The redox-driven structural transition of a cell cycle regulator protein in zebrafish

In mammals, cell division is driven by the cyclin–CDK complex, whose activity is tightly controlled by cyclin-dependent kinase inhibitors. In human, the INK4 family of proteins represents a group of inhibitors. 

It was recently demonstrated that oxidation of a cysteine residue in human INK4 protein called p16^INK4a induces the transition into amyloid structures. While amyloids are typically associated with neurodegenerative diseases such as Alzheimer’s and Parkinson’s, the amyloid formation observed for p16^INK4a occurs under physiological conditions and is reversible upon reduction of a disulfide bond, suggesting that this transition may represent a functional property rather than a pathological event. 

This study focuses on the zebrafish (Danio rerio) ink4 member P18 (drP18) and investigates the impact of oxidation on the protein. In contrast to the reported human p16^INK4a, drP18 is the only INK4 family member in both zebrafish and humans that contains two cysteine residues (C50 and C128). This distinctive composition raises the possibility of multiple oxidation-induced modifications and alternative structural outcomes. 

Exposure of recombinant drP18 to different oxidants such as diamide, hydrogen peroxide (H₂O₂), or peroxymonocarbonate (HCO₄^-) primarily resulted in the formation of an intramolecular disulfide bond, blocking the protein in a compact monomeric state. In contrast, treatment with hypothiocyanous acid (HOSCN) at a defined concentration promoted a mixed response, including the formation of intermolecular dimers that subsequently aggregated into large amyloid-like species. 

Mutational analyses showed that while the drP18 C50S variant readily formed dimers and ThT-positive aggregates, the drP18 C128S variant remained largely unreactive. The C50 residue of drP18 was found to undergo glutathionylation, and this modification was sufficient to trigger formation of ThT-positive aggregates upon oxidation, highlighting the role of C50 as the redox regulator in drP18.

To assess the potential biological relevance, a cdkn2c-/- (drP18) knockout zebrafish line was generated. The knockout fish line developed normally and was viable and fertile, indicating that loss of drP18 does not cause overt developmental defects under basal conditions. However, RNA-seq analysis of four-day-old larvae revealed transcriptional alterations in circadian rhythm and metabolic pathways, suggesting subtle physiological effects of drP18 depletion. In addition, functional rescue experiments were performed in both wild-type and knockout embryos, with either a wild-type or a double-cysteine mutant drP18 mRNA. Body length measurements showed no significant phenotypic differences between wild-type and cysteine-mutant rescues. However, western blot analysis of embryo lysates detected potential dimeric drP18 species in embryos, a potential building block of amyloid structures. The consistent presence of dimers in injected embryos suggests that basal oxidative stress during early development may be sufficient to promote drP18 oxidation in vivo. Furthermore, complementary assays in established zebrafish embryonic cell cultures showed that oxidant exposure reduced cellular viability, and highlighting the susceptibility to oxidants.

In summary, drP18 is likely redox-active, showing a complex interplay between its two cysteine residues, which respond differently under oxidising conditions. This behaviour reveals a unique redox-based regulatory mechanism within the INK4 family, offering new insight into how subtle chemical changes can influence protein structure and function.