Oxidation of the tumour suppressor p15INK4b triggers aggregation into an amyloid-like state

The human protein p15^INK4b regulates the cell cycle, making it critical for the suppression of tumour development. Here I aimed to investigate how oxidation alters p15^INK4b structure and function. Our lab has previously shown that monomeric p16^INK4a, a homologue to p15^INK4b, undergoes a thiol-dependent oxidation mechanism to form amyloid fibrils. These are protein aggregates that are characterised by their cross-beta secondary structure and fibrillar morphology. p15^INK4b and p16^INK4a both include a conserved cysteine residue, the critical amino acid which drives the transition of p16^INK4a into the amyloid state. I investigated p15^INK4b amyloid fibril structures and formation kinetics using a range of biophysical techniques with recombinant protein. I also investigated ectopically expressed p15^INK4b in human cell line models with fluorescence microscopy and amyloid-specific dyes. I found that recombinant p15^INK4b forms amyloid fibrils faster than its counterpart, p16^INK4a. Cellular models also showed localised aggregation of p15^INK4b which bound to amyloid-specific dyes and also formed at a faster rate than p16^INK4a. Overall, this work shows that oxidation-driven fibril formation is a common mechanism within the family of INK4 tumour suppressor proteins. It is conceivable that the aggregation of p15^INK4b into amyloid fibrils could be a pathological misfold contributing to cancer progression, or a functional mechanism that is exploited for oncogenic consequences.