p16ink4a is a tumour suppressor protein that blocks the G1/S phase transition in the cell cycle. We recently reported that p16ink4a, can undergo rapid structural rearrangement from a small, alpha-helical protein into amyloid fibrils [1]. The onset of this transition is triggered by an oxidation event where a cysteine disulfide bond formation is leading to homodimers that subsequently fold into amyloid.
We find that the amyloid formation and stability rely on the presence of the disulfide bond and addition of reducing agents leads to disassembly of the amyloids. When testing different physiological oxidants, only some lead to the transition into amyloid whereas others do not trigger fibril formation. This suggests that the structural conversion is favoured by a specific oxidation mechanism.In a cellular environment, our oxidation experiments of p16ink4a suggest that the amyloid state is a type of loss-of-function state because the protein can no longer inhibit cyclin-dependent kinases to block the G1/S phase transition during cell division. When localizing the oxidation-induced protein aggregates, we observe a rapid re-localization in specific cellular compartments.
p16ink4a is amongst the most mutated proteins in cancer and studying the amyloid transition of reported single-point cancer mutations shows greatly increased propensity for the amyloid state. In contrast, introduction of single mutations that stabilize the fold inhibit transition into amyloid fibrils, highlighting that individual amino acid contributions greatly control the protein state.
Here, we present a novel pathway of amyloid formation that can be strictly triggered by oxidation. Individual mutations have a dramatic impact on the ability to form amyloids and this transition might constitute a novel loss-of-function mechanism.