References of "Dumoulin, Mireille"
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See detailSwitching to the Dark Side: Repositioning of Polyglutamine Repeat Promotes Amyloid Fibril Formation by the Model Protein, β-Lactamase BlaP
Thorn, David ULg; Pain, Coralie ULg; Scarafone, Natacha et al

Conference (2013, November 20)

Background: The expansion of polyglutamine (polyQ) repeats is associated with an increased propensity of the protein to aggregate into amyloid fibrils. There are ten human proteins presently known within ... [more ▼]

Background: The expansion of polyglutamine (polyQ) repeats is associated with an increased propensity of the protein to aggregate into amyloid fibrils. There are ten human proteins presently known within which polyQ expansion above a threshold length, e.g. 35-50 residues, leads to ten distinct neurodegenerative disorders [1], the most well-known being Huntington’s disease. While repeat length, aggregation, and disease are well correlated, recent studies suggest the non-polyQ regions of these proteins can also play a significant role, both preventative and facilitative, in the aggregation process. With the aim of exploring this role in more detail, we have engineered chimeric proteins via the insertion of polyQ repeats of various length (23, 30, 55, 79 Q) into two sites of antibiotic resistance enzyme BlaP β-lactamase from Bacillus licheniformis 749/C [2]. Questions addressed: How does polyQ repeat position affect the structure, stability and aggregation of polyQ proteins? Methods: Aggregation kinetics determined by monitoring the decrease in soluble protein fraction over time. Aggregate morphology examined by transmission electron microscopy. Protein stability derived from thermal or chemical unfolding transitions monitored by far-UV CD and intrinsic fluorescence. Results and discussion: PolyQ insertion at either of the two positions led to a decrease in thermodynamic stability that was largely independent of polyQ length. Chimeras with polyQ insertions at position 216 were destabilised to a much greater extent than those with insertions at position 197. The reduced stability of the 216 chimeras was associated with an increased aggregation propensity: (i) the minimum polyQ length leading to aggregation was lower, and (ii) the aggregation rate was significantly higher than that observed by 197 chimeras with equivalent polyQ lengths. Remarkably, the two sites of polyQ insertion are indeed very similar, both residing within flexible loop regions between stable α-helices. Moreover, the 216 chimeras exhibited a higher aggregation propensity than their 197 counterparts even under denaturing conditions, suggesting the disparity between the two chimeras cannot be accounted for by structural differences alone. These findings highlight the strong and complex influence of the overall protein context on polyQ-mediated aggregation. The molecular basis for the observed changes in stability and aggregation propensity is the subject of on-going work. [less ▲]

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See detailClass A β -Lactamases as Versatile Scaffolds to Create Hybrid Enzymes: Applications from Basic Research to Medicine
Huynen, Céline ULg; Filée, Patrice; Matagne, André ULg et al

in BioMed Research International (2013), 2013

Designing hybrid proteins is a major aspect of protein engineering and covers a very wide range of applications frombasic research to medical applications. This review focuses on the use of class A 𝛽 ... [more ▼]

Designing hybrid proteins is a major aspect of protein engineering and covers a very wide range of applications frombasic research to medical applications. This review focuses on the use of class A 𝛽-lactamases as versatile scaffolds to design hybrid enzymes (referred to as 𝛽-lactamase hybrid proteins, BHPs) in which an exogenous peptide, protein or fragment thereof is inserted at various permissive positions.We discuss how BHPs can be specifically designed to create bifunctional proteins, to produce and to characterize proteins that are otherwise difficult to express, to determine the epitope of specific antibodies, to generate antibodies against nonimmunogenic epitopes, and to better understand the structure/function relationship of proteins. [less ▲]

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See detailA Nanobody Binding to Non-amyloidogenic Regions of the Protein Human Lysozyme Enhances Partial Unfolding but Inhibits Amyloid Fibril Formation.
de Genst, EJ; Chan, PH; Pardon, Els et al

in Journal of Physical Chemistry B (2013)

