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 promotes the conversion of the α-helical monomer to a β-sheet based amyloid (1). Importantly, the amyloid state of p16ᴵᴺᴷ⁴ᵃ is unable to inhibit CDK4/6, suggesting a loss-of-function amyloid phenotype.
Here we describe the accumulation of p16ᴵᴺᴷ⁴ᵃ in human cell cultures 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. We further identified that these aggregates have a typical amyloid-like structure using the amyloidogenic dye Thioflavin-S. We show the absence of these p16ᴵᴺᴷ⁴ᵃ aggregates from typical degradation compartments, such as the nucleolus or lysosome, which may suggest a functional role of this protein conversion into amyloid.
A stabilising mutant of p16ᴵᴺᴷ⁴ᵃ, W15D, was found to slow the amyloid formation resulting in smaller aggregates, leaving majority of the p16ᴵᴺᴷ⁴ᵃ distributed throughout the cell. We further introduced three stabilising mutations to p16ᴵᴺᴷ⁴ᵃ, W15D, L37S and L121R, which abolished its ability to form amyloid-like aggregates within cells. Since a loss-of-function event may be occurring due to the amyloid phenotype, it would be vital to study these non-amyloid forming variants to understand the role amyloids may play in the progression of uncontrolled cell division by leaving p16ᴵᴺᴷ⁴ᵃ unable to inhibit CDK4/6.
This study illuminates the structural flexibility of an important tumour suppressor protein and paves the way for further functional analysis of this oxidation-based structural and functional transition.