References of "Galleni, Moreno"
     in
Bookmark and Share    
Full Text
Peer Reviewed
See detailIncreased antimicrobial resistance in a novel CMY-54 AmpC-type enzyme with a GluLeu217-218 insertion in the Ω-loop
Pérez-Llarena, Francisco José; Vázquez-Ucha, Juan Carlos; Kerff, Frédéric ULg et al

in Microbial Drug Resistance : Mechanism, Epidemiology, & Disease (in press)

Detailed reference viewed: 57 (0 ULg)
Full Text
Peer Reviewed
See detailCrystal Structure and Kinetic Analysis of the Class B3 Di-Zinc Metallo-β-Lactamase LRA-12 from an Alaskan Soil Metagenome
Rodríguez, María Margarita; Herman, Raphaël ULg; Ghiglione, Barbara et al

in PLoS ONE (in press)

Detailed reference viewed: 23 (2 ULg)
Full Text
Peer Reviewed
See detailThe Lys-Asp-Tyr Triad within the Mite Allergen Der p 1 Propeptide Is a Critical Structural Element for the pH-Dependent Initiation of the Protease Maturation.
Chevigne, Andy; Campizi, Vincenzo; Szpakowska, Martyna et al

in International Journal of Molecular Sciences (2017), 18(5),

The major house dust mite allergen, Der p 1, is a papain-like cysteine protease expressed as an inactive precursor, proDer p 1, carrying an N-terminal propeptide with a unique structure. The maturation of ... [more ▼]

The major house dust mite allergen, Der p 1, is a papain-like cysteine protease expressed as an inactive precursor, proDer p 1, carrying an N-terminal propeptide with a unique structure. The maturation of the zymogen into an enzymatically-active form of Der p 1 is a multistep autocatalytic process initiated under acidic conditions through conformational changes of the propeptide, leading to the loss of its inhibitory ability and its subsequent gradual cleavage. The aims of this study were to characterize the residues present in the Der p 1 propeptide involved in the initiation of the zymogen maturation process, but also to assess the impact of acidic pH on the propeptide structure, the activity of Der p 1 and the fate of the propeptide. Using various complementary enzymatic and structural approaches, we demonstrated that a structural triad K17p-D51p-Y19p within the N-terminal domain of the propeptide is essential for its stabilization and the sensing of pH changes. Particularly, the protonation of D51p under acidic conditions unfolds the propeptide through disruption of the K17p-D51p salt bridge, reduces its inhibition capacity and unmasks the buried residues K17p and Y19p constituting the first maturation cleavage site of the zymogen. Our results also evidenced that this triad acts in a cooperative manner with other propeptide pH-responsive elements, including residues E56p and E80p, to promote the propeptide unfolding and/or to facilitate its proteolysis. Furthermore, we showed that acidic conditions modify Der p 1 proteolytic specificity and confirmed that the formation of the first intermediate represents the limiting step of the in vitro Der p 1 maturation process. Altogether, our results provide new insights into the early events of the mechanism of proDer p 1 maturation and identify a unique structural triad acting as a stabilizing and a pH-sensing regulatory element. [less ▲]

Detailed reference viewed: 19 (3 ULg)
Full Text
See detailExploring potentialities of RanbP2-type Zinc Fingers in RNA-binding proteins design
De Franco, Simona ULg; Vandenameele, Julie ULg; Galleni, Moreno ULg et al

Poster (2016, June 29)

Transcriptomes consist of several classes of RNAs, whose roles are central to innumerable biological processes as well as diseases. These observations justify the increasing interest in the engineering of ... [more ▼]