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See detailNanobodies as structural probes to investigate the mechanism of fibril formation by the amyloidogenic variants of human lysozyme
Dumont, Janice ULg; pardon, Els; Aumont-Nicaise, Magali et al

Poster (2012, June)

Six variants of human lysozyme (single-point mutatants I56T, F57I, W64R, D67H and double mutants F57I/T70N, W112R/T70N) are associated with a hereditary non-neuropathic systemic amyloidosis. These ... [more ▼]

Six variants of human lysozyme (single-point mutatants I56T, F57I, W64R, D67H and double mutants F57I/T70N, W112R/T70N) are associated with a hereditary non-neuropathic systemic amyloidosis. These proteins form extracellular amyloid fibrils that deposit in a wide range of tissues and organs such as liver, spleen and kidneys where they cause damages [1]. It was shown that the D67H and I56T mutations cause a loss in stability and more particularly a loss of global cooperativity of protein [1]. 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. We have also shown that the binding of three variable domain of camelid antibodies (VHHs) - raised against the wild type human lysozyme inhibit in vitro the formation of amyloid fibrils by the lysozyme variants. These three VHHs bind on different regions of lysozyme and act as amyloid fibril inhibitor through different mechanisms [2, 3, and unpublished results]. In the present work, sixteen new VHHs specific of human lysozyme have been generated. Competition experiments have shown that they bind to five non-overlapping epitopes. We have demonstrated that five of these VHHs are able to bind lysozyme in conditions used for amyloid fibril formation, and interestingly two of them recognize two epitopes that are different from those of the three VHHs previously characterized [2, 3, and unpublished results]. The effects of these new VHHs on the properties of lysozyme variants such as stability, cooperativity and aggregation will be discussed. [1] Dumoulin, M., J.R. Kumita, and C.M. Dobson, Normal and aberrant biological self-assembly: Insights from studies of human lysozyme and its amyloidogenic variants. Acc Chem Res, 2006, 39(9), 603-610. [2] Dumoulin, M., et al., A camelid antibody fragment inhibits the formation of amyloid fibrils by human lysozyme. Nature, 2003, 424, 783-788. [3] Chan, P.H., et al., Engineering a camelid antibody fragment that binds to the active site of human lysozyme and inhibits its conversion into amyloid fibrils. Biochemistry, 2008, 47, 11041-11054. [less ▲]

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See detailDisease-related amyloidogenic variants of human lysozyme trigger the unfolded protein response and disturb eye development in Drosophila melanogaster
Kumita, Janet R.; Helmfors, Linda; Williams, Jocy et al

in FASEB Journal (2012)

We have created a Drosophila model of lysozyme amyloidosis to investigate the in vivo behavior of disease-associated variants. To achieve this objective, wild-type (WT) protein and the amyloidogenic ... [more ▼]

We have created a Drosophila model of lysozyme amyloidosis to investigate the in vivo behavior of disease-associated variants. To achieve this objective, wild-type (WT) protein and the amyloidogenic variants F57I and D67H were expressed in Drosophila melanogaster using the UAS-gal4 system and both the ubiquitous and retinal expression drivers Act5C-gal4 and gmr-gal4. The nontransgenic w(1118) Drosophila line was used as a control throughout. We utilized ELISA experiments to probe lysozyme protein levels, scanning electron microscopy for eye phenotype classification, and immunohistochemistry to detect the unfolded protein response (UPR) activation. We observed that expressing the destabilized F57I and D67H lysozymes triggers UPR activation, resulting in degradation of these variants, whereas the WT lysozyme is secreted into the fly hemolymph. Indeed, the level of WT was up to 17 times more abundant than the variant proteins. In addition, the F57I variant gave rise to a significant disruption of the eye development, and this correlated to pronounced UPR activation. These results support the concept that the onset of familial amyloid disease is linked to an inability of the UPR to degrade completely the amyloidogenic lysozymes prior to secretion, resulting in secretion of these destabilized variants, thereby leading to deposition and associated organ damage.-Kumita, J. R., Helmfors, L., Williams, J., Luheshi, L. M., Menzer, L., Dumoulin, M., Lomas, D. A., Crowther, D. C., Dobson, C. M., Brorsson, A.-C. Disease-related amyloidogenic variants of human lysozyme trigger the unfolded protein response and disturb eye development in Drosophila melanogaster. [less ▲]

