The Interplay between PolyQ and Protein Context Delays Aggregation by Forming a Reservoir of Protofibrils

Abstract

Polyglutamine (polyQ) diseases are inherited neurodegenerative disorders caused by the expansion of CAG codon repeats, which code for polyQ in the corresponding gene products. These diseases are associated with the presence of amyloid-like protein aggregates, induced by polyQ expansion. It has been suggested that the soluble aggregates rather than the mature fibrillar aggregates are the toxic species, and that the aggregation properties of polyQ can be strongly modulated by the surrounding protein context. To assess the importance of the protein carrier in polyQ aggregation, we have studied the misfolding pathway and the kinetics of aggregation of polyQ of lengths above (Q41) and below (Q22) the pathological threshold fused to the well-characterized protein carrier glutathione S-transferase (GST). This protein, chosen as a model system, is per se able to misfold and aggregate irreversibly, thus mimicking the behaviour of domains of naturally occurring polyQ proteins. We prove that, while it is generally accepted that the aggregation kinetics of polyQ depend on its length and are faster for longer polyQ tracts, the presence of GST alters the polyQ aggregation pathway and reverses this trend. Aggregation occurs through formation of a reservoir of soluble intermediates whose populations and kinetic stabilities increase with polyQ length. Our results provide a new model that explains the toxicity of expanded polyQ proteins, in which the interplay between polyQ regions and other aggregation-prone domains plays a key role in determining the aggregation pathway.

In-cell Aggregation of a Polyglutamine-containing Chimera Is a Multistep Process Initiated by the Flanking Sequence

“…Thus, the role of the polyQ stretch in the structure of the aggregate core ranges from peripheral in the early spherical detergent-soluble prefibrillar species to central in the second stage, the formation of detergent-resistant fibrils. Strikingly, our results on in-cell aggregation of this polyQ-containing chimeric system are entirely analogous to those observed for ataxin-3 in vitro in the Bottomley laboratory (22,23) and for a chimera of glutathione S-transferase and exon 1 of huntingtin (17). This emphasizes the potential general significance of the effect of polyQ segments on their neighboring domains and the resulting impact on the time-dependent character of aggregates and suggests that the complex aggregation mechanisms previously observed in vitro can also occur in the cellular environment.…”

“…In contrast, studies on full-length Htt exon 1 fragments have identified non-amyloid aggregates (5-7) in addition to mature amyloid. Moreover, studies of natural multidomain polyQ-containing proteins (22,23), as well as an artificial chimera like the one studied here (17), also reveal multistage aggregation with early prefibrillar species. In particular, the extensive recent studies of ataxin-3 aggregation in vitro have established that this multidomain protein follows a two-stage aggregation process with the first step dominated by the Josephin domain, and not the polyQ-rich domain (18,22,23).…”

“…This emphasizes the potential general significance of the effect of polyQ segments on their neighboring domains and the resulting impact on the time-dependent character of aggregates and suggests that the complex aggregation mechanisms previously observed in vitro can also occur in the cellular environment. As pointed out by Pastore and co-workers (17), such mechanistic insights are critical to understanding pathology, because the early, potentially more toxic prefibrillar aggregates could form in a variety of systems via indirect effects of elongated polyQ tracts.…”

“…Indeed, profound effects of polyQ context have been reported on aggregation propensity (17)(18)(19) and cytotoxicity (20,21). Based on their conclusion from in vitro results that polyQ extensions in ataxin-3 alter the conformation of the adjacent Josephin domain so as to favor its aggregation, Pastore and colleagues (19) proposed the "induced misfit" model, which argued for an indirect as well as a direct role of the polyQ sequence.…”

The Interaction of Polyglutamine Peptides with Lipid Membranes Is Regulated by Flanking Sequences Associated with Huntingtin

“…Flanking sequences directly adjacent to the poly(Q) domain within htt strongly influence aggregation (8,9). The first 17 amino acids of htt exon1 (Nt17) has been shown to promote oligomer formation in synthetic poly(Q) peptides (10,11), and targeting the Nt17 domain is an effective way of altering the aggregates formed and the kinetics of formation (10,12).…”