The tumor suppressor protein p16INK4a (p16) shows a unique behavior in transitioning between structured monomeric and amyloid states through oxidative changes [1]. This redox-sensitive transformation is initiated by the oxidation of a single cysteine residue, resulting in the formation of a disulfide-bonded dimer that shifts p16 from its α-helical structure to a β-sheet amyloid conformation. We find that this process is reversible and reducing agents disassemble the amyloid, allowing p16 to revert to its original monomeric state.
We provide detailed insights into this novel redox-regulated structural switch using a combination of biophysical techniques and observations of amyloid formation in human cell culture and Danio rerio (zebrafish). p16 is one of the most frequently mutated proteins in cancer, and our data indicate that various single-point variants transition into amyloids more rapidly than the wild-type protein. We also engineered stabilized p16 variants incapable of transitioning into amyloids to gain insights into the mechanism of this dramatic structural transition.
In cellular models, we observed that p16 aggregates rapidly into amyloids, forming dense foci that can be visualized through multiple imaging techniques. This redox-modulated switch impacts the role of p16 in cell cycle regulation, as the amyloid state lost its well-established CDK4/6 inhibitory function. Our studies include the generation of p16 knockout zebrafish, which display altered sensitivity to oxidative stress; and their phenotype was rescued by reintroducing wild-type p16. The cellular and in vivo experiments suggest a functional role for the structural flexibility of p16.
We recently extended these findings by performing a proteolytic mass-spectrometry approach which revealed a novel amyloid-forming motif within p16 [2]. Identification of homologous regions in other human proteins showed that they are also prone to oxidation-induced amyloid transitions.
Our data collectively highlight the existence of redox-switches in several ankyrin-repeat proteins, that significantly influencing their structure and function.