References of "Bouillenne, Fabrice"
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See detailInsulin aggregation assessment by capillary gel electrophoresis without sodium dodecyl sulfate: Comparison with size-exclusion chromatography
Demelenne, Alice ULiege; NAPP, Aurore ULiege; Bouillenne, Fabrice ULiege et al

in Talanta (2019), 199

Size-exclusion chromatography (SEC) is a method of choice for the analysis of protein aggregates in pharmaceuticals. The United States and European Pharmacopoeias currently use a SEC method with an acidic ... [more ▼]

Size-exclusion chromatography (SEC) is a method of choice for the analysis of protein aggregates in pharmaceuticals. The United States and European Pharmacopoeias currently use a SEC method with an acidic pH mobile phase to assess the content of aggregates in insulin formulations. In this article, we analyzed aggregated human insulin samples and demonstrated that both methods under neutral conditions, namely neutral pH SEC (nSEC) and capillary gel electrophoresis (CGE), yield to similar aggregate content contrary to SEC under acidic conditions (aSEC). aSEC showed polymeric complexes that were not observed in nSEC and CGE. During method development, the effect on SEC profiles of arginine and acetonitrile were highlighted. In CGE, the effect of SDS on disruption of non-covalent insulin aggregates was confirmed and the benefit of sodium deoxycholate addition in sieving gel was discussed. The three methods were applied to the analysis of an insulin formulation and similar results to those obtained for human insulin as raw material were observed. Finally, the CGE method was used to study the stability of human insulin under different storage conditions. In view of the obtained results one may question the relevance of the current pharmacopoeia method to study insulin aggregates by emphasizing the importance of the mobile phase composition and pH in SEC. The new CGE method developed is an easy method for studying non-covalent aggregates of insulin, which could be applied to other proteins. © 2019 Elsevier B.V. [less ▲]

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See detailThe atypical subunit composition of respiratory complexes I and IV is associated with original extra structural domains in Euglena gracilis.
Miranda-Astudillo, H. V.; Yadav, K. N. S.; Colina-Tenorio, L. et al

in Scientific Reports (2018), 8(1), 9698

In mitochondrial oxidative phosphorylation, electron transfer from NADH or succinate to oxygen by a series of large protein complexes in the inner mitochondrial membrane (complexes I-IV) is coupled to the ... [more ▼]

In mitochondrial oxidative phosphorylation, electron transfer from NADH or succinate to oxygen by a series of large protein complexes in the inner mitochondrial membrane (complexes I-IV) is coupled to the generation of an electrochemical proton gradient, the energy of which is utilized by complex V to generate ATP. In Euglena gracilis, a non-parasitic secondary green alga related to trypanosomes, these respiratory complexes totalize more than 40 Euglenozoa-specific subunits along with about 50 classical subunits described in other eukaryotes. In the present study the Euglena proton-pumping complexes I, III, and IV were purified from isolated mitochondria by a two-steps liquid chromatography approach. Their atypical subunit composition was further resolved and confirmed using a three-steps PAGE analysis coupled to mass spectrometry identification of peptides. The purified complexes were also observed by electron microscopy followed by single-particle analysis. Even if the overall structures of the three oxidases are similar to the structure of canonical enzymes (e.g. from mammals), additional atypical domains were observed in complexes I and IV: an extra domain located at the tip of the peripheral arm of complex I and a "helmet-like" domain on the top of the cytochrome c binding region in complex IV. [less ▲]

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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 ULiege; Ghiglione, Barbara et al

in PLoS ONE (2017), 12(7), 0182043

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See detailAtypical composition and structure of the mitochondrial dimeric ATP synthase from Euglena gracilis
Yadav, K.N. Sathish; Miranda Astudillo, Héctor Vicente ULiege; Colina-Tenorio, Lili et al

in Biochimica et Biophysica Acta. Bioenergetics (2017), 1858(4), 267-275

Mitochondrial respiratory-chain complexes from Euglenozoa comprise classical subunits described in other eukaryotes (i.e. mammals and fungi) and subunits that are restricted to Euglenozoa (e.g. Euglena ... [more ▼]

