Abstract
The tumour suppressor protein p16INK4a is a negative regulator of the cell division, inhibiting the cyclin dependent kinases 4 and 6 and preventing progression of the cell cycle. The loss of p16INK4a activity is widely believed to be a common and important event in the development of cancer. Amyloid fibrils are protein aggregates with a characteristic ß-sheet structure that are commonly associated with neurodegenerative diseases. Previous studies have found that oxidation of p16INK4a leads to the formation of disulfide-bridged dimers that subsequently fold into amyloid fibrils. While amyloid fibrils typically form spontaneously, p16INK4a is the first example of strictly oxidation-induced amyloid formation. Previous studies have highlighted the oxidation of p16INK4a by hydrogen peroxide, however, there are several reactive oxygen species present during physiological processes.
This thesis studies the impact of five physiological oxidants, including peroxymonocarbonate and hypothiocyanous acid, on the structural state of p16INK4a. The chemical modifications of p16INK4a that occur upon oxidation were investigated using mass spectrometry, and amyloid formation was monitored using a fluorescent diagnostic dye and electron microscopy. These effects were studied in detail using recombinantly expressed p16INK4a, where cysteine-based dimerisation and amyloid formation rates of p16INK4a were found to differ between physiological oxidants. In particular, peroxymonocarbonate, a product of hydrogen peroxide in the presence of bicarbonate, was found to induce dimerisation and subsequent amyloid formation of p16INK4a more efficiently than hydrogen peroxide itself. p16INK4a was then ectopically expressed in Jurkat cells and initial results indicate that hydrogen peroxide can induce the formation of p16INK4a dimer in a cellular environment. The oxidation induced conversion of p16INK4a into the amyloid state is a novel mechanism that could potentially lead to loss of function of p16INK4a as a tumour suppressor.