Tumour suppressors are proteins that are critical in preventing the development of cancers. They protect the cell by blocking cell division and cell growth, and they are therefore frequently mutated in all types of cancers. One important family of tumour suppressors is the INK4 inhibitors. p18INK4c is a tumour suppressor protein of the INK4 family that is associated with many lethal blood and brain cancers. p18INK4c specifically inhibits cyclin-dependent kinase (CDK) 4/6-cyclin-D complexes at the G1 to S phase transition to arrest cell division.
We have discovered that under oxidizing conditions, p18INK4c undergoes homo-dimerization by the formation of a disulfide bridge via its cysteine amino acid (C141). p18INK4c is an all α‑helical protein, however, upon cysteine-dependent dimerization, p18INK4c can undergo structural changes into amyloid fibrils with an underlying cross β-sheet structure. Amyloid fibrils is a specific kind of protein aggregate that are most often associated with neurodegenerative diseases. This is one of the first known cases of a cancer-related protein that can form amyloid fibrils and oxidation-induced amyloid formation is a novel mechanism.
We have characterized the p18INK4c oxidation-induced structural transition using purified protein combined with biophysical methods, including thioflavin-T fluorescence assays, transmission electron microscopy and gel electrophoresis. We find that oxidation occurs under relatively mild oxidizing conditions at physiological pH and temperature. HEK293 cells have been used as a cellular model to further characterise p18INK4c oxidation. These combined methods provide mechanistic details and kinetic insights into this unique protein transformation.
This novel thiol-based oxidant-driven amyloid fibril formation of p18INK4c introduces a new concept to tumour biology, establishing the importance of amyloid fibril formation in the INK4 family of tumour suppressors.