(157a) 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.