Eligible Student Poster 49th Lorne Conference on Protein Structure and Function 2024

Unravelling the functional impact of the zebrafish p16INK4a transition into amyloid fibrils (#128)

Aakriti Sethi 1 , Hannah Darroch 2 , Nick Magon 1 , Briana Smith 1 , Julia Horsfield 2 , Vanessa Morris 1 3 , Christoph Goebl 1 3
  1. Centre for Redox Biology and Medicine, University of Otago Christchurch, Christchurch, New Zealand
  2. Department of Pathology , University of Otago , Dunedin , New Zealand
  3. Biomolecular Interaction Centre , University of Canterbury , Christchurch, New Zealand

The continuous cell division in mammals is modulated by cell cycle regulators such as cyclins and the cyclin-dependent kinases (CDKs). They promote phosphorylation of Rb (Retinoblastoma) and the release of E2F transcription factors to enable transcription of genes essential for cell division. However, this complex is inactivated by the INK4 family of inhibitor proteins, whose best studied example is the tumour suppressor p16INK4a1,2.

Recent evidence suggests that oxidation of the human p16INK4a protein leads to a disulphide-dependent dimer that subsequently forms β-sheet based amyloids fibrils. There is a considerable amount of literature on association of amyloids with neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. However, the characteristics of oxidation induced p16INK4a amyloids, such as rapid formation, low monomer stability, and transition at physiological conditions, led us to explore their potentially functional nature rather than a disease related transition3.

This project studies the homologue INK4 family of Danio rerio (zebrafish). We recombinantly expressed zebrafish p18 protein and explored its propensity to form amyloid. The protein harbours two cysteine residues which upon oxidation forms an intra-molecular disulphide bond that unfolds the protein but locks it in a monomeric state. In contrast, a single cysteine variant is highly prone to oxidation and rapidly folds into amyloid. We characterized this transition using several methods including thioflavin-T fluorescence measurements and solution nuclear magnetic resonance spectroscopy.

We are also using Danio rerio (Zebrafish) as a model organism where we generated INK4A knockout variants using Crispr-Cas9 for investigation of this transition in in vivo studies.

This study aims to reveal a potential functional role of oxidation-induced amyloid formation.

 

References:

  1. Weintraub, S. J., Prater, C. A. & Dean, D. C. Retinoblastoma protein switches the E2F site from positive to negative element. Nature 358, 259–261 (1992).
  2. Kim, W. Y. & Sharpless, N. E. The Regulation of INK4/ARF in Cancer and Aging. Cell 127, 265–275 (2006).
  3. Göbl, C. et al. Cysteine oxidation triggers amyloid fibril formation of the tumor suppressor p16INK4A. Redox Biol. 28, 101316 (2020).