Abstract
The human tumour suppressor protein p16 negatively regulates cell division. This protein is therefore one of the main factors necessary to prevent tumour formation, with its inactivation observed in numerous types of cancers. It was recently discovered that oxidation causes the monomeric p16 protein to form amyloid fibrils through a unique structural change mechanism. Here, I present the first characterisation of these large aggregate amyloid structures, which represent a novel loss-of-function state. The structural differences between the alpha-helical monomeric and the beta-sheet based amyloid state were probed using a limited digestion mass spectrometry approach. This technique utilises an enzyme to partially digest a protein sample for varying lengths of time, allowing the identification of particular regions in the sequence that displayed different surface accessibility to the enzyme between the monomer and amyloid conformations. Heavy isotope labelled p16 protein was also expressed to enable monomeric and amyloid samples to be combined and simultaneously analysed, reducing experimental variation and therefore increasing accuracy and confidence in the mass spectrometry results.
Through this method, I gained information on the specific peptide regions that are key to the amyloid fibril structure. These identified peptide regions from the limited digest mass spectrometry technique were subsequently tested for their ability to form amyloids themselves using Thioflavin T assays. This combined approach enabled the discovery of the peptide regions, including a proposed novel dual motif, that seems involved in p16 amyloid formation. Despite some tumour diagnosis techniques already utilising p16 presence, these first insights into the unique p16 oxidation-induced amyloid mechanism system may contribute to improving diagnostic methods. The long-term aim is to make the amyloid state of p16 measurable and potentially improve cancer diagnoses for patients.