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See detailHeavy-chain antibody fragments as model proteins to investigate the molecular mechanism of formation of amyloid fibrils
Chavignon, Chloé ULg

Doctoral thesis (2015)

Amyloidoses are rather common and widespread debilitating and/or life-threatening diseases. They display structurally identical amyloid fibrils, and consequently appears to share a common, but still ... [more ▼]

Amyloidoses are rather common and widespread debilitating and/or life-threatening diseases. They display structurally identical amyloid fibrils, and consequently appears to share a common, but still poorly understood, mechanism of fibrillogenesis. Variable domains of camelid heavy-chain antibodies, referred to as VHHs or nanobodies, were chosen to investigate the factors that favour amyloid fibrillogenesis and to get a better understanding of the mechanism of Ig fibrillogenesis. Our study focused on the contribution of protein regions, i.e. the framework (FR) and the complementary determining regions (CDRs) of VHHs, to the formation of amyloid fibrils. Three VHHs were selected: cAb HuL6, cAb BcII10 and cAb BcII10 H H H, a chimera bearing the FR of cAb BcII10 and the CDRs of cAb HuL6. We first established experimental conditions suitable for fibrillogenesis: even the especially stable and soluble VHHs can form amyloid fibrils in denaturing conditions. This comforts the proposal that amyloid conformation is a generic structural form that all proteins can adopt in the appropriate conditions. Amyloid fibrils were characterised by transmission electron microscopy (TEM), circular dichroism (CD), thioflavin T (ThT) fluorescence and X-ray fibre diffraction. The kinetics of fibril formation were monitored by turbidity measurements. We then determined conditions suitable for characterising self-seeding and cross-seeding kinetics, and ascertained that the three VHHs convert into amyloid fibrils according to a nucleation-dependent process. Fibril elongation rates in self-seeding and cross-seeding experiments suggest that amyloid fibril elongation occurs through sequential addition of protein monomers to preformed fibrils. The kinetics of aggregation and self-seeding of cAb-HuL6 and cAb-BcII10 differ significantly, and a remarkable specificity for cross-seeding was observed, with cAb-BcII10 being unable to cross-seed the formation of fibrils by the two other VHHs, while cAb-HuL6 and cAb BcII10 H-H-H mutually cross-seed each other’s fibril formation. With the help of three prediction algorithms (AGGRESCAN, Zyggregator and 3D-profile), we showed that various peptides of the VHHs had the ability to form fibrils by themselves. Our results strongly suggest that the sequences of the regions forming the core of the fibrils essentially correspond to the CDRs. Concurrently, we set up experimental conditions for interrupting the fibril formation process and we characterised the multimer and fibril species formed in the early stages of fibrillogenesis by analytical ultracentrifugation (AUC) and small angle neutron scattering (SANS) ex situ. [less ▲]

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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 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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See detailVHHs as model proteins to investigate amyloid fibril formation: Fibril stability and effect of seeding and cross-seeding on aggregation kinectics
Chavignon, Chloé ULg

Poster (2010, June)

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 (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 unfolding 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. We will discuss the kinetics of aggregation obtained in the absence and the presence of seeding/cross-seeding. [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 ▲]

Detailed reference viewed: 31 (3 ULg)
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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 ▲]

Detailed reference viewed: 34 (6 ULg)