Abstract
The tumour suppressor protein p16ᴵᴺᴷ⁴ᵃ is a master regulator of cell proliferation. Frequently observed mutations have been associated with cancer initiation and progression. p16ᴵᴺᴷ⁴ᵃ regulates the cell cycle through the inactivation of downstream effectors of the retinoblastoma pathway. This slows cell division by inhibiting the progression of the cell cycle from the G1- to the S-phase. Recently, it was suggested that oxidation triggers the formation of an amyloid fibril state of p16ᴵᴺᴷ⁴ᵃ which has been proposed to eliminate p16ᴵᴺᴷ⁴ᵃ’s ability to directly inhibit cyclin-dependent kinases 4/6. Amyloid fibrils are typically composed of monomeric proteins that have aggregated by adopting a characteristic cross-β-sheet structure, which have been associated with a great variety of human diseases, including Alzheimer’s and type-II diabetes.
Here we explored the plausibility of fluorescent protein-labelled p16ᴵᴺᴷ⁴ᵃ to allow for the characterisation of p16ᴵᴺᴷ⁴ᵃ’s conversion to the amyloid state in a cellular environment. To validate the viability of this approach, we confirmed that p16ᴵᴺᴷ⁴ᵃ coupled with an enhanced green fluorescent protein (eGFP) is still able to form amyloids upon oxidation. Just like wild-type p16ᴵᴺᴷ⁴ᵃ, a disulfide-dependent dimerisation of p16ᴵᴺᴷ⁴ᵃ-eGFP leads to a dramatic structural rearrangement and forms aggregates that have characteristic features of amyloid fibrils, including binding of diagnostic dyes and typical dimensions found in electron microscopy. Utilising fluorescence microscopy and ectopic expression we were able to study the impact of oxidation of p16ᴵᴺᴷ⁴ᵃ-eGFP in a cellular environment. We observed a fast aggregation process of p16ᴵᴺᴷ⁴ᵃ-eGFP upon oxidation, suggesting a potential functional role of the protein oxidation event.