The tumour suppresser protein p16INK4a plays an integral role in cell cycle regulation. P16INK4a prevents progression of the cell cycle from G1 to S phase by inhibiting the cyclin dependent kinases 4/6, thereby preventing inappropriate cell division. Loss of p16INK4a activity is widely believed to be a common and important event in the development of cancer and the protein is frequently over-expressed in aging cells. Oxidation of p16INK4a facilitates the formation of disulphide-linked homodimers that subsequently fold into amyloid fibrils. Amyloid fibrils are protein aggregates with a cross β-sheet structure that are most commonly associated with neurodegenerative diseases. While we have previously established that p16INK4a forms amyloid fibrils under oxidising conditions, it is not clear which physiological reactive oxygen species are able to induce the structural change from an α-helical protein into β-sheet based amyloids.
Here, we study the impact of five physiological oxidants, including peroxymonocarbonate and hypochlorous acid, on the structural state of p16INK4a. We studied these effects first using recombinantly expressed p16INK4a, and then translated these findings into the cellular environment of Jurkat cells. Rates of p16INK4a oxidation, dimerization and amyloid fibril formation were found to greatly differ between oxidants. These oxidant-dependent differences suggest that the transition of p16INK4a into the amyloid state may have physiological relevance. Mass spectrometry revealed the chemical modifications of p16INK4a that occur upon oxidation and the subsequent mechanism of dimerization. The oxidation induced conversion of the otherwise stable, α-helical p16INK4a monomer into the amyloid state is a novel mechanism that could potentially constitute a loss of function of p16INK4a as a tumour suppressor.