Transcriptomes consist of several classes of RNAs, whose roles are central to innumerable biological processes as well as diseases. These observations justify the increasing interest in the engineering of functionalized RNA-binding proteins (RBPs) to specifically manipulate RNA function. In this context, the RanBP2-type Zinc Finger (ZF) domain emerged as a suitable scaffold for single-stranded RBPs design. The present study aimed to identify the sequence-specificity of several naturally occurring RanBP2-type ZFs by in vitro selection and use the natural variation in their substrate to create arrays of ZF domains displaying customized specificity. A parallel kinetic and thermodynamic characterization of ssRNA-ZFs interaction was performed by Isothermal Titration Calorimetry (ITC) and BioLayer Interferometry (BLI), respectively. Although our data showed that only little variations in the recognized sequence could be observed for the studied ZFs, they contribute to elucidate the molecular basis for the RanBP2-type ZF-ssRNA interaction. [less ▲]

Detailed reference viewed: 35 (9 ULg)
Full Text
See detailThe use of protein-polysaccharide complexes to enhance protein delivery in CaCO3 microparticles
Ramalapa, Bathabile ULg; Crasson, Oscar ULg; Vandevenne, Marylène ULg et al

Conference (2016, March 22)

The role of protein therapeutics in modern medicine has increased considerably due to their potential to perform complex functions in the body that cannot be mimicked by simple chemical compounds. To ... [more ▼]

The role of protein therapeutics in modern medicine has increased considerably due to their potential to perform complex functions in the body that cannot be mimicked by simple chemical compounds. To increase the safety, stability and efficacy of protein therapeutics; there exists a need for delivery systems that are biocompatible, prevent premature degradation of bioactives and allow targeted delivery and controlled release. Calcium carbonate (CaCO3) has gained great favour for employment in protein delivery due to possibilities of controlling size, morphology and crystalline forms of particles by tuning the synthesis conditions. The aim of the present work was to assess the significance of polysaccharide-protein complexes in enhancing the encapsulation of proteins in CaCO3 microspheres. A Chitin Binding Domain (ChBD), reported to have affinity for hyaluronic acid, was inserted on β-lactamase enzyme to develop a chimeric protein. The chimeric protein retained the activity of the enzyme and the binding properties and was encapsulated in CaCO3 microspheres by a super critical CO2 (ScCO2) process using hyaluronic acid as a templating agent. The particles were characterised in terms of size, zeta potential, morphology and protein loading. The results obtained confirmed the affinity of the ChBD to hyaluronic acid towards the production of stable, vaterite microparticles. Protein assays demonstrated that the ChBD enhanced the encapsulation of protein by up to 10 fold. Confocal images also suggested high encapsulation of the chimeric protein compared to native protein. Thus the production of polysaccharide-protein complexes seems effective in enhancing the encapsulation of proteins in CaCO3 microparticles using the ScCO2 process. [less ▲]

Detailed reference viewed: 71 (7 ULg)
Full Text
Peer Reviewed
See detailKinetics of the Interaction between BAL29880 and LK157 and the Class C β-Lactamase CHE-1
Fernea, Adriana; Galleni, Moreno ULg; Frère, Jean-Marie ULg

in Antimicrobial Agents and Chemotherapy (2016), 60

Detailed reference viewed: 19 (4 ULg)
Full Text
Peer Reviewed
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 ▲]

Detailed reference viewed: 135 (29 ULg)
Full Text
Peer Reviewed
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 ▲]

Detailed reference viewed: 37 (10 ULg)
Full Text
Peer Reviewed
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 ▲]

Detailed reference viewed: 28 (13 ULg)
Full Text
See detailDesign of new delivery systems for therapeutic proteins based on calcium carbonate microspheres
Ramalapa, Bathabile ULg; Crasson, Oscar ULg; Vandevenne, Marylène ULg et al

Poster (2015, December 10)

There exists a constant need for delivery systems that are biocompatible, offer bioactives protection from premature degradation and allow for targeted delivery and controlled release. Calcium carbonate ... [more ▼]

