Amyloid-Like Fibril Formation by PolyQ Proteins: A Critical Balance between the PolyQ Length and the Constraints Imposed by the Host Protein

Abstract

Nine neurodegenerative disorders, called polyglutamine (polyQ) diseases, are characterized by the formation of intranuclear amyloid-like aggregates by nine proteins containing a polyQ tract above a threshold length. These insoluble aggregates and/or some of their soluble precursors are thought to play a role in the pathogenesis. The mechanism by which polyQ expansions trigger the aggregation of the relevant proteins remains, however, unclear. In this work, polyQ tracts of different lengths were inserted into a solvent-exposed loop of the β-lactamase BlaP and the effects of these insertions on the properties of BlaP were investigated by a range of biophysical techniques. The insertion of up to 79 glutamines does not modify the structure of BlaP; it does, however, significantly destabilize the enzyme. The extent of destabilization is largely independent of the polyQ length, allowing us to study independently the effects intrinsic to the polyQ length and those related to the structural integrity of BlaP on the aggregating properties of the chimeras. Only chimeras with 55Q and 79Q readily form amyloid-like fibrils; therefore, similarly to the proteins associated with diseases, there is a threshold number of glutamines above which the chimeras aggregate into amyloid-like fibrils. Most importantly, the chimera containing 79Q forms amyloid-like fibrils at the same rate whether BlaP is folded or not, whereas the 55Q chimera aggregates into amyloid-like fibrils only if BlaP is unfolded. The threshold value for amyloid-like fibril formation depends, therefore, on the structural integrity of the β-lactamase moiety and thus on the steric and/or conformational constraints applied to the polyQ tract. These constraints have, however, no significant effect on the propensity of the 79Q tract to trigger fibril formation. These results suggest that the influence of the protein context on the aggregating properties of polyQ disease-associated proteins could be negligible when the latter contain particularly long polyQ tracts.

Camelid single-domain antibody fragments: Uses and prospects to investigate protein misfolding and aggregation, and to treat diseases associated with these phenomena

“…Deletion of one of the oligomerisation domains prevents the pathological aggregation of the mutant form of PABPN1 containing 17 alanines (mPABPN1-Ala17) into KCl-resistant aggregates [130,131]. This observation indicates that, like for the aggregation of proteins containing a polyglutamine tract, the regions outside the alanine repeat have their own role to play [132] and suggests a multi-domain aggregation mechanism. Differences in the aggregation kinetics and/or pathway by the wild-type and mPABPN1 could explain their differences in toxicity [133].…”

1H, 13C and 15N backbone resonance assignments of the β-lactamase BlaP from Bacillus licheniformis 749/C and two mutational variants

“…BlaP has been chosen as a protein scaffold because: (i) it is a relatively small and stable protein (~ 30 kDa), readily overexpressed in E.coli (Huynen et al 2013); (ii) its threedimensional structure is well-characterized: the protein is composed of two structural domains (an α domain and an α/β domain) at the interface of which the catalytic site is located; and most importantly, (iii) it tolerates peptide insertions at several positions (Vandevenne et al 2007;Scarafone et al 2012;Huynen et al 2013).…”

“…The correspondence between the two numbering systems is given in Table 1. The ABL numbering has been previously used to describe the characterization of BlaP-based hybrid proteins (Vandevenne et al 2007;Scarafone et al 2012;Huynen et al 2013Huynen et al , 2015 and is therefore used here in the introduction and methods sections; in the remainder of the paper dealing with NMR, only the sequence number (underlined number) will be used. Note also that the BlaP protein used to create the polyQ chimeras is the secreted form following complete N-terminal proteolytic processing and therefore starting at position 27 (Vandevenne et al 2007) (Table 1).…”

“…between sequences coding for the amino acids 197-198 or 216-217) (Scarafone et al 2012;Thorn et al in preparation) (Table 1). The insertion of this restriction site at the gene level leads to translation of the dipeptide proline-glycine at position 197 and 216 respectively; these proteins are referred to as BlaP197Q0 and BlaP216Q0.…”

“…The aggregation properties of the BlaP polyQ-chimeras are characteristic of proteins associated with polyQ diseases (Scarafone et al 2012;Thorn et al in preparation); namely: (i) there is a Q-threshold length above which the polyQ sequence triggers the aggregation of proteins into amyloid fibrils, and (ii) above this threshold, the longer the polyQ sequence, the faster the aggregation. Moreover, the aggregation propensity of BlaP-polyQ chimeras depends on the location of the polyQ sequence within BlaP.…”