Abstract
Amyloids represent a distinct protein state characterised by the self-assembly of polypeptides into highly ordered β-sheet–rich structures, which have been linked to a broad spectrum of pathologies. This study focuses on one amyloid-prone protein, p16INK4A (p16) that has been shown to possess a unique aggregation mechanism. Unlike most amyloidogenic proteins, p16 undergoes a strictly redox-controlled aggregation pathway, wherein oxidation of its single cysteine residue triggers dimerization and subsequent fibril formation. Remarkably, these fibrils can be fully depolymerized back to the functional monomeric state upon addition of reducing agent, providing a reversible system to probe amyloid formation.
Using the strictly inducible properties of p16 amyloid formation, this study investigates the mechanistic pathway of protein aggregation, primarily by ultracentrifugation. The presence of a previously proposed stable dimeric intermediate was further confirmed, and a novel intermediate species with a sedimentation coefficient of approximately 13 S was identified. This highly stable species delineates the boundary between smaller transient oligomers and the larger, stable amyloid assemblies, which lead to the creation of an aggregation model in which this oligomeric nucleus seeds fibril elongation.
Further structural investigations revealed an unexpected role for the flexible C-terminal tail of p16 in regulating aggregation kinetics. Truncation of this C-terminal region accelerated the amyloid formation, and an NMR study revealed its interaction with other regions of the protein, including the loop harbouring the reactive cysteine residue. These findings highlighted the importance of intramolecular interaction outside the amyloid core in controlling protein aggregation.
Beyond mechanistic studies, this work addresses the functional implications of p16 aggregation. Amyloid formation abolishes the ability of p16 to inhibit CDK4/6, potentially impacting its role as a tumor suppressor. To selectively detect p16 amyloids in cellular contexts, a conformation-specific nanobody was developed, which binds preferentially to amyloid fibrils without interacting with the monomer.
Finally, the influence of microgravity on p16 aggregation was explored during an experiment aboard the International Space Station. Despite the potential for residual cosmic radiation to generate reactive oxygen species and trigger amyloid formation, only limited dimer formation was observed. Nevertheless, aggregates with distinct morphologies from conventional p16 fibrils were detected, and these structures lacked the ability to inhibit CDK4/6 activity, suggesting altered functional properties.
Overall, this thesis advances our understanding of p16 amyloid formation by elucidating the role of two discrete intermediate species. These key elements allowed us to propose an aggregation model, that may encompass other amyloid forming proteins and therefore broaden the understanding of these critical protein aggregates. The development of a conformation-specific nanobody provides a versatile tool for detecting amyloid species in complex biological environments, with implications for studying the physiological and pathological roles of p16 aggregation.