References of "Galleni, Moreno"
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See detailKinetic Studies on CphA Mutants Reveal the Role of the P158-P172 Loop in Activity versus Carbapenems.
Bottoni, Carlo; Perilli, Mariagrazia; Marcoccia, Francesca et al

in Antimicrobial Agents and Chemotherapy (2016), 60(5), 3123-6

Site-directed mutagenesis of CphA indicated that prolines in the P158-P172 loop are essential for the stability and the catalytic activity of subclass B2 metallo-beta-lactamases against carbapenems. The ... [more ▼]

Site-directed mutagenesis of CphA indicated that prolines in the P158-P172 loop are essential for the stability and the catalytic activity of subclass B2 metallo-beta-lactamases against carbapenems. The sequential substitution of proline led to a decrease of the catalytic efficiency of the variant compared to the wild-type (WT) enzyme but also to a higher affinity for the binding of the second zinc ion. [less ▲]

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See detailMetal binding to the N-terminal cytoplasmic domain of the PIB ATPase HMA4 is required for metal transport in Arabidopsis.
Laurent, Clémentine ULg; Lekeux, Gilles ULg; Ukuwela, Ashwinie A et al

in Plant Molecular Biology (2016), 90

PIB ATPases are metal cation pumps that transport metals across membranes. These proteins possess N- and C-terminal cytoplasmic extensions that contain Cys- and His-rich high affinity metal binding ... [more ▼]

PIB ATPases are metal cation pumps that transport metals across membranes. These proteins possess N- and C-terminal cytoplasmic extensions that contain Cys- and His-rich high affinity metal binding domains, which may be involved in metal sensing, metal ion selectivity and/or in regulation of the pump activity. The PIB ATPase HMA4 (Heavy Metal ATPase 4) plays a central role in metal homeostasis in Arabidopsis thaliana and has a key function in zinc and cadmium hypertolerance and hyperaccumulation in the extremophile plant species Arabidopsis halleri. <br />Here, we examined the function and structure of the N-terminal cytoplasmic metal-binding domain of HMA4. We mutagenized a conserved CCTSE metal-binding motif in the domain and assessed the impact of the mutations on protein function and localization in planta, on metal-binding properties in vitro and on protein structure by Nuclear Magnetic Resonance spectroscopy. <br />The two Cys residues of the motif are essential for the function, but not for localization, of HMA4 in planta, whereas the Glu residue is important but not essential. These residues also determine zinc coordination and affinity. Zinc binding to the N-terminal domain is thus crucial for HMA4 protein function, whereas it is not required to maintain the protein structure. <br />Altogether, combining in vivo and in vitro approaches in our study provides insights towards the molecular understanding of metal transport and specificity of metal P-type ATPases. [less ▲]

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See detailKinetic Study of Laboratory Mutants of NDM-1 Metallo-beta-Lactamase and the Importance of an Isoleucine at Position 35.
Marcoccia, Francesca; Bottoni, Carlo; Sabatini, Alessia et al

in Antimicrobial agents and chemotherapy (2016), 60(4), 2366-72

Two laboratory mutants of NDM-1 were generated by replacing the isoleucine at position 35 with threonine and serine residues: the NDM-1(I35T)and NDM-1(I35S)enzymes. These mutants were well characterized ... [more ▼]

Two laboratory mutants of NDM-1 were generated by replacing the isoleucine at position 35 with threonine and serine residues: the NDM-1(I35T)and NDM-1(I35S)enzymes. These mutants were well characterized, and their kinetic parameters were compared with those of the NDM-1 wild type. Thekcat,Km, andkcat/Kmvalues calculated for the two mutants were slightly different from those of the wild-type enzyme. Interestingly, thekcat/Kmof NDM-1(I35S)for loracarbef was about 14-fold higher than that of NDM-1. Far-UV circular dichroism (CD) spectra of NDM-1 and NDM-1(I35T)and NDM-1(I35S)enzymes suggest local structural rearrangements in the secondary structure with a marked reduction of alpha-helix content in the mutants. [less ▲]

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See detailEnzymatic functionalization of a nanobody using protein insertion technology
Crasson, Oscar ULg; Rhazi, Noureddine; Jacquin, Olivier et al

in Protein Engineering, Design & Selection (2015), 28(10), 451-460

Antibody-based products constitute one of the most attractive biological molecules for diagnostic, medical imagery and therapeutic purposes with very few side effects. Their development has be- come a ... [more ▼]

