Hierarchical cysteine oxidation controls reversible amyloid formation in an ankyrin repeat protein
- 1University of Otago, Christchurch and University of Canterbury, Christchurch
Abstract
The formation of amyloids, including functional amyloids, is observed for an increasing number of proteins but the molecular mechanisms that control this structural transition remain poorly understood. Here we report that the kinase inhibitor protein P18 (drP18) from Danio rerio (zebrafish), which contains two cysteine residues, undergoes a complex and hierarchical redox switch that strictly governs reversible amyloid formation. We identify cysteine 50 (C50) acting as a regulatory residue. Upon oxidation, C50 forms an intramolecular disulfide bond with the executioner cysteine 128 (C128), thereby blocking it. C50 can become S-glutathionylated, and upon oxidation, C128 then forms intermolecular disulfides that lead to rapid transition into amyloid fibrils. S-glutathionylation of C50 therefore enables amyloid formation of drP18 and the outcome is oxidant-dependent with diamide, hydrogen peroxide, peroxymonocarbonate and hypothiocyanous acid each leading to amyloid assembly with distinct kinetics and morphologies. These amyloids are fully reversible, where disulfide reduction is leading to disassembly. Whereas monomeric drP18 inhibits CDK4-mediated retