References of "Dumoulin, Mireille"
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See detailComparative study of mature and zymogen mite cysteine protease stability and pH unfolding.
Chevigne, A.; Dumez, Marie-Eve ULg; Dumoulin, Mireille ULg et al

in Biochimica et Biophysica Acta (2010), 1800(9), 937-945

BACKGROUND: Papain-like proteases (CA1) are synthesized as inactive precursors carrying an N-terminal propeptide, which is further removed under acidic conditions to generate active enzymes. METHODS: To ... [more ▼]

BACKGROUND: Papain-like proteases (CA1) are synthesized as inactive precursors carrying an N-terminal propeptide, which is further removed under acidic conditions to generate active enzymes. METHODS: To have a better insight into the mechanism of activation of this protease family, we compared the pH unfolding of the zymogen and the mature form of the mite cysteine protease Der p 1. RESULTS: We showed that the presence of the propeptide does not significantly influence the pH-induced unfolding of the catalytic domain but does affect its fluorescence properties by modifying the exposure of the tryptophan 192 to the solvent. In addition, we demonstrated that the propeptide displays weaker pH stability than the protease domain confirming that the unfolding of the propeptide is the key event in the activation process of the zymogen. GENERAL SIGNIFICANCE: Finally, we show, using thermal denaturation and enzymatic activity measurements, that whatever the pH value, the propeptide does not stabilize the structure of the catalytic domain but very interestingly, prevents its autolysis. [less ▲]

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See detailUnderstanding the Mechanism of Amyloid Fibrils Formation by Human Lysozyme
Dumoulin, Mireille ULg

Conference (2009, July 04)

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See detailVHHs as model proteins to investigate amyloid fibril formation
Chavignon, Chloé ULg; Pardon, Els; Wyns, Lode et al

Poster (2009, July)

The term "amyloidosis" covers up a group of diseases associated with deposition in different organs of protein aggregates organized into amyloid fibrils. About twenty-five amyloidosis are known so far ... [more ▼]

The term "amyloidosis" covers up a group of diseases associated with deposition in different organs of protein aggregates organized into amyloid fibrils. About twenty-five amyloidosis are known so far, amongst which Alzheimer's disease, type II diabetes and immunoglobulin amyloidosis [1]. Although the mechanism of amyloid fibrils formation at the molecular level is not yet completely understood, it has been shown that the capacity to form amyloid fibrils in vitro is an intrinsic property of all polypeptide chains [1]. The choice of model proteins to investigate the aggregation process in vitro is therefore no more restrained to proteins involved in amyloidosis but can be settled on a wide variety of proteins. In this study, we have chosen two variable domains of camelid heavy-chain antibodies (referred to as VHHs or nanobodies), cAb-HuL6 and cAb-BcII10, and this choice was motivated by the following reasons: - First, they are small monomeric domains (~14 kDa) presenting high stability and high solubility [2], which permits their expression with a high yield (20-40 mg.L-1). - Second, a wide range of stable mutants of these two VHHs is available. Mutations located at the disulfide bond [3,4], the CDRs [3] and the framework have been introduced. Characterisation of the aggregating properties of these mutants will allow the investigation of the impact of these structural elements on the process of fibril formation. In order to determine conditions in which cAb-HuL6 and cAb-BcII10 are more susceptible to form intermediates and thus amyloid fibrils, heat induced infolding experiments at pHs comprised in a range from 2,5 to 9,5 have been monitored by intrinsic fluorescence, ANS binding and circular dichroism. Then, aggregation experiments have been performed in the selected conditions and the presence of amyloid fibrils has been acknowledged by thioflavineT fluorescence experiments and electronic microscopy. [1] Chiti, F. and Dobson, C. M., Protein misfolding, functional amyloid, and human disease, Annu. Rev. Biochem., 75, 2006, 333-366. [2] Dumoulin, M., Conrath, K., Van Meirhaeghe, A., Meersman, F., Heremans, K., Frenken, L. G., Muyldermans, S., Wyns, L. & Matagne, A., Single-domain antibody fragments with high conformational stability, Protein Sci., 11, 2002, 500-515. [3] Saerens, D., Pellis, M., Loris, R., Pardon, E., Dumoulin, M., Matagne, A., Wyns, L., Muyldermans, S., Conrath, K., Identification of a universal VHH framework to graft non-canonical antigen-binding loops of camel single-domain antibodies, J. Mol. Biol., 352, 2005, 597-607. [4] Saerens D., Conrath K., Govaert J., Muyldermans S., Disulfide bond introduction for general stabilization of immunoglobulin heavy-chain variable domains, J Mol Biol., 377, 2008, 478-488. [less ▲]

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See detailThe Zinc Center Influences the Redox and Thermodynamic Properties of Escherichia coli Thioredoxin 2
El Hajjaji, Hayat; Dumoulin, Mireille ULg; Matagne, André ULg et al

in Journal of Molecular Biology (2009), 386(1), 60-71

Thioredoxins are small, ubiquitous redox enzymes that reduce protein disulfide bonds by using a pair of cysteine residues present in a strictly conserved WCGPC catalytic motif. The Escherichia coli ... [more ▼]