There exists a constant need for delivery systems that are biocompatible, offer bioactives protection from premature degradation and allow for targeted delivery and controlled release. Calcium carbonate (CaCO3) is one such system that has gained great favour for employment in the biomedical field due to possibilities of controlling size, morphology and crystalline forms of particles by tuning the synthesis conditions. CaCO3 has demonstrated ability to increase safety, stability and overall efficiency of protein therapeutics. The aim of the present work was to assess the significance of polysaccharide-protein complexes in enhancing the encapsulation of proteins in CaCO3 microspheres. A Chitin Binding Domain (ChBD), reported to have affinity for hyaluronic acid, was inserted on β-lactamase enzyme to develop a chimeric protein. The chimeric protein retained the activity of the enzyme and the binding properties and was encapsulated in CaCO3 microspheres by a super critical CO2 (ScCO2) process using hyaluronic acid as a templating agent. The particles were characterised in terms of size, zeta potential, morphology and protein loading. The results obtained confirmed the affinity of the ChBD to hyaluronic acid towards the production of stable, vaterite microparticles. Protein assays demonstrated that the ChBD enhanced the encapsulation of protein by up to 10 fold. Confocal images also suggested high encapsulation of the chimeric protein compared to native protein. Thus the production of polysaccharide-protein complexes seems effective in enhancing the encapsulation of proteins in CaCO3 microparticles using the ScCO2 process. Moreover this method will further be used to enhance encapsulation of therapeutic proteins such as growth factors for bone and cartilage regeneration. [less ▲]

Detailed reference viewed: 59 (5 ULg)
Full Text
Peer Reviewed
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 ▲]

Detailed reference viewed: 60 (19 ULg)
Full Text
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 ULg 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 ▲]

Detailed reference viewed: 118 (31 ULg)
Full Text
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 ▲]

Detailed reference viewed: 82 (8 ULg)
Full Text
Peer Reviewed
See detailDevelopment of recombinant stable house dust mite allergen Der p 3 molecules for component resolved diagnosis and specific immunotherapy
Bouaziz, Ahlem ULg; Galleni, Moreno ULg; Louis, Renaud ULg

in Clinical and Experimental Allergy (2015), 45

Abstract BACKGROUND: The allergen Der p 3 is underrepresented in house dust mite (HDM) extracts probably due to autolysis. Recombinant stable molecule of the allergen is thus needed to improve the ... [more ▼]

Abstract BACKGROUND: The allergen Der p 3 is underrepresented in house dust mite (HDM) extracts probably due to autolysis. Recombinant stable molecule of the allergen is thus needed to improve the diagnosis of allergy and the safety and efficacy of immunotherapy. OBJECTIVE: The current study reports the immunological characterization of two recombinant molecules of the HDM allergen Der p 3 as useful tools for diagnosis and immunotherapy. METHODS: Recombinant mature (rDer p 3) and immature (proDer p 3) Der p 3 and their corresponding S196A mutants were produced in Pichia pastoris and purified. The stability, IgE-binding capacity and allergenicity of the different proteins were analysed and compared with those of the major mite allergen Der p 1 used as a reference. Additionally, the immunogenicity of the different allergens was evaluated in a murine model of Der p 3 sensitization. RESULTS: Compared to the IgE reactivity to recombinant and natural Der p 3 (nDer p 3), the mean IgE binding of patient's sera to rDer p 3-S196A (50%) was higher. The poorly binding to nDer p 3 or rDer p 3 was due to autolysis of the allergen. Contrary to Der p 3, proDer p 3 displayed very weak IgE reactivity, as measured by sandwich ELISA and competitive inhibition, rat basophil leukaemia degranulation and human basophil activation assays. Moreover, proDer p 3 induced a TH 1-biased immune response that prevented allergic response in mice but retained Der p 3-specific T-cell reactivity. CONCLUSION: rDer p 3-S196A should be used for the diagnosis of HDM allergy elicited by Der p 3, and proDer p 3 may represent a hypoallergen of Der p 3. [less ▲]

Detailed reference viewed: 15 (6 ULg)
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 ▲]

Detailed reference viewed: 162 (17 ULg)
Full Text
Peer Reviewed
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

Detailed reference viewed: 21 (7 ULg)