Amyloids are protein aggregates that display a characteristic cross β-sheet structure, and several well-characterized examples form the hallmark of neurodegenerative diseases. We recently discovered that the tumour suppressor protein p16INK4A can form amyloids under mild oxidative conditions1. This protein plays a critical role in tumour proliferation and has been found to be frequently mutated in many cancers.
Unlike the great majority of amyloid-prone proteins, such as the well-defined amyloid beta or α-synuclein, p16INK4A cannot spontaneously fold into amyloids. Instead, we have found that the amyloid conversion is initiated by oxidation and the resulting disulfide-bond dependent dimerization of the protein2.
We use this strictly inducible system as a model for studying amyloid fibril growth by monitoring the entire transition, starting from the stable, alpha-helical monomeric protein via the dimeric species to higher order and the fully-grown amyloids. Using a combination of biophysical and biochemical techniques including fluorescence spectroscopy, small-angle X-ray scattering, nuclear magnetic resonance, and analytical ultracentrifugation (AUC), we further aim to gain insights into the intermediate, oligomeric species arising in the amyloid formation course.
Our AUC data show that early-stage oligomers are short-lived, as they fold quickly into larger species. We further identified several mutants that alter the stability of individual species, enabling us to gain insights into lowly-populated and elusive oligomers.