Thioredoxins are small, ubiquitous redox enzymes that reduce protein disulfide bonds by using a pair of cysteine residues present in a strictly conserved WCGPC catalytic motif. The Escherichia coli cytoplasm contains two thioredoxins, Trx1 and Trx2. Trx2 is special because it is induced under oxidative stress conditions and it has an additional N-terminal zinc-binding domain. We have determined the redox potential of Trx2, the pKa of the active site nucleophilic cysteine, as well as the stability of the oxidized and reduced form of the protein. Trx2 is more oxidizing than Trx1 (–221 mV versus –284 mV, respectively), which is in good agreement with the decreased value of the pKa of the nucleophilic cysteine (5.1 versus 7.1, respectively). The difference in stability between the oxidized and reduced forms of an oxidoreductase is the driving force to reduce substrate proteins. This difference is smaller for Trx2 (ΔΔG°H2O = 9 kJ/mol and ΔTm = 7. 4 °C) than for Trx1 (ΔΔG°H2O = 15 kJ/mol and ΔTm = 13 °C). Altogether, our data indicate that Trx2 is a significantly less reducing enzyme than Trx1, which suggests that Trx2 has a distinctive function. We disrupted the zinc center by mutating the four Zn2+-binding cysteines to serine. This mutant has a more reducing redox potential (–254 mV) and the pKa of its nucleophilic cysteine shifts from 5.1 to 7.1. The removal of Zn2+ also decreases the overall stability of the reduced and oxidized forms by 3.2 kJ/mol and 5.8 kJ/mol, respectively. In conclusion, our data show that the Zn2+-center of Trx2 fine-tunes the properties of this unique thioredoxin. [less ▲]

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See detailCharacterization of Oligomeric Species on the Aggregation Pathway of Human Lysozyme
Frare, Erica; Mossuto, Maria F.; Polverino de Laureto, Patrizia et al

in Journal of Molecular Biology (2009)

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See detail(1)H, (13)C and (15)N assignments of a camelid nanobody directed against human alpha-synuclein.
Vuchelen, Anneleen; O'Day, Elizabeth; De Genst, Erwin et al

in Biomolecular NMR Assignments (2009), 3(2), 231-3

Nanobodies are single chain antibodies that are uniquely produced in Camelidae, e.g. camels and llamas. They have the desirable features of small sizes (Mw < 14 kDa) and high affinities against antigens ... [more ▼]

Nanobodies are single chain antibodies that are uniquely produced in Camelidae, e.g. camels and llamas. They have the desirable features of small sizes (Mw < 14 kDa) and high affinities against antigens (Kd approximately nM), making them ideal as structural probes for biomedically relevant motifs both in vitro and in vivo. We have previously shown that nanobody binding to amyloidogenic human lysozyme variants can effectively inhibit their aggregation, the process that is at the origin of systemic amyloid disease. Here we report the NMR assignments of a new nanobody, termed NbSyn2, which recognises the C-terminus of the intrinsically disordered protein, human alpha-synuclein (aS), whose aberrant self-association is implicated in Parkinson's disease. [less ▲]

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See detailProduction of four amyloidogenic variants of human lysozyme as inclusion bodies in Escherichia coli
Dumont, Janice ULg; Menzer, Linda ULg; Scarafone, Natacha ULg et al

Poster (2009)

Six variants of human lysozyme (I56T, F57I, W64R, D67H, F57I/T70N and W112R/T70N) are associated with a hereditary non-neuropathic systemic amyloidosis. This disease involved an extra cellular deposition ... [more ▼]

Six variants of human lysozyme (I56T, F57I, W64R, D67H, F57I/T70N and W112R/T70N) are associated with a hereditary non-neuropathic systemic amyloidosis. This disease involved an extra cellular deposition of amyloid fibrils made of lysozyme variants in a wide range of organs such as liver, spleen and kidneys [1]. The characterisation at the molecular level of two variants, I56T and D67H, has shown that these mutations reduce the stability and more particularly the global cooperativity of the protein. Consequently, under physiologically relevant conditions, these variants can transiently populate a partially unfolded state in which the beta-domain and the C-helix are cooperatively unfolded while the rest of the protein remains native like [1]. The formation of intermolecular interactions between the regions that are unfolded in this intermediate state is likely to be a fundamental trigger of the aggregation process that ultimately leads to the formation and deposition of fibrils in tissues. In order to study the effects of the other amyloidogenic mutations on the properties of lysozyme and thus to get more insight in the mechanism of amyloid formation, it is necessary to produce them in large quantities. The D67H, I56T and F57I variants are currently produced in Aspergillus niger; the expression in this organism is, however, time consuming and the yield is very low. The attempts to use alternative systems such as Pichia pastoris [2], Saccharomyces cerevisiae, and Arabidopsis thaliana have not been conclusive so far. In this work, we have produced the four single-point lysozyme variants as inclusion bodies in Escherichia coli and explored the possibility to refold them. [1] Dumoulin & al., (2006) Acc. Chem. Res., 39, 603 - 610 [2] Kumita & al., (2006) FEBS J., 273, 711-720 [less ▲]

