p16INK4a is a tumour suppressor protein that regulates cell cycle progression by binding to and inhibiting the cyclin-dependent kinases 4 and 6 (CDK4/6). It is described as a tumour suppressor as it induces cell cycle arrest and senescence in response to oncogenic transformation and it’s function is therefore frequently lost in cancer.
p16 is a 16 kDa protein comprised of four ankyrin repeats joined by three loop regions. In loop 2 there is a single solvent-accessible cysteine residue located on the surface facing away from the CDK4/6 binding site. Recently, our group has discovered that this single cysteine in p16 is able to undergo oxidation under relatively mild oxidizing conditions which leads to disulfide-dependent homo-dimerization. This dimerization leads to subsequent structural rearrangement into amyloid fibrils. In this amyloid fibril state p16 is unable to perform its normal function as a CDK4/6 inhibitor1.
In order to further understand the mechanism of p16 amyloid fibril formation, as well as to gain a better understanding of how p16 is inactivated in cancer, we have screened cancer-associated single-residue mutations. These p16 variants have been characterised using thioflavin-T aggregation assays, SDS-PAGE analysis of dimerization, electron microscopy and nuclear magnetic resonance (NMR) spectroscopy. Interestingly, most of these mutations result in a significant increase in the rate of dimer and/or fibril formation. Overall, our analysis has determined some of the key influences in p16 dimer and amyloid fibril formation.
In our current work we are exploring the impact of four stabilising mutations2 on dimer and amyloid fibril formation via NMR spectroscopy. This will aid in structural investigation of the short-lived dimer species and overall structure of p16 amyloid fibrils. Overall, this work will lead us to a greater understanding of the mechanism by which these unique aggregate species form and their role in pathogenic processes.