven ND proteins. Immunopurification detected several subcomplexes. One of these, containing the 23, 30 and 49 kDa subunits, also contained prohibitin. This is the first description of prohibitin interaction with complex I subunits and suggests that this protein might play a role in the assembly or degradation of mitochondrial complex I. [less ▲]

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See detailSecondary-structure characterization by far-UV CD of highly purified uncoupling protein 1 expressed in yeast
Douette, Pierre ULg; Navet, Rachel ULg; Bouillenne, Fabrice ULg et al

in Biochemical Journal (2004), 380(Pt 1), 139-145

The rat UCP1 (uncoupling protein 1) is a mitochondrial inner-membrane carrier involved in energy dissipation and heat production. We expressed UCP1 carrying a His(6) epitope at its C-terminus in ... [more ▼]

The rat UCP1 (uncoupling protein 1) is a mitochondrial inner-membrane carrier involved in energy dissipation and heat production. We expressed UCP1 carrying a His(6) epitope at its C-terminus in Saccharomyces cerevisiae mitochondria. The recombinant-tagged UCP1 was purified by immobilized metal-ion affinity chromatography to homogeneity (>95 %). This made it suitable for subsequent biophysical characterization. Fluorescence resonance energy transfer experiments showed that n-dodecyl-beta-D-maltoside-solubilized UCPI-His(6) retained its PN (purine nucleotide)-binding capacity. The far-UV CD spectrum of the functional protein clearly indicated the predominance of a-helices in the UCP1 secondary structure. The UCP1 secondary structure exhibited an alpha-helical degree of approx. 68 %, which is at least 25 % higher than the previously reported estimations based on computational predictions. Moreover, the helical content remained unchanged in free and PN-loaded UCP1. A homology model of the first repeat of UCP1, built on the basis of X-ray-solved close parent, the ADP/ATP carrier, strengthened the CD experimental results. Our experimental and computational results indicate that (i) alpha-helices are the major component of UCP1 secondary structure; (ii) PN-binding mechanism does not involve significant secondary-structure rearrangement; and (iii) UCP1 shares similar secondary-structure characteristics with the ADP/ATP carrier, at least for the first repeat. [less ▲]

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See detailMutational analysis of the catalytic centre of the Citrobacter freundii AmpD N-acetylmuramyl-L-alanine amidase
Genereux, Catherine ULg; Dehareng, Dominique ULg; Devreese, Bart et al

in Biochemical Journal (2004), 377(Pt 1), 111-120

Citrobacter freundii AmpD is an intracellular 1,6-anhydro-N-acetylmuramyl-L-alanine amidase involved in both peptidoglycan recycling and beta-lactamase induction. AmpD exhibits a strict specificity for 1 ... [more ▼]

Citrobacter freundii AmpD is an intracellular 1,6-anhydro-N-acetylmuramyl-L-alanine amidase involved in both peptidoglycan recycling and beta-lactamase induction. AmpD exhibits a strict specificity for 1,6-anhydromuropeptides and requires zinc for enzymic activity. The AmpD three-dimensional structure exhibits a fold similar to that of another Zn2+ N-acetylmuramyl-L-alanine amidase, the T7 lysozyme, and these two enzymes define a new family of Zn-amidases which can be related to the eukaryotic PGRP (peptidoglycan-recognition protein) domains. In an attempt to assign the different zinc ligands and to probe the catalytic mechanism of AmpD amidase, molecular modelling based on the NMR structure and site-directed mutagenesis were performed. Mutation of the two residues presumed to act as zinc ligands into alanine (H34A and D164A) yielded inactive proteins which had also lost their ability to bind zinc. By contrast, the active H154N mutant retained the capacity to bind the metal ion. Three other residues which could be involved in the AmpD catalytic mechanism have been mutated (Y63F, E116A, K162H and K162Q). The E116A mutant was inactive, but on the basis of the molecular modelling this residue is not directly involved in the catalytic mechanism, but rather in the binding of the zinc by contributing to the correct orientation of His-34. The K162H and K162Q mutants retained very low activity (0.7 and 0.2% of the wildtype activity respectively), whereas the Y63F mutant showed 16% of the wild-type activity. These three latter mutants exhibited a good affinity for Zn ions and the substituted residues are probably involved in the binding of the substrate. We also describe a new method for generating the N-acetylglucosaminyl-1,6-anhydro-N-acetylmuramyl-tripeptide AmpD substrate from purified peptidoglycan by the combined action of two hydrolytic enzymes. [less ▲]

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See detailOn the substrate specificity of bacterial DD-peptidases: evidence from two series of peptidoglycan-mimetic peptides
Anderson, J. W.; Adediran, S. A.; Charlier, Paulette ULg et al

in Biochemical Journal (2003), 373(Part 3), 949-955

The reactions between bacterial DD-peptidases and beta-lactam antibiotics have been studied for many years. Less well understood are the interactions between these enzymes and their natural substrates ... [more ▼]

The reactions between bacterial DD-peptidases and beta-lactam antibiotics have been studied for many years. Less well understood are the interactions between these enzymes and their natural substrates, presumably the peptide moieties of peptidoglycan. In general, remarkably little activity has previously been demonstrated in vitro against potential peptide substrates, although in many cases the peptides employed were non-specific and not homologous with the relevant peptidoglycan. In this paper, the specificity of a panel of DD-peptidases against elements of species-specific D-alanyl-D-alanine peptides has been assessed. In two cases, those of soluble, low-molecular-mass DD-peptidases, high activity against the relevant peptides has been demonstrated. In these cases, the high specificity is towards the free N-terminus of the peptidoglycan fragment. With a number of other enzymes, particularly high-molecular-mass DD-peptidases, little or no activity against these peptides was observed. In separate experiments, the reactivity of the enzymes against the central, largely invariant, peptide stem was examined. None of the enzymes surveyed showed high activity against this structural element although weak specificity in the expected direction towards the one structural variable (D-gammaGln versus D-gammaGlu) was observed. The current state of understanding of the activity of these enzymes in vitro is discussed. [less ▲]

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