Robert Walters, PhD 2011

Thesis Title: Mechanistic Insights into the Aggregation of Polyglutamine Peptides

Abnormally expanded polyglutamine domains are associated with at least nine neurodegenerative diseases, including Huntington's disease. Expansion of the glutamine region facilitates aggregation of the impacted protein into neuronal inclusions, and aggregation has been linked to neurotoxicity. Additionally, a strong inverse correlation exists between the length of the expanded polyQ domain and the age of onset of symptoms for each disease. In the proteins implicated in polyQ diseases, no known homologies exist outside the expanded polyQ region. Due to the strong link between expanded polyQ regions, aggregation, and cellular toxicity, many studies have probed the aggregation properties of expanded polyQ regions. Both proteins and peptides containing expanded polyQ domains have been employed as model systems to better understand the connection between expanded polyQ domains and aggregation.

Studies of the aggregation of synthetic polyQ peptides have led to the proposal of a mechanism of aggregation based on a nucleation elongation (NE) model which has been widely adopted by the field. In this mechanism, a β-sheet rich, thermodynamically rare conformation of monomer serves as a nucleus of aggregation, which is elongated by monomer which consolidates to the structure of the nucleus, resulting in the formation of fibrillar aggregates. However, the results of recent experiments and computational simulations have called a number of aspects of this mechanism into question. Therefore, this study sought to establish a better mechanistic understanding of polyQ peptide aggregation, especially with regard to how peptide length and structure influence aggregation behavior.

To examine the impact of polyglutamine length on aggregation, peptides containing 8 to 24 glutamines were synthesized, and their conformational and aggregation properties were examined. All peptides possessed disordered secondary structure. Fluorescence resonance energy transfer (FRET) studies revealed that the peptides became increasingly collapsed as the number of glutamine residues increased. A comparison of our data with theoretical results suggests that phosphate-buffered saline is a good solvent for Q8 and Q12, a theta solvent for Q16, and a poor solvent for Q20 and Q24. By dynamic light scattering, we observed that Q16, Q20 and Q24, but not Q8 or Q12, immediately formed soluble aggregates upon dilution into phosphate buffered saline at 37°C. Thus, Q16 stands at the transition point between good and poor solvent, and between stable and aggregation-prone peptide. Examination of aggregates by transmission electron microscopy, along with kinetic assays for sedimentation, provided evidence indicating that soluble aggregates mature into sedimentable aggregates. Together, the data support a mechanism of aggregation in which monomer collapse is accompanied by formation of soluble oligomers; these soluble species lack regular secondary structure but appear morphologically similar to the sedimentable aggregates into which they eventually mature. We have termed this mechanism the association conformational conversion (ACC) mechanism.

The impact of peptide structure on polyQ peptide aggregation was examined by inserting interrupting residues into a Q20 peptide. A peptide with 2 alanine residues formed laterally-aligned fibrillar aggregates that were similar to the uninterrupted Q20 peptide, indicating interruption of the glutamine region per se does not alter aggregation behavior. 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 D PG rapidly accelerated aggregation and resulted in a fibrillar aggregate morphology that lacked the lateral alignment between fibrils observed in Q20. Consistent with the ACC mechanism, these results indicate nonspecific interactions between glutamines lead to the formation of soluble oligomers, while insoluble fibrils form following an increase in β-sheet content and dehydration. Additionally, soluble oligomers dynamically interact with each other, while insoluble fibrils are relatively inert. Further, kinetic analysis revealed that the increase in aggregation caused by the D PG insert is inconsistent with the NE mechanism of aggregation featuring a monomeric β-sheet nucleus.

The elongation of polyQ fibrils by a variety of polyQ peptides was examined using quartz crystal microbalance with dissipation monitoring and optical waveguide lightmode spectroscopy. Results indicated that longer polyQ peptides elongate fibrils more rapidly than shorter polyQ peptides. Peptide structure also played an important role in fibril elongation. The peptide with the D PG insert was able to efficiently elongate polyQ fibrils with no change in the structure of the growing fibril. Q20 and Q24 were also able to elongate polyQ fibrils, but the structure of the elongated fibrils varied as they grew, resulting in fibrils that were more susceptible to bending. Additionally, fibrils elongated by the peptide with the D PG insert were found to have little water associated with the growing structure, while fibrils elongated by Q20 contained much more water. Consistent with the ACC mechanism, the structure of the fibril can vary as it elongates, and dehydration of newly added monomer is a feature of fibril maturation.

The research presented in this dissertation has a broad impact on the understanding of polyQ-mediated aggregation. For polyQ peptides, the ACC mechanism provides a practical explanation of the length dependence of polyQ peptide aggregation, accounts for the presence of soluble oligomers, and can explain the toxicity of soluble oligomers compared to insoluble fibrils. Therefore, the association conformational conversion mechanism represents a significant improvement over the nucleation elongation mechanism of polyQ peptide elongation.