Antibody-based products constitute one of the most attractive biological molecules for diagnostic, medical imagery and therapeutic purposes with very few side effects. Their development has be- come a major priority of biotech and pharmaceutical industries. Recently, a growing number of modified antibody-based products have emerged including fragments, multi-specific and conjugate antibodies. In this study, using protein engineering, we have functionalized the anti-hen egg-white lysozyme (HEWL) camelid VHH antibody fragment (cAb-Lys3), by insertion into a solvent-exposed loop of the Bacillus licheniformis β-lactamase BlaP. We showed that the generated hybrid protein conserved its enzymatic activity while the displayed nanobody retains its ability to inhibit HEWL with a nanomolar affinity range. Then, we successfully implemented the functionalized cAb-Lys3 in enzyme-linked immunosorbent assay, potentiometric biosensor and drug screening assays. The hybrid protein was also expressed on the surface of phage particles and, in this context, was able to interact specifically with HEWL while the β-lactamase activity was used to monitor phage interactions. Finally, using thrombin-cleavage sites surrounding the permissive insertion site in the β-lactamase, we reported an expression system in which the nanobody can be easily separated from its carrier protein. Altogether, our study shows that insertion into the BlaP β-lactamase consti- tutes a suitable technology to functionalize nanobodies and allowsthe creation of versatile tools that can be used in innovative biotechnological assays. [less ▲]

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See detailThe hidden face of the human macrophage chitotriosidase: taking a new look at this enzyme based on the biochemical and phylogenomic analysis of its chitin binding domain
Crasson, Oscar ULg; Legrand, François; Léonard, Raphaël et al

Poster (2015, August)

Carbohydrates recognition is a critical process involved in numerous aspects of the cell biology such as inflammation, innate immune responses and proliferation. Chitin is an homopolysaccharide composed ... [more ▼]

Carbohydrates recognition is a critical process involved in numerous aspects of the cell biology such as inflammation, innate immune responses and proliferation. Chitin is an homopolysaccharide composed of β-1,4-linked N-acetylglucosamine (GlcNAc) units that is an abundant structural component of various infectious organisms like protozoans, nematodes and fungi. As there is no endogenous chitin produced by mammals, this polymer appeared to be a strategic target for innate immune agents which is why various carbohydrate binding proteins, associated or not with catalytic domains, are synthetized by plants and animals and are known to play a crucial role in innate immunity. The macrophage chitotriosidase (HCHT) is one of the three active chitinases synthetized by humans and has triggered significant attention recently due to its association with various inflammatory disorders. HCHT belongs to the Glycosyl Hydrolase family 18 (GH18) and is known to be involved in innate immunity. Nevertheless, its precise physiological function remains unclear. As numerous GHs, HCHT is a modular protein composed of a catalytic domain (GH18) associated to a Carbohydrate Binding Module (CBM) which is essential to hydrolyse crystalline chitin. If the catalytic domain GH18 is highly common in other GHs from animals, plants, fungi, bacteria, archea and viruses, its CBM (named ChBD) is much less conserved which makes the association between these two domains particularly intriguing. This work aims to demystify HCHT’s physiological function. Firstly, using competitive inhibition assays, we have highlighted the ability of ChBD to interact with chitooligosaccharides (GlcNAc1-2-4-6) which suggests that ChBD can potentially act as a lectin domain. Secondly, to better understand the molecular basis for chitin recognition, we have used homology modelling to build, with high confidence, the 3D structure model of ChBD. Based on this model, a specific set of residues has been selected for alanine scan mutagenesis which has allowed us to define the minimum chitin binding interface of the protein. Thirdly, Phylogenomic studies were performed to analyse the evolutionary history of the isolated catalytic and ChBD domains and understand how these domains were combined. Based on all these results, we discuss a new way of looking at HCHT where its ChBD would be the key determinant that has guided the catalytic domain from a basic metabolic function to a critical component of innate immunity in human. Finally, we propose a mechanism that explains how this enzyme could act at the molecular level to defend us against chitin-containing pathogens. [less ▲]

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See detailSPECIFICITY OF CLASS I TAGATOSE 1,6-BISPHOSPHATE ALDOLASE ENHANCED TOWARD TAGATOSE 1,6-BISPHOPHATE
Freichels, Régine ULg; Delmarcelle, Michaël ULg; Colarusso, Andrea et al

Poster (2015, July)

Class I tagatose 1,6-bisphosphate aldolase catalyzes the reversible condensation of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate to produce four D-ketohexoses 1,6-bisphosphate: D-tagatose 1,6 ... [more ▼]

Class I tagatose 1,6-bisphosphate aldolase catalyzes the reversible condensation of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate to produce four D-ketohexoses 1,6-bisphosphate: D-tagatose 1,6-bisphosphate, D-fructose 1,6-bisphosphate, D-psicose 1,6-bisphosphate and D-sorbose 1,6-bisphosphate. These four sugars are diastereoisomers and differs from each other in their stereochemistry at carbons 3 and 4. The structure determination of three class I tagatose 1,6-bisphosphate aldolases has afforded new insight into their catalytic mechanism as well as their evolution. However, the determinant(s) that allow(s) the enzyme to be so unspecific at carbon 4 have remains unknown. The aim of this project is focused on the characterization of the structural features of tagatose 1,6-bisphosphate aldolases that determine the specificity of the enzyme towards tagatose versus fructose 1,6-bisphosphate (carbon C4). [less ▲]

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See detailRésistance aux antibiotiques : le retour à l’ère prébiotique ?
Galleni, Moreno ULg; Kerff, Frédéric ULg; Rigali, Sébastien ULg

Conference (2015, February 05)

Aujourd’hui, la résistance des bactéries aux antibiotiques relève d'une préoccupation croissante dans le domaine de la santé publique et s'impose plus que jamais comme une priorité. Ce problème a un coût ... [more ▼]

