Influence of protein context on the propensity of polyglutamine tracts to induce protein aggregation into amyloid fibrils

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 pathogenic 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 big and relatively insoluble, they are difficult to produce and manipulate. We have
therefore decided to study the aggregating 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 at position 197 of BlaP. The aggregating properties of these BlaP chimeras recapitulate those of proteins
associated with polyQ diseases. We indeed observed that there is a minimal number of glutamines (threshold) required
for 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 the role of oligomers in the process of aggregation 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). Preliminary QCM-D results
indicate that in native condition there is a minimal number of glutamines required for the elongation of BlaP197(Gln)79
fibrils by BlaP chimeras. This threshold is different to that observed for the aggregation monitored in solution in absence
of seed. Most interestingly it corresponds to the threshold observed in denaturing conditions. These observations
suggest that the conformation of BlaP is the limiting step for amyloid fibril formation by interfering with the nucleation
step. Preliminary DLS experiments indicate that the oligomers formed by BlaP197(Gln)79 in native condition are on the
pathway of amyloid fibril formation and could act as aggregation nuclei.