References of "Dumoulin, Mireille"
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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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See detailHuman lysozyme amyloidosis
Dumoulin, Mireille ULg; Johnson, Russell J.K.; Bellotti, Vittorio et al

in Uversky, V.; Fink, A. L. (Eds.) Protein Misfolding, Aggregation and Conformational Diseases. II. Molecular Basis of Conformational Diseases. (2007)

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See detailImpact of the native-state stability of human lysozyme variants on protein secretion by Pichia pastoris
Kumita, Janet; Johnson, Russel; Alcocer, Marcos et al

in FEBS Journal (2006), 273

We report the secreted expression by Pichia pastoris of two human lysozyme variants F57I and W64R, associated with systemic amyloid disease, and describe their characterization by biophysical methods ... [more ▼]

We report the secreted expression by Pichia pastoris of two human lysozyme variants F57I and W64R, associated with systemic amyloid disease, and describe their characterization by biophysical methods. Both variants have a substantially decreased thermostability compared with wild-type human lysozyme, a finding that suggests an explanation for their increased propensity to form fibrillar aggregates and generate disease. The secreted yields of the F57I and W64R variants from P. pastoris are 200- and 30-fold lower, respectively, than that of wild-type human lysozyme. More comprehensive analysis of the secretion levels of 10 lysozyme variants shows that the low yields of these secreted proteins, under controlled conditions, can be directly correlated with a reduction in the thermostability of their native states. Analysis of mRNA levels in this selection of variants suggests that the lower levels of secretion are due to post-transcriptional processes, and that the reduction in secreted protein is a result of degradation of partially folded or misfolded protein via the yeast quality control system. Importantly, our results show that the human disease-associated mutations do not have levels of expression that are out of line with destabilizing mutations at other sites. These findings indicate that a complex interplay between reduced native-state stability, lower secretion levels, and protein aggregation propensity influences the types of mutation that give rise to familial forms of amyloid disease. [less ▲]

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See detailNormal and aberrant biological self-assembly: Insights from studies of human lysozyme and its amyloidogenic variants
Dumoulin, Mireille ULg; Kumita, Janet; Dobson, Christopher M

in Accounts of Chemical Research (2006), 39

Studies of lysozyme have played a major role over several decades in defining the general principles underlying protein structure, folding, and stability. Following the discovery some 10 years ago that ... [more ▼]

Studies of lysozyme have played a major role over several decades in defining the general principles underlying protein structure, folding, and stability. Following the discovery some 10 years ago that two mutational variants of lysozyme are associated with systemic amyloidosis, these studies have been extended to investigate the mechanism of amyloid fibril formation. This Account describes our present knowledge of lysozyme folding and misfolding, and how the latter can give rise to amyloid disease. It also discusses the significance of these studies for our general understanding of normal and aberrant protein folding in the context of human health and disease. [less ▲]

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See detailMethods to study protein aggregation and amyloid formation
Dumoulin, Mireille ULg; Bader, R.

in Protein & Peptide Letters (2006), 13

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See detailIdentification of the Core Structure of Lysozyme Amyloid Fibrils by Proteolysis
Frare, Erica; Mossuto, Maria F.; Polverino de Laureto, Patrizia et al

in Journal of Molecular Biology (2006), 361

Human lysozyme variants form amyloid fibrils in individuals suffering from a familial non-neuropathic systemic amyloidosis. In vitro, wild-type human and hen lysozyme, and the amyloidogenic mutants can be ... [more ▼]

Human lysozyme variants form amyloid fibrils in individuals suffering from a familial non-neuropathic systemic amyloidosis. In vitro, wild-type human and hen lysozyme, and the amyloidogenic mutants can be induced to form amyloid fibrils when incubated under appropriate conditions. In this study, fibrils of wild-type human lysozyme formed at low pH have been analyzed by a combination of limited proteolysis and Fouriertransform infrared (FTIR) spectroscopy, in order to map conformational features of the 130 residue chain of lysozyme when embedded in the amyloid aggregates. After digestion with pepsin at low pH, the lysozyme fibrils were found to be composed primarily of N and C-terminally truncated protein species encompassing residues 26–123 and 32–108, although a significant minority of molecules was found to be completely resistant to proteolysis under these conditions. FTIR spectra provide evidence that lysozyme fibrils contain extensive β-sheet structure and a substantial element of non β-sheet or random structure that is reduced significantly in the fibrils after digestion. The sequence 32–108 includes the β-sheet and helix C of the native protein, previously found to be prone to unfold locally in human lysozyme and its pathogenic variants. Moreover, this core structure of the lysozyme fibrils encompasses the highly aggregation-prone region of the sequence recently identified in hen lysozyme. The present proteolytic data indicate that the region of the lysozyme molecule that unfolds and aggregates most readily corresponds to the most highly protease-resistant and thus highly structured region of the majority of mature amyloid fibrils. Overall, the data show that amyloid formation does not require the participation of the entire lysozyme chain. The majority of amyloid fibrils formed from lysozyme under the conditions used here contain a core structure involving some 50% of the polypeptide chain that is flanked by proteolytically accessible N and C-terminal regions. [less ▲]

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