Mitochondrial respiratory-chain complexes from Euglenozoa comprise classical subunits described in other eukaryotes (i.e. mammals and fungi) and subunits that are restricted to Euglenozoa (e.g. Euglena gracilis and Trypanosoma brucei). Here we studied the mitochondrial F1FO-ATP synthase (or Complex V) from the photosynthetic eukaryote E. gracilis in detail. The enzyme was purified by a two-step chromatographic procedure and its subunit composition was resolved by a three-dimensional gel electrophoresis (BN/SDS/SDS). Twenty-two different subunits were identified by mass-spectrometry analyses among which the canonical alpha, beta, gamma, delta, epsilon and OSCP subunits, and at least seven subunits previously found in Trypanosoma. The ADP/ATP carrier was also associated to the ATP synthase into a dimeric ATP synthasome. Single-particle analysis by transmission electron microscopy of the dimeric ATP synthase indicated that the structures of both the catalytic and central rotor parts are conserved while other structural features are original. These new features include a large membrane-spanning region joining the monomers, an external peripheral stalk and a structure that goes through the membrane and reaches the inter membrane space below the c-ring, the latter having not been reported for any mitochondrial F-ATPase. [less ▲]

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See detailProtein Factory: A Protein Production and Purification Facility
Bouillenne, Fabrice ULiege; Matton, Anne-Marie ULiege; Thamm, Iris ULiege et al

Poster (2016, November 16)

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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 ULiege; Bouillenne, Fabrice ULiege; Farnir, Frédéric ULiege 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 ULiege 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 detailDERP6 (ELP5) and C3ORF75 (ELP6) regulate tumorigenicity and migration of melanoma cells as subunits of Elongator
Close, Pierre ULiege; Gillard, Magali; Ladang, Aurélie ULiege et al

in Journal of Biological Chemistry (2012)

The Elongator complex is composed of 6 subunits (Elp1-Elp6) and promotes RNAPII transcript elongation through histone acetylation in the nucleus as well as tRNA modification in the cytoplasm. This ... [more ▼]

The Elongator complex is composed of 6 subunits (Elp1-Elp6) and promotes RNAPII transcript elongation through histone acetylation in the nucleus as well as tRNA modification in the cytoplasm. This acetyltransferase complex directly or indirectly regulates numerous biological processes ranging from exocytosis and resistance to heat shock in yeast to cell migration and neuronal differentiation in higher eukaryotes. The identity of human ELP1 through ELP4 has been reported but human ELP5 and ELP6 have remained uncharacterized. Here, we report that DERP6 (ELP5) and C3ORF75 (ELP6) encode these subunits of human Elongator. We further investigated the importance and function of these two subunits by a combination of biochemical analysis and cellular assays. Our results show that DERP6/ELP5 is required for the integrity of Elongator and directly connects ELP3 to ELP4. Importantly, the migration and tumorigenicity of melanomaderived cells are significantly decreased upon Elongator depletion through ELP1 or ELP3. Strikingly, DERP6/ELP5 and C3ORF75/ELP6-depleted melanoma cells have similar defects, further supporting the idea that DERP6/ELP5 and C3ORF75/ELP6 are essential for Elongator function. Together, our data identify DERP6/ELP5 and C3ORF75/ELP6 as key players for migration, invasion and tumorigenicity of melanoma cells, as integral subunits of Elongator. [less ▲]

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See detailCritical role of tryptophan 154 for the activity and stability of class D beta-lactamases.
Baurin, Stephane; Vercheval, Lionel ULiege; Bouillenne, Fabrice ULiege et al

in Biochemistry (2009), 48(47), 11252-63

The catalytic efficiency of the class D beta-lactamase OXA-10 depends critically on an unusual carboxylated lysine as the general base residue for both the enzyme acylation and deacylation steps of ... [more ▼]

The catalytic efficiency of the class D beta-lactamase OXA-10 depends critically on an unusual carboxylated lysine as the general base residue for both the enzyme acylation and deacylation steps of catalysis. Evidence is presented that the interaction between the indole group of Trp154 and the carboxylated lysine is essential for the stability of the posttranslationally modified Lys70. Substitution of Trp154 by Gly, Ala, or Phe yielded noncarboxylated enzymes which displayed poor catalytic efficiencies and reduced stability when compared to the wild-type OXA-10. The W154H mutant was partially carboxylated. In addition, the maximum values of k(cat) and k(cat)/K(M) were shifted toward pH 7, indicating that the carboxylation state of Lys70 is dependent on the protonation level of the histidine. A comparison of the three-dimensional structures of the different proteins also indicated that the Trp154 mutations did not modify the overall structures of OXA-10 but induced an increased flexibility of the Omega-loop in the active site. Finally, the deacylation-impaired W154A mutant was used to determine the structure of the acyl-enzyme complex with benzylpenicillin. These results indicate a role of the Lys70 carboxylation during the deacylation step and emphasize the importance of Trp154 for the ideal positioning of active site residues leading to an optimum activity. [less ▲]

