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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 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
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 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 detailReduced global copperativity is a common feature underlying the amyloidogenicity of pathogenic lysozyme mutations
Dumoulin, Mireille ULg; Canet, Denis; Last, Alexander M. et al

in Journal of Molecular Biology (2005), 346(3), 773-788

One of the 20 or so human amyloid diseases is associated with the deposition in vital organs of full-length mutational variants of the antibacterial protein lysozyme. Here, we report experimental data ... [more ▼]

One of the 20 or so human amyloid diseases is associated with the deposition in vital organs of full-length mutational variants of the antibacterial protein lysozyme. Here, we report experimental data that permit a detailed comparison to be made of the behaviour of two of these amyloidogenic variants, I56T and D67H, under identical conditions. Hydrogen/deuterium exchange experiments monitored by NMR and mass spectrometry reveal that, despite their different locations and the different effects of the two mutations on the structure of the native state of lysozyme, both mutations cause a cooperative destabilisation of a remarkably similar segment of the structure, comprising in both cases the beta-domain and the adjacent C-helix. As a result, both variant proteins populate transiently a closely similar, partially unstructured intermediate in which the beta-domain and the adjacent C-helix are substantially and simultaneously unfolded, whereas the three remaining a-helices that form the core of the a-domain still have their native-like structure. We show, in addition, that the binding of a camel antibody fragment, cAb-HuL6, which was raised against wild-type lysozyme, restores to both variant proteins the stability and cooperativity characteristic of the wild-type protein; as a consequence, it inhibits the formation of amyloid fibrils by both variants. These results indicate that the reduction in global cooperativity, an associated ability to populate transiently a specific, partly unfolded intermediate state under physiologically relevant conditions, is a common feature underlying the behaviour of these two pathogenic mutations. The formation of intermolecular interactions between lysozyme molecules that are in this partially unfolded state is therefore likely to be the fundamental trigger of the aggregation process that ultimately leads to the formation and deposition in tissue of amyloid fibrils. (C) 2004 Elsevier Ltd. All rights reserved. [less ▲]

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See detailIdentification of a universal VHH framework to graft non-canonical antigen-binding loops of camel single-domain antibodies
Saerens, Dirk; Pellis, Mireille; Loris, Remy et al

in Journal of Molecular Biology (2005), 352

Camel single-domain antibody fragments (VHHs) are promising tools in numerous biotechnological and medical applications. However, some conditions under which antibodies are used are so demanding that they ... [more ▼]

Camel single-domain antibody fragments (VHHs) are promising tools in numerous biotechnological and medical applications. However, some conditions under which antibodies are used are so demanding that they can be met by only the most robust VHHs. A universal framework offering the required properties for use in various applications (e.g. as intrabody, as probe in biosensors or on micro-arrays) is highly valuable and might be further implemented when employment of VHHs in human therapy is envisaged. We identified the VHH framework of cAbBCII10 as a potential candidate, useful for the exchange of antigen specificities by complementarity determining region (CDR) grafting. Due to the large number of CDRH loop structures present on VHHs, this grafting technique was expected to be rather unpredictable. Nonetheless, the plasticity of the cAbBCII10 framework allows successful transfer of antigen specificity from donor VHHs onto its scaffold. The cAbBCII10 was chosen essentially for its high level of stability (47 kJ/mol), good expression level (5 mg/l in E. coli) and its ability to be functional in the absence of the conserved disulfide bond. All five chimeras generated by grafting CDR-Hs, from donor VHHs belonging to subfamily 2 that encompass 75% of all antigen-specific VHHs, on the framework of cAbBCII10 were functional and generally had an increased thermodynamic stability. The grafting of CDR-H loops from VHHs belonging to other subfamilies resulted in chimeras of reduced antigen-binding capacity. [less ▲]

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See detailSingle-domain antibody fragments with high conformational stability.
Dumoulin, Mireille ULg; Conrath, Katja; Van Meirhaeghe, Annemie et al

in Protein Science : A Publication of the Protein Society (2002), 11(3), 500-15

A variety of techniques, including high-pressure unfolding monitored by Fourier transform infrared spectroscopy, fluorescence, circular dichroism, and surface plasmon resonance spectroscopy, have been ... [more ▼]

A variety of techniques, including high-pressure unfolding monitored by Fourier transform infrared spectroscopy, fluorescence, circular dichroism, and surface plasmon resonance spectroscopy, have been used to investigate the equilibrium folding properties of six single-domain antigen binders derived from camelid heavy-chain antibodies with specificities for lysozymes, beta-lactamases, and a dye (RR6). Various denaturing conditions (guanidinium chloride, urea, temperature, and pressure) provided complementary and independent methods for characterizing the stability and unfolding properties of the antibody fragments. With all binders, complete recovery of the biological activity after renaturation demonstrates that chemical-induced unfolding is fully reversible. Furthermore, denaturation experiments followed by optical spectroscopic methods and affinity measurements indicate that the antibody fragments are unfolded cooperatively in a single transition. Thus, unfolding/refolding equilibrium proceeds via a simple two-state mechanism (N <--> U), where only the native and the denatured states are significantly populated. Thermally-induced denaturation, however, is not completely reversible, and the partial loss of binding capacity might be due, at least in part, to incorrect refolding of the long loops (CDRs), which are responsible for antigen recognition. Most interestingly, all the fragments are rather resistant to heat-induced denaturation (apparent T(m) = 60-80 degrees C), and display high conformational stabilities (DeltaG(H(2)O) = 30-60 kJ mole(-1)). Such high thermodynamic stability has never been reported for any functional conventional antibody fragment, even when engineered antigen binders are considered. Hence, the reduced size, improved solubility, and higher stability of the camelid heavy-chain antibody fragments are of special interest for biotechnological and medical applications. [less ▲]

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