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See detailVHHs as structural probes to investigate the mechanis of fibril formation by the amyloidogenic variants of human lysozyme
Dumont, Janice; Kumita, Janet; Menzer, Linda et al

Scientific conference (2011, August 26)

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See detailVHHs as model proteins to investigate amyloid fibril formation: effect of seeding and cross-seeding on the stability of fibrils
Chavignon, Chloé ULg; Pardon, Els; Wyns, Lode et al

Poster (2011, August)

The term "amyloidosis" covers a group of diseases associated with the deposition of protein aggregates organized into amyloid fibrils in different organs. About forty amyloidoses are known so far, amongst ... [more ▼]

The term "amyloidosis" covers a group of diseases associated with the deposition of protein aggregates organized into amyloid fibrils in different organs. About forty amyloidoses are known so far, amongst which Alzheimer's disease, type II diabetes and immunoglobulin amyloidosis [1]. Although the mechanism of amyloid fibril 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 not restrained to proteins involved in amyloidoses but can be settled on a wide variety of proteins. In this study, we have chosen to investigate the mechanism of amyloid fibril formation by two variable domains of camelid heavy-chain antibodies (referred to as VHHs or nanobodies), cAb-HuL6 and cAb-BcII10, for which variants with mutations located at the disulfide bond [3,4] and the CDRs [3] are available. 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 amyloid fibrils, heat-induced unfolding experiments at several pHs have been monitored by intrinsic fluorescence 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 electron microscopy. We will discuss the kinetics of aggregation obtained in the absence and the presence of seeding/cross-seeding and the stability of the formed fibrils. [1] Chiti and Dobson, Annu. Rev. Biochem., 75, 2006, 333-366 ; [2] Dumoulin et al., Protein Sci., 11, 2002, 500-515 ; [3] Saerens et al., J. Mol. Biol., 352, 2005, 597-607 ; [4] Saerens et al., J. Mol. Biol., 377, 2008, 478-488. [less ▲]

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See detailMechanism of amyloid fibril formation by human lysozyme and VHHs
Dumoulin, Mireille ULg; Chavignon, Chloé ULg

Conference (2011, January)

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See detailEffects of monopropanediamino-beta-cyclodextrin on the denaturation process of the hybrid protein BlaPChBD.
Vandevenne, Marylène ULg; GASPARD, Genevieve ULg; Belgsir, E. M. et al

in Biochimica et biophysica acta (2011)

Irreversible accumulation of protein aggregates represents an important problem both in vivo and in vitro. The aggregation of proteins is of critical importance in a wide variety of biomedical situations ... [more ▼]

Irreversible accumulation of protein aggregates represents an important problem both in vivo and in vitro. The aggregation of proteins is of critical importance in a wide variety of biomedical situations, ranging from diseases (such as Alzheimer's and Parkinson's diseases) to the production (e.g. inclusion bodies), stability, storage and delivery of protein drugs. beta-Cyclodextrin (beta-CD) is a circular heptasaccharide characterized by a hydrophilic exterior and a hydrophobic interior ring structure. In this research, we studied the effects of a chemically modified beta-CD (BCD07056), on the aggregating and refolding properties of BlaPChBD, a hybrid protein obtained by inserting the chitin binding domain of the human macrophage chitotriosidase into the class A beta-lactamase BlaP from Bacillus licheniformis 749/I during its thermal denaturation. The results show that BCD07056 strongly increases the refolding yield of BlaPChBD after thermal denaturation and constitutes an excellent additive to stabilize the protein over time at room temperature. Our data suggest that BCD07056 acts early in the denaturation process by preventing the formation of an intermediate which leads to an aggregated state. Finally, the role of beta-CD derivatives on the stability of proteins is discussed. [less ▲]