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See detailCharacterization of the proteins encoded by the Bacillus subtilis yoxA-dacC operon.
Duez, Colette ULiege; Zervosen, Astrid ULiege; Teller, Nathalie et al

in FEMS Microbiology Letters (2009), 300

Abstract In Bacillus subtilis, the yoxA and dacC genes were proposed to form an operon. The yoxA gene was overexpressed in Escherichia coli and its product fused to a polyhistidine tag was purified. An ... [more ▼]

Abstract In Bacillus subtilis, the yoxA and dacC genes were proposed to form an operon. The yoxA gene was overexpressed in Escherichia coli and its product fused to a polyhistidine tag was purified. An aldose-1-epimerase or mutarotase activity was measured with the YoxA protein that we propose to rename as GalM by analogy with its counterpart in E. coli. The peptide d-Glu-delta-m-A(2)pm-d-Ala-m-A(2)pm-d-Ala mimicking the B. subtilis and E. coli interpeptide bridge was synthesized and incubated with the purified dacC product, the PBP4a. A clear dd-endopeptidase activity was obtained with this penicillin-binding protein, or PBP. The possible role of this class of PBP, present in almost all bacteria, is discussed. [less ▲]

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See detailEngineering a camelid antibody fragment that binds to the active site of human lysozyme and inhibits its conversion into amyloid fibrils
Chan, Pak Ho; Pardon, Els; Menzer, Linda ULiege et al

in Biochemistry (2008), 47

single-domain fragment, cAb-HuL22, of a camelid heavy-chain antibody specific for the active site of human lysozyme has been generated, and its effects on the properties of the I56T and D67H amyloidogenic ... [more ▼]

single-domain fragment, cAb-HuL22, of a camelid heavy-chain antibody specific for the active site of human lysozyme has been generated, and its effects on the properties of the I56T and D67H amyloidogenic variants of human lysozyme, which are associated with a form of systemic amyloidosis, have been investigated by a wide range of biophysical techniques. Pulse-labeling hydrogen-deuterium exchange experiments monitored by mass spectrometry reveal that binding of the antibody fragment strongly inhibits the locally cooperative unfolding of the I56T and D67H variants and restores their global cooperativity to that characteristic of the wild-type protein. The antibody fragment was, however, not stable enough under the conditions used to explore its ability to perturb the aggregation behavior of the lysozyme amyloidogenic variants. We therefore engineered a more stable version of cAb-HuL22 by adding a disulfide bridge between the two beta-sheets in the hydrophobic core of the protein. The binding of this engineered antibody fragment to the amyloidogenic variants of lysozyme inhibited their aggregation into fibrils. These findings support the premise that the reduction in global cooperativity caused by the pathogenic mutations in the lysozyme gene is the determining feature underlying their amyloidogenicity. These observations indicate further that molecular targeting of enzyme active sites, and of protein binding sites in general, is an effective strategy for inhibiting or preventing the aberrant self-assembly process that is often a consequence of protein mutation and the origin of pathogenicity. Moreover, this work further demonstrates the unique properties of camelid single-domain antibody fragments as structural probes for studying the mechanism of aggregation and as potential inhibitors of fibril formation. [less ▲]

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

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

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

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

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See detailThe ponA gene of Enterococcus faecalis JH2-2 codes for a low-affinity class a penicillin-binding protein
Duez, Colette ULiege; Hallut, Séverine; Rhazi, Noureddine ULiege et al

in Journal of Bacteriology (2004), 186(13), 4412-4416

soluble derivative of the Enterococcus faecalis JH2-2 class A PBP1 (*PBP1) was overproduced and purified. It exhibited a glycosyltransferase activity on the Escherichia coli (14)C(-)labeled lipid 11 ... [more ▼]

soluble derivative of the Enterococcus faecalis JH2-2 class A PBP1 (*PBP1) was overproduced and purified. It exhibited a glycosyltransferase activity on the Escherichia coli (14)C(-)labeled lipid 11 precursor. As a DD-peptidase, it could hydrolyze thiolester substrates with efficiencies similar to those of other class A penicillin-binding proteins (PBPs) and bind beta-lactams, but with k(2)/K (a parameter accounting for the acylation step efficiency) values characteristic of penicillin-resistant PBPs. [less ▲]