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See detailCharacterization of Oligomeric Species on the Aggregation Pathway of Human Lysozyme
Frare, Erica; Mossuto, Maria F.; Polverino de Laureto, Patrizia et al

in Journal of Molecular Biology (2009), 387

has been analyzed by characterizing a series of distinct species formed on the aggregation pathway, specifically the amyloidogenic monomeric precursor protein, the oligomeric soluble prefibrillar ... [more ▼]

has been analyzed by characterizing a series of distinct species formed on the aggregation pathway, specifically the amyloidogenic monomeric precursor protein, the oligomeric soluble prefibrillar aggregates, and the mature fibrils. Particular attention has been focused on the analysis of the structural properties of the oligomeric species, since recent studies have shown that the oligomers formed by lysozyme prior to the appearance of mature amyloid fibrils are toxic to cells. Here, soluble oligomers of human lysozyme have been analyzed by a range of techniques including binding to fluorescent probes such as thioflavin T and 1-anilino-naphthalene-8-sulfonate, Fourier transforminfrared spectroscopy, and controlled proteolysis. Oligomers were isolated after 5 days of incubation of the protein and appear as spherical particles with a diameter of 8–17 nm when observed by transmission electron microscopy. Unlike the monomeric protein, oligomers have solventexposed hydrophobic patches able to bind the fluorescent probe 1-anilinonaphthalene- 8-sulfonate. Fourier transforminfrared spectroscopy spectra of oligomers are indicative of misfolded species when compared to monomeric lysozyme, with a prevalence of random structure but with significant elements of the β-sheet structure that is characteristic of the mature fibrils. Moreover, the oligomeric lysozyme aggregates were found to be more susceptible to proteolysis with pepsin than both the monomeric protein and the mature fibrils, indicating further their less organized structure. In summary, this study shows that the soluble lysozyme oligomers are locally unfolded species that are present at low concentration during the initial phases of aggregation. The nonnative conformational features of the lysozyme molecules of which they are composed are likely to be the factors that confer on them the ability to interact inappropriately with a variety of cellular components including membranes. [less ▲]

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See detailOptimization of the Production of the Amyloidogenic Variants of Human Lysozyme
Menzer, Linda ULg; Tocquin, Pierre ULg; Dony, Nicolas et al

Poster (2008, February 16)

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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 ULg 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 detailThe Bacillus licheniformis BlaP beta-lactamase as a model protein scaffold to study the insertion of protein fragments.
Vandevenne, Marylène ULg; Filée, Patrice ULg; Scarafone, Natacha ULg et al

in Protein Science : A Publication of the Protein Society (2007), 16(10), 2260-71

Using genetic engineering technologies, the chitin-binding domain (ChBD) of the human macrophage chitotriosidase has been inserted into the host protein BlaP, a class A beta-lactamase produced by Bacillus ... [more ▼]

Using genetic engineering technologies, the chitin-binding domain (ChBD) of the human macrophage chitotriosidase has been inserted into the host protein BlaP, a class A beta-lactamase produced by Bacillus licheniformis. The product of this construction behaved as a soluble chimeric protein that conserves both the capacity to bind chitin and to hydrolyze beta-lactam moiety. Here we describe the biochemical and biophysical properties of this protein (BlaPChBD). This work contributes to a better understanding of the reciprocal structural and functional effects of the insertion on the host protein scaffold and the heterologous structured protein fragments. The use of BlaP as a protein carrier represents an efficient approach to the functional study of heterologous protein fragments. [less ▲]

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See detailPlant-based production of human lysozyme mutants
Tocquin, Pierre ULg; Dumoulin, Mireille ULg; Dony, Nicolas ULg et al

Poster (2007)

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See detailThe extracellular chaperone clusterin potently inhibits human lysozyme amyloid formation by interacting with prefibrillar species
Kumita, Janet R.; Poon, Stephen; Caddy, Gemma L. et al

in Journal of Molecular Biology (2007), 369

We have studied the effects of the extracellular molecular chaperone, clusterin, on the in vitro aggregation of mutational variants of human lysozyme, including one associated with familial amyloid ... [more ▼]

We have studied the effects of the extracellular molecular chaperone, clusterin, on the in vitro aggregation of mutational variants of human lysozyme, including one associated with familial amyloid disease. The aggregation of the amyloidogenic variant I56T is inhibited significantly at clusterin to lysozyme ratios as low as 1:80 (i.e. one clusterin molecule per 80 lysozyme molecules). Experiments indicate that under the conditions where inhibition of aggregation occurs, clusterin does not bind detectably to the native or fibrillar states of lysozyme, or to the monomeric transient intermediate known to be a key species in the aggregation reaction. Rather, it seems to interact with oligomeric species that are present at low concentrations during the lag (nucleation) phase of the aggregation reaction. This behavior suggests that clusterin, and perhaps other extracellular chaperones, could have a key role in curtailing the potentially pathogenic effects of the misfolding and aggregation of proteins that, like lysozyme, are secreted into the extracellular environment. [less ▲]

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