Reference : Influence of the protéin context on the propensity of polyglutamine tracts to trigger pr...
Scientific congresses and symposiums : Unpublished conference
Life sciences : Biochemistry, biophysics & molecular biology
Influence of the protéin context on the propensity of polyglutamine tracts to trigger protein aggregation into amyloid fibrils
Huynen, Céline[Université de Liège - ULg > Département des sciences de la vie > Enzymologie et repliement des protéines >]
Sofia School of Protein Science: Structure and dynamics of Biological Macromolecules.
du 9 septembre 2012 au 14 septembre 2012
FEDERATION OF EUROPEAN BIOCHEMICAL SOCIETIES
[en] Nine neurodegenerative diseases, referred to as polyglutamine diseases, are associated with nine proteins containing an expanded polyglutamine (polyQ) tract. PolyQ tracts are encoded by a repetition of the CAG codon in the corresponding genes, and are present in proteins of healthy people. They are however pathogenic when their length, due to mutations, becomes higher than a threshold generally comprised between 35 and 45Q. Such pathological tracts trigger the aggregation of the proteins into amyloid-like aggregates that could play an important role in the disease. It is therefore necessary to investigate at a molecular level the aggregation process of polyQ proteins. Since proteins associated with polyQ diseases are generally large and relatively insoluble, they are difficult to produce and handle experimentally. We have therefore decided to study the aggregation properties of polyQ proteins by designing and characterizing model proteins made of a well-characterized host protein, the β-lactamase BlaP, and polyQ tracts of different lengths (23 to 79Q) inserted either at positions 197 or 216 of BlaP. The aggregation behaviour of BlaP chimeras recapitulate those of proteins associated with polyQ diseases. We indeed observed that there is a minimal number of glutamines (i.e. a threshold) required for the chimeras’ aggregation into amyloid-like fibrils and that the kinetics of aggregation are faster with longer glutamine repeats. Most importantly, the value of the threshold for amyloid-like fibril formation seems to critically depend on the structural integrity of BlaP and thus on the constraints applied to the polyQ tract. In the present work, we investigate more deeply the role of the protein context and of oligomers on the aggregation process of BlaP chimeras. This study is mainly based on the use of two techniques: the quartz crystal microbalance with dissipation (QCM-D) in combination with atomic force microscopy (AFM), and the dynamic light scattering (DLS). QCM-D studies indicate that, under native condition, there is a minimal number of glutamines required for the elongation of BlaP197(Gln)79 fibrils by BlaP chimeras containing a polyQ tract in position 197. This threshold is lower than that observed for the aggregation assays monitored in solution in the absence of seed; it corresponds however to the threshold for fibril formation observed under denaturing conditions. These observations suggest that the conformation of BlaP is the limiting step for amyloid fibril formation by interfering with the nucleation step. BlaP chimeras containing 55Q or more in position 216 of BlaP also elongate BlaP197(Gln)79 fibrils. Interestingly this rate of cross-elongation is faster than the elongation rate observed with BlaP197(Gln)55 and BlaP197(Gln)79. This observation demonstrates that the protein environment of the polyQ tract influences its propensity to form amyloid fibrils. Finally, preliminary DLS experiments indicate that oligomers formed by BlaP197(Gln)79 under native condition are on-pathway of amyloid fibril formation and could act as aggregation nuclei.
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
Mécanisme d'agrégation de protéines chimères composées de la béta-lactamase BlaP et de séquences polyglutamine: étude des contraintes stériques et/ou conformationelles imposées par BlaP sur les propriétés des stretchs polyglutamine