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See detailSecondary-structure characterization by far-UV CD of highly purified uncoupling protein 1 expressed in yeast
Douette, Pierre ULiege; Navet, Rachel ULiege; Bouillenne, Fabrice ULiege 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 detailAdenylation-dependent conformation and unfolding pathways of the NAD(+)-dependent DNA ligase from the thermophile Thermus scotoductus
Georlette, D.; Blaise, Vinciane ULiege; Bouillenne, Fabrice ULiege et al

in Biophysical Journal (2004), 86(2), 1089-1104

In the last few years, an increased attention has been focused on NAD(+)-dependent DNA ligases. This is mostly due to their potential use as antibiotic targets, because effective inhibition of these ... [more ▼]

In the last few years, an increased attention has been focused on NAD(+)-dependent DNA ligases. This is mostly due to their potential use as antibiotic targets, because effective inhibition of these essential enzymes would result in the death of the bacterium. However, development of an efficient drug requires that the conformational modifications involved in the catalysis of NAD(+)-dependent DNA ligases are understood. From this perspective, we have investigated the conformational changes occurring in the thermophilic Thermus scotoductus NAD(+)-DNA ligase upon adenylation, as well as the effect of cofactor binding on protein resistance to thermal and chemical (guanidine hydrochloride) denaturation. Our results indicate that cofactor binding induces conformational rearrangement within the active site and promotes a compaction of the enzyme. These data support an induced "open-closure" process upon adenylation, leading to the formation of the catalytically active enzyme that is able to bind DNA. These conformational changes are likely to be associated with the protein function, preventing the formation of nonproductive complexes between deadenylated ligases and DNA. In addition, enzyme adenylation significantly increases resistance of the protein to thermal denaturation and GdmCl-induced unfolding, establishing a thermodynamic link between ligand binding and increased conformational stability. Finally, chemical unfolding of deadenylated and adenylated enzyme is accompanied by accumulation of at least two equilibrium intermediates, the molten globule and premolten globule states. Maximal populations of these intermediates are shifted toward higher GdmCl concentrations in the case of the adenylated ligase. These data provide further insights into the properties of partially folded intermediates. [less ▲]

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See detailCofactor binding modulates the conformational stabilities and unfolding patterns of NAD(+)-dependent DNA ligases from Escherichia coli and Thermus scotoductus
Georlette, D.; Blaise, Vinciane ULiege; Dohmen, C. et al

in Journal of Biological Chemistry (2003), 278(50), 49945-49953

DNA ligases are important enzymes required for cellular processes such as DNA replication, recombination, and repair. NAD(+)-dependent DNA ligases are essentially restricted to eubacteria, thus ... [more ▼]

DNA ligases are important enzymes required for cellular processes such as DNA replication, recombination, and repair. NAD(+)-dependent DNA ligases are essentially restricted to eubacteria, thus constituting an attractive target in the development of novel antibiotics. Although such a project might involve the systematic testing of a vast number of chemical compounds, it can essentially gain from the preliminary deciphering of the conformational stability and structural perturbations associated with the formation of the catalytically active adenylated enzyme. We have, therefore, investigated the adenylation-induced conformational changes in the mesophilic Escherichia coli and thermophilic Thermus scotoductus NAD(+)-DNA ligases, and the resistance of these enzymes to thermal and chemical (guanidine hydrochloride) denaturation. Our results clearly demonstrate that anchoring of the cofactor induces a conformational rearrangement within the active site of both mesophilic and thermophilic enzymes accompanied by their partial compaction. Furthermore, the adenylation of enzymes increases their resistance to thermal and chemical denaturation, establishing a thermodynamic link between cofactor binding and conformational stability enhancement. Finally, guanidine hydrochloride-induced unfolding of NAD(+)-dependent DNA ligases is shown to be a complex process that involves accumulation of at least two equilibrium intermediates, the molten globule and its precursor. [less ▲]

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