Abstract :
[en] Nine neurodegenerative disorders, referred to as polyglutamine diseases and including Huntington’s disease, are associated with the abnormal expansion of a polyglutamine tract inside nine unrelated proteins. This polyQ expansion is thought to be the major determinant in the development of neurotoxicity, triggering protein aggregation into amyloid fibrils. A large body of evidence however suggests that non-polyQ regions modulate the aggregation process triggered by polyQ expansions. The interplay between the polyQ tract and non-polyQ regions is complex and still not fully understood. In order to better understand it, we previously designed and characterized model polyQ proteins made of the beta-lactamase BlaP with 23, 30, 55 and 79Q inserted at position 197 or 216. Our first results had indicated that our model is relevant to study polyQ aggregation since it recapitulates the aggregation properties of polyQ disease-associated proteins: there is a Q-threshold for the spontaneous formation of amyloid fibrils in solution, and above the threshold, the longer the polyQ, the faster the aggregation. Moreover, the structure of BlaP and the position of insertion of the polyQ tract influence their aggregation properties in solution. This work aims to better understand, at the molecular level, (i) the precise role of the polyQ length (23, 30, 55, 61, 67, 73 and 79Q), (ii) the conformation of the host protein (native or unfolded BlaP), (iii) the location of the polyQ tract within BlaP (197 or 216), (iv) the flexibility of the polyQ flanking sequences, and (v) the origin of constraints applied by BlaP to the inserted polyQ tract (at its N- or C-terminal end) on the structural, thermodynamic and aggregation properties of BlaP-polyQ chimeras, using a wide range of biophysical techniques (e.g., spectroscopy methods, quartz crystal microbalance, atomic force microscopy and dynamic light scattering). The effect on the aggregation properties will be determined on the spontaneous aggregation into amyloid fibrils in solution, and on the nucleation and on the elongation steps of amyloid fibril formation. For this purpose, new chimeras containing 61, 67 and 73Q at position 197, or 55Q inserted at position 197 in between two different protease’s cleavage sites, that are relatively flexible, will be moreover created. Our results first demonstrate that the spontaneous aggregation into amyloid fibrils in solution is correlated to the polyQ length with an exponential growth function, and that the elongation rate is linearly correlated to the polyQ length, independently of the protein context (i.e., conformation of BlaP, and/or location of the polyQ tract, and/or polyQ peptides inserted or not within BlaP). However, the location of the polyQ tract inside BlaP, and/or its conformational state, and/or the flexibility of polyQ flanking sequences, and/or the origin of constraints applied to the polyQ tract drastically influence the ability of a polyQ tract to trigger the nucleation and/or the elongation step of amyloid fibrils (variation in the Q-threshold and in the absolute rate of both steps). Altogether, our results suggest that non-polyQ regions constitute an additional potential therapeutic target, more specific than drugs targeting the polyQ sequence, to interfere with the nucleation and/or the elongation of amyloid fibrils, associated to neurotoxicity. A possible drug could be constituted by a ligand specific to non-polyQ regions of disease-associated proteins, which further increases the constraints applied to the polyQ expansion to prevent the disease onset and/or progression.