Aujourd’hui, la résistance des bactéries aux antibiotiques relève d'une préoccupation croissante dans le domaine de la santé publique et s'impose plus que jamais comme une priorité. Ce problème a un coût, sociétal et économique en Europe: 25.000 décès annuels par septicémie et 1,5 milliard d’euros d’augmentation du coût des traitements. Bien que le besoin de nouveaux agents antibactériens en milieu clinique est urgent, seules deux nouvelles classes d’antibiotiques ont pu être proposées durant ces 30 dernières années. En effet, la découverte et le développement de nouveaux antibiotiques posent des défis scientifiques, cliniques et financiers importants. [less ▲]

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See detailGeneration of a soluble recombinant trimeric form of bovine CD40L and its potential use as a vaccine adjuvant in cows
Pujol, Julien ULg; Bouillenne, Fabrice ULg; Farnir, Frédéric ULg et al

in Veterinary immunology and immunopathology (2015), 168(1), 1-13

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See detailStructural and Kinetic Insights into the "Ceftazidimase" Behavior of the Extended-Spectrum beta-Lactamase CTX-M-96.
Ghiglione, Barbara; Rodriguez, Maria Margarita; Herman, Raphaël ULg et al

in Biochemistry (2015), 54(32), 5072-82

Diversification of the CTX-M beta-lactamases led to the emergence of variants responsible for decreased susceptibility to ceftazidime, like the Asp240Gly-harboring "ceftazidimases". We solved the ... [more ▼]

Diversification of the CTX-M beta-lactamases led to the emergence of variants responsible for decreased susceptibility to ceftazidime, like the Asp240Gly-harboring "ceftazidimases". We solved the crystallographic structure of the Asp240Gly variant CTX-M-96 at 1.2 A and evaluated the role of Asp240 in the activity toward oxyimino-cephalosporins through simulated models and kinetics. There seem to be subtle changes in the conformation of the active site cavity of CTX-M-96, compared to enzyme variants harboring the Asp240, and these small rearrangements could be due to localized shifts in the environment of the beta3 strand. According to the crystallographic evidence, CTX-M-96 presents a "compact" active site, which in spite of its reduced cavity seems to allow the proper interaction with oxyimino-cephalosporins, as suggested by simulated models. The term "ceftazidimases" that is currently applied for the Asp240Gly-harboring CTX-M variants should be used carefully. Structural differences between CTX-M harboring the Asp240Gly mutation (and also probably others like those at Pro167) do not seem to be conclusive to determine the "ceftazidimase" behavior observed in vivo, which is in turn partially supported by the mild improvement in the catalytic efficiency toward ceftazidime by CTX-M-96 and similar enzymes, compared to "parental" Asp240-harboring variants. In addition, it is observed that alterations in OmpF expression could act synergistically with CTX-M-96 for yielding clinical resistance toward ceftazidime. We therefore propose that the observed resistance in vivo is due to the sum of synergic mechanisms, and the term "cefotaximases associated with ceftazidime resistance" could be conveniently used to describe CTX-M harboring the Asp240Gly substitution. [less ▲]

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See detailKinetics of the interaction between avibactam and the CHE-1 class C beta-lactamase
Fernea, Adriana; Galleni, Moreno ULg; Frère, Jean-Marie ULg

in Journal of Antimicrobial Chemotherapy (2015), 70(3), 951--953

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See detailStructural and functional analysis of the HMA4 protein
Lekeux, Gilles ULg; Laurent, Clémentine ULg; Damblon, Christian ULg et al

Poster (2014, September 09)

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See detailSynthesis of N-fluoroalkyl-tryptophan and study of their biological activity as potential substrates for indoleamine 2,3-dioxygenase
Henrottin, Jean ULg; Zervosen, Astrid ULg; Lemaire, Christian ULg et al

Poster (2014, June)

Indoleamine 2,3-dioxygenase (rhIDO) is an enzyme mainly expressed in brain and tumor cells and catalyzing the oxidative cleavage of the indole ring of L-tryptophan through the kynurenine pathway ... [more ▼]

Indoleamine 2,3-dioxygenase (rhIDO) is an enzyme mainly expressed in brain and tumor cells and catalyzing the oxidative cleavage of the indole ring of L-tryptophan through the kynurenine pathway. Furthermore this enzyme could be responsible for the eventual suppression of immune responses by blocking locally T-lymphocyte proliferation. The syntheses of 1-(2-fluoroethyl)-tryptophan (1-[19F]FETrp) and 1-((1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl)methyl)-tryptophan, two N-fluoroalkylated tryptophan derivatives, are described here. In vitro enzymatic assays with these two new potential substrates of rhIDO show that 1-[19F]FETrp is a good and specific substrate of hIDO. [less ▲]

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See detailHow to build a biological linker dedicated to the engineering of novel drug delivery systems
Crasson, Oscar ULg; Galleni, Moreno ULg; Parente, Raffaella et al

Poster (2014, May 15)

Detailed reference viewed: 99 (20 ULg)