What a Difference a Letter Makes: Aggregation of PolyA and PolyQ

Polyglutamine (polyQ) and polyalanine (polyA) are two of the three most prevalent homopeptide repeats in eukaryotes. Abnormally expanded polyQ domains are associated with at least nine neurodegenerative diseases, including Huntington's disease. Abnormal expansion of the polyA repeat is linked to at least 9 human diseases, eight of which are congenital disorders. In both cases, expansion of the glutamine or alanine domain facilitates aggregation of the impacted protein, and the disease mechanism likely involves the aggregation triggered by the increase in length of the repeat unit. As with other aggregation-related disorders, soluble oligomers are suspected to serve as intermediates in the aggregation process, and to be more toxic than mature aggregates.

Expanded polyA diseases share some characteristics with the better-known expanded polyQ-mediated disorders, but there are distinct differences as well. All known polyQ diseases are late-onset disorders, while symptoms in all but one polyA disease appear at birth. The normal length of polyA, and the threshold expansion for disease phenotype, tend to be smaller than polyQ (normally 9-20 for polyA with +1-14 expansion, compared to 4-44 with +1-271 expansion for polyQ). Some evidence suggests that expansion of polyA may be more damaging than expansion of polyQ.

Studies of synthetic peptides have contributed substantially to our understanding of the mechanism of aggregation. We have previously presented work in which we examined the length-dependent aggregation of polyQ peptides. In work presented in this talk, we inserted interrupting residues into a peptide containing 20 glutamines, and examined the impact on conformational and aggregation properties. A peptide with 2 alanine residues formed laterally-aligned fibrillar aggregates which were similar to the uninterrupted Q20 peptide. Insertion of 2 proline residues resulted in soluble, nonfibrillar aggregates, which did not mature into insoluble aggregates. In contrast, insertion of the β-turn template _DPG rapidly accelerated aggregation and resulted in a fibrillar aggregate morphology that lacked the lateral alignment between fibrils observed in Q20. These results were interpreted to indicate that (a) nonspecific interactions between glutamines lead to the formation soluble oligomers, while insoluble oligomers form following an increase in β-sheet content and dehydration, and (b) that soluble oligomers dynamically interact with each other, while insoluble oligomers are relatively inert. Kinetic analysis revealed that the increase in aggregation caused by the _DPG insert is inconsistent with the nucleation-elongation mechanism of aggregation featuring a monomeric nucleus. Rather, the data support a mechanism of polyglutamine aggregation by which monomer collapse drives formation of soluble oligomers, which then undergo slow structural rearrangement to form sedimentable aggregates.

We also synthesized polyA peptides containing 6 to 24 alanines, and characterized their length-dependent conformation and aggregation properties. Helical content increased with increasing length. Measurements of end-to-end distance demonstrated that physiological buffers are theta solvents for shorter polyA peptides and poor solvents for longer peptides.  At moderate concentrations, some soluble aggregates were observed in all peptides, with a sharp transition in the aggregate physical properties between 18 and 24 alanines. All aggregates were globular, none progressed to fibrillar morphologies, and none were insoluble, in sharp contrast to the behavior of polyglutamine peptides. The data suggest that, under physiologically relevant conditions, polyA peptides assemble into soluble oligomers due to hydrophobic collapse, but do not undergo structural rearrangement to form fibrils. The data were interpreted in light of a simple thermodynamic model, to explain why polyQ sometimes forms fibrils while polyA does not.

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