Reference : VHHs as model proteins to investigate amyloid fibril formation: effect of seeding and cr...
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Life sciences : Biochemistry, biophysics & molecular biology
VHHs as model proteins to investigate amyloid fibril formation: effect of seeding and cross-seeding on aggregation kinetics and stability of fibrils
Chavignon, Chloé mailto [Université de Liège - ULg > Département des sciences de la vie > Enzymologie et repliement des protéines >]
Dumoulin, Mireille mailto [Université de Liège - ULg > Département des sciences de la vie > Enzymologie et repliement des protéines >]
Pardon, Els []
Wyns, Lode []
Muyldermans, Serge []
Single domain antibodies, come of age
du 14 octobre au 15 octobre 2010
Muyldermans Serge (VUB-BIB), Brussels, Belgium
Revets Hilde (Ablynx, Ghent, Belgium)
[en] amyloid fibrils ; camelid heavy-chain antibody (VHH)
[en] 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.
Centre d'Ingénierie des Protéines - CIP
Fonds pour la formation à la Recherche dans l'Industrie et dans l'Agriculture (Communauté française de Belgique) - FRIA ; Fonds de la Recherche Scientifique (Communauté française de Belgique) - F.R.S.-FNRS
Etude du mécanisme moléculaire de la formation de fibres amyloïdes à l'aide de fragments d'anticorps à chaînes lourdes comme protéines modèles
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