Amyloid formation and depolymerization of tumor suppressor p16INK4a are regulated by a thiol-dependent redox mechanism.

The conversion of a soluble protein into polymeric amyloid structures is a process that is poorly understood. Here, we describe a fully redox-regulated amyloid system in which cysteine oxidation of the tumor suppressor protein p16INK4a leads to rapid amyloid formation. We identify a partially-structured disulfide-bonded dimeric intermediate species that subsequently assembles into fibrils. The stable amyloid structures disassemble when the disulfide bond is reduced. p16INK4a is frequently mutated in cancers and is considered highly vulnerable to single-point mutations. We find that multiple cancer-related mutations show increased amyloid formation propensity whereas mutations stabilizing the fold prevent transition into amyloid. The complex transition into amyloids and their structural stability is therefore strictly governed by redox reactions and a single regulatory disulfide bond.

Characterizing the amyloid core region of the tumor suppressor protein p16INK4a using a limited proteolysis and peptide-based approach.

The human tumor suppressor p16INK4a is a small monomeric protein that can form amyloid structures. Formation of p16INK4a amyloid fibrils is induced by oxidation which creates an intermolecular disulfide bond. The conversion into amyloid is associated with a change from an all α-helical structure into ß-sheet fibrils. Currently, structural insights into p16INK4a amyloid fibrils are lacking. Here, we investigate the amyloid-forming regions of this tumor suppressor using isotope-labeling limited-digestion mass spectrometry analysis. We discover two key regions that likely form the structured core of the amyloid. Further investigations using thioflavin-T fluorescence assays, electron microscopy, and solution nuclear magnetic resonance spectroscopy of shorter peptide regions confirm the self-assembly of the identified sequences that include methionine and leucine repeat regions. This work describes a simple approach for studying protein motifs involved in the conversion of monomeric species into aggregated fibril structures. It provides insight into the polypeptide sequence underlying the core structure of amyloid p16INK4a formed after a unique oxidation-driven structural transition.