Abstract
p16ᴵᴺᴷ⁴ᵃ is a small, monomeric protein that acts as a checkpoint mediator in the cell cycle by inhibiting cyclin-dependent kinases 4/6 thereby preventing cell division. It was previously found that oxidation of p16ᴵᴺᴷ⁴ᵃ triggers a structural rearrangement to an amyloid state, which abolishes its ability to inhibit cyclin-dependent kinases 4/6.
The conversion of soluble proteins into highly-ordered amyloid structures is not yet well understood but it is known to play a key role in a variety of diseases, such as Alzheimer’s disease and Type-II diabetes. In a unique mechanism, the oxidation of p16ᴵᴺᴷ⁴ᵃ was reported to lead to the formation of disulfide-dependent dimers that subsequently fold into amyloids. Typical amyloid fibrils form spontaneously, but p16ᴵᴺᴷ⁴ᵃ appears to be the first example of oxidation-induced amyloid formation. Previous studies describe the amyloid formation of p16ᴵᴺᴷ⁴ᵃ using recombinant protein, however, this transition is not explored within a cellular environment.
This thesis describes the impact of the physiological oxidant hydrogen peroxide on p16ᴵᴺᴷ⁴ᵃ within human embryonic kidney cells. Here, it was found that overexpressed wild-type p16ᴵᴺᴷ⁴ᵃ rapidly forms localised deposits upon exogenous oxidant addition to cells. These deposits were identified to be composed of higher-order aggregates and are preceded by disulfide-dependent dimers of wild-type p16ᴵᴺᴷ⁴ᵃ that form upon oxidation. Additional experiments performed with purified protein revealed that increased thermostability of p16ᴵᴺᴷ⁴ᵃ through single-point-mutations appear to lower or abolish amyloid fibril formation. Further exploring this in a cellular environment, it was identified that increased thermostability of p16ᴵᴺᴷ⁴ᵃ slows the formation of deposits.
The oxidation-induced conversion of a protein into amyloid is a novel concept and this is the first study to explore the response of p16ᴵᴺᴷ⁴ᵃ to oxidants in a cellular environment.