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See detailNanobodies as structural probes to investigate the mechanism of fibril formation by the amyloidogenic variants of human lysozyme
Dumont, Janice ULg; Pardon, Els; Aumont-Nicaise, Magalie et al

Poster (2011)

Six variants of human lysozyme (single-point mutations I56T, F57I, W64R, D67H and double mutations F57I/T70N, W112R/T70N) are associated with a hereditary non-neuropathic systemic amyloidose. These ... [more ▼]

Six variants of human lysozyme (single-point mutations I56T, F57I, W64R, D67H and double mutations F57I/T70N, W112R/T70N) are associated with a hereditary non-neuropathic systemic amyloidose. These proteins form extracellular amyloid fibrils that deposit in a wide range of tissues and organs such as liver, spleen and kidneys where they cause damages [1]. It was shown that the D67H and I56T mutations cause a loss in stability and more particularly a loss of global cooperativity of protein [1]. 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. We have also shown that the binding of three variable domain of camelid antibodies or (VHHs) - raised against the wild type human lysozyme inhibit in vitro the formation of amyloid fibrils by the lysozyme variants. These three VHHs bind on different regions of lysozyme and act as amyloid fibrils inhibitor through different mechanisms [2, 3, and unpublished results]. In the present work, sixteen new VHHs specific of human lysozyme have been generated. Competition experiments have shown that they bind to five non overlapping epitopes. We have demonstrated that five of these new VHHs are able to bind lysozyme in conditions used for amyloid fibril formation, and interestingly two of them recognize two epitopes that are different from those of the three VHHs previously characterized [2, 3, and unpublished results]. The effects of these new VHHs on the properties of lysozyme variants such as activity, stability, cooperativity and aggregation will be discussed. [less ▲]

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See detailPopulation of nonnative States of lysozyme variants drives amyloid fibril formation.
Buell, Alexander K.; Dhulesia, Anne; Mossuto, Maria F. et al

in Journal of the American Chemical Society (2011), 133(20), 7737-43

The propensity of protein molecules to self-assemble into highly ordered, fibrillar aggregates lies at the heart of understanding many disorders ranging from Alzheimer's disease to systemic lysozyme ... [more ▼]

The propensity of protein molecules to self-assemble into highly ordered, fibrillar aggregates lies at the heart of understanding many disorders ranging from Alzheimer's disease to systemic lysozyme amyloidosis. In this paper we use highly accurate kinetic measurements of amyloid fibril growth in combination with spectroscopic tools to quantify the effect of modifications in solution conditions and in the amino acid sequence of human lysozyme on its propensity to form amyloid fibrils under acidic conditions. We elucidate and quantify the correlation between the rate of amyloid growth and the population of nonnative states, and we show that changes in amyloidogenicity are almost entirely due to alterations in the stability of the native state, while other regions of the global free-energy surface remain largely unmodified. These results provide insight into the complex dynamics of a macromolecule on a multidimensional energy landscape and point the way for a better understanding of amyloid diseases. [less ▲]

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See detailDisulfide Bonds Reduce the Toxicity of the Amyloid Fibrils Formed by an Extracellular Protein.
Mossuto, M. F.; Bolognesi, B.; Guixer, B. et al

in Angewandte Chemie (International ed. in English) (2011)

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See detailGeneration of camelid single-domain antibody fragments raised against proteins containing polyglutamine expansions
Pain, Coralie ULg; Scarafone, Natacha; Jaspar, Aurélie et al

Poster (2010, October 14)

Nine progressive neurodegenerative diseases are associated with the expansion of a polyglutamine (polyQ) tract above a threshold size (~ 35-45 residues) into nine different proteins [1]. These proteins ... [more ▼]

Nine progressive neurodegenerative diseases are associated with the expansion of a polyglutamine (polyQ) tract above a threshold size (~ 35-45 residues) into nine different proteins [1]. These proteins with expanded polyQ repeats have been found to form intranuclear amyloid-like aggregates, and the formation of these aggregates could play an important role in the pathogenesis [2-4]. The polyQ expansion is the only common feature among the proteins involved, suggesting it may be responsible for the aggregation phenomenon. Understanding the molecular mechanism by which the polyQ expansions promote aggregation is therefore crucial for the development of therapeutic strategies. The nine proteins associated with polyQ diseases are difficult to express recombinantly due to their big size and/or their insoluble character. In order to get further insights into the mechanism by which polyQ tracts promote aggregation, we have therefore decided to insert polyQ sequences into a well studied protein, the b-lactamase BlaP from B. licheniformis [5-6]. We have created chimeras containing 23, 30, 55, and 79 glutamines and we have investigated the effects of the insertions on the activity, the structure, the stability of BlaP as well as on its aggregating properties. Preliminary results indicate that BlaP is a good framework to study the molecular mechanism of aggregation associated with expanded polyglutamine tracts. On another hand, our previous work on the amyloidogenic variants of human lysozyme has shown that camelid single domain antibody fragments are very powerful structural probes to understand, at the molecular level, the mechanism of amyloid fibril formation [7]. Moreover, a recent study has suggested that expanded polyQ strectches adopt multiple conformations in solution that can be readily distinguished by monoclonal antibodies [8]. Altogether these results have encouraged us to generate VHHs against our different chimeras and we present here our preliminary results. References [1] Orr and Zoghbi (2007) Annu Rev Neurosci 30, 575-621. [2] DiFiglia et al. (1997) Science 277, 1990-1993. [3] Paulson HL (2000) Brain Pathol 10, 293-299. [4] Sanchez I. et al. (2003) Nature 421, 373-379. [5] Scarafone N. (2008) Mémoire de DEA en Sciences. Université de Liège. [6] Pain C. (2009) Mémoire de Master en Biochimie. Université de Liège. [7] Dumoulin et al. (2003) Nature 424, 783-788. [8] Legleiter J. et al. (2009) J Biol Chem 284, 21647-21648. [less ▲]

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See detailVHHs as model proteins to investigate amyloid fibril formation: effect of seeding and cross-seeding on aggregation kinetics and stability of fibrils
Chavignon, Chloé ULg; Dumoulin, Mireille ULg; Pardon, Els et al

Poster (2010, October)

The term "amyloidosis" covers a group of diseases associated with the deposition of protein aggregates organized into amyloid fibrils in different organs. About forty amyloidosis are known so far, amongst ... [more ▼]

The term "amyloidosis" covers a group of diseases associated with the deposition of protein aggregates organized into amyloid fibrils in different organs. About forty 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 to investigate the mechanism of amyloid fibrils formation by 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, VHHs are small monomeric proteins (~14 kDa) presenting a high stability and a high solubility [2], which permits their expression with a high yield (5-20 mg.L-1). - Second, a wide range of stable mutants of these two VHHs is available. Mutations located at the disulfide bond [3,4] and the CDRs [3] 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 unfolding experiments at pHs comprised in a range from 2,5 to 9,5 have been monitored by intrinsic fluorescence, ANS binding and far-UV circular dichroism. Then, aggregation experiments have been performed in the selected conditions and the presence of amyloid fibrils has been observed by thioflavin T fluorescence experiments and electron microscopy. The kinetics of aggregation obtained in the absence and the presence of seeding/cross-seeding allowed to identify the regions of the protein which could be involved in the formation of fibrils. [1] Chiti and Dobson, Annu. Rev. Biochem., 75, 2006, 333-366. [2] Dumoulin et al., Protein Sci., 11, 2002, 500-515. [3] Saerens et al., J. Mol. Biol., 352, 2005, 597-607. [4] Saerens et al., J. Mol. Biol., 377, 2008, 478-488. [less ▲]

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