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Switching to the Dark Side: Repositioning of Polyglutamine Repeat Promotes Amyloid Fibril Formation by the Model Protein, β-Lactamase BlaP
Thorn, David; Pain, Coralie; Scarafone, Natacha et al.
20134th Scandinavian Meeting on Amyloid Proteins and Disease
 

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Keywords :
polyQ protein; aggregation; neurodegenerative disease
Abstract :
[en] 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.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Thorn, David ;  Université de Liège - ULiège > Département des sciences de la vie > Enzymologie et repliement des protéines
Pain, Coralie ;  Université de Liège - ULiège > Département des sciences de la vie > Enzymologie et repliement des protéines
Scarafone, Natacha
Huynen, Céline ;  Université de Liège - ULiège > Département des sciences de la vie > Enzymologie et repliement des protéines
Preumont, Stéphane ;  Université de Liège - ULiège > Département des sciences de la vie > Enzymologie et repliement des protéines
Menzer, Linda ;  Université de Liège - ULiège > Centre d'ingénierie des protéines
Duez, Colette ;  Université de Liège - ULiège > Centre d'ingénierie des protéines
Dumoulin, Mireille  ;  Université de Liège - ULiège > Département des sciences de la vie > Enzymologie et repliement des protéines
Language :
English
Title :
Switching to the Dark Side: Repositioning of Polyglutamine Repeat Promotes Amyloid Fibril Formation by the Model Protein, β-Lactamase BlaP
Publication date :
20 November 2013
Event name :
4th Scandinavian Meeting on Amyloid Proteins and Disease
Event organizer :
University of Lund
Event place :
Lund, Sweden
Event date :
from 18-11-2013 to 20-11-2013
Audience :
International
Funders :
F.R.S.-FNRS - Fonds de la Recherche Scientifique [BE]
Biochemical Society
ULiège - Université de Liège [BE]
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since 06 January 2014

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