Poster Presentation 49th Lorne Conference on Protein Structure and Function 2024

The tumour suppressor protein p16ink4a forms fully redox-controlled, reversible amyloids (#411)

Christoph Goebl 1 2 , Vanessa K. Morris 2 3 , Alexander K. Buell 4 , Julia Horsfield 5 , Sarah G. Heath 1 , Shelby G. Gray 3 , Emilie M. Hamzah 3 , Karina M. O'Connor 1 , Stephanie M. Bozonet 1 , Pierre de Cordovez 1 , Aakriti Sethi 1 , Alex Botha 1 , Briana R. Smith 1 , Abigail Schwartfeger 3 , Hannah Darroch 5 , Emma Kloss 1
  1. Mātai Hāora - Centre for Redox Biology and Medicine, University of Otago Christchurch, Christchurch, CANTERBURY, New Zealand
  2. Biomolecular Interaction Centre, University of Canterbury, Christchurch, Canterbury, New Zealand
  3. School of Biology, University of Canterbury, Christchurch, Canterbury, New Zealand
  4. Department of Biotechnology and Biomedicine, Technical University of Denmark, Lyngby, Denmark
  5. Department of Pathology at the University of Otago, University of Otago, Otago, Dunedin, New Zealand

Amyloid fibrils are beta-sheet based protein aggregates that can be formed by a range of different proteins. Various factors contribute to the conversion from the native species to the amyloid which complicates detailed studies on the underlying mechanisms.

We recently reported that the tumour suppressor protein p16ink4a is a stable, monomeric protein that rapidly converts into amyloid when oxidized. This transition takes place through the oxidation of a cysteine residue, leading to formation of a dimeric species that subsequently folds into amyloid (1).

Here, we present that the amyloid state of p16ink4a is controlled by oxidation and reduction of the cysteine. This creates a uniquely regulated amyloid system whose structural state can easily be switched through a simple redox treatment. By studying multiple single-point variants that slow or accelerate different stages of the transition, we have characterised the fibril formation and show that it is strongly correlated with the stability of the monomer (2).

The main cellular function of p16ink4a is to inhibit cyclin-dependent kinases (CDK4/6). Monomeric p­16ink4a tightly binds to CDK4/6, but we show that the amyloid structure loses its inhibitory effect. When reduced, p16ink4a disassembles into monomeric species that fully regain CDK4/6 inhibition, suggesting that the kinase regulation is fully redox-controlled via the amyloid transition. We are further investigating p16ink4a amyloids in cell cultures, where we find that the protein structural state changes during the different stages of the cell cycle. To investigate details of the potential functional roles of the amyloid state, we also study protein homologues in other organisms. We have further generated a zebrafish knock-out model, where we rescue the function with variants of p16ink4a that harbour intact or impaired fibril formation ability.

Overall, this work unravels the underlying mechanism and function of a novel oxidation-induced amyloid fibril system.

 

  1. Göbl, C. et al. Cysteine oxidation triggers amyloid fibril formation of the tumor suppressor p16INK4A. Redox Biology 28, 101316 (2020)
  2. Heath, S. G. et al. Amyloid formation and depolymerization of the tumor suppressor protein p16INK4a is strictly controlled by an oxidative thiol-based mechanism. Under review