The tumour suppressor protein p16ᴵᴺᴷ⁴ᵃ plays an executional role in cell proliferation through inhibition of cyclin-dependent kinases 4/6 (CDK4/6), thereby preventing the progression of the cell cycle from the G1- to S-phase. This central role of cell cycle regulation makes p16ᴵᴺᴷ⁴ᵃ amongst the most frequently mutated proteins in cancer. We recently discovered that p16ᴵᴺᴷ⁴ᵃ accumulates under conditions of oxidative stress and converts into an amyloid fibril structure. This unique mechanism allows the induction of an α-helical monomer to a β-sheet based amyloid transition. Importantly, the amyloid state of p16ᴵᴺᴷ⁴ᵃ is unable to inhibit CDK4/6, suggesting a novel loss-of-function amyloid phenotype.
Here we describe the use of fluorescence methods to study the accumulation of p16ᴵᴺᴷ⁴ᵃ in cells during physiological conditions of oxidative stress. Oxidising conditions lead to disulphide-dependent dimerization of p16ᴵᴺᴷ⁴ᵃ in cells and the protein undergoes a spatial rearrangement into fast-forming aggregates. The absence of these p16 aggregates from typical degradation compartments, such as the nucleolus or lysosome, may suggest a functional role of this protein oxidation event in cells. This is further supported by the structural rearrangement of p16ᴵᴺᴷ⁴ᵃ observed during different phases of the cell cycle. During the S-phase, p16ᴵᴺᴷ⁴ᵃ forms aggregates amongst itself without the addition of oxidant. This unique system may be a form of functional control mediating the different stages of the cell cycle. This study illuminates the structural flexibility of an important tumour suppressor protein and paves the way for further functional analysis of this transition.