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

The human tumor suppressor p16IN1(4a is a small monomeric protein that can form amyloid structures. Formation of p16IN1(4a amyloid fibrils is induced by oxidation which creates an intermolecular disulfide bond. The conversion into amyloid is associated with a change from an all c6-helical structure into (3-sheet fibrils. Currently, structural insights into p16IN1(4a amyloid fibrils are lacking. Here, we investigate the amyloidforming regions of this tumor suppressor using isotopelabeling 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 p16IN1(4a formed after a unique oxidation-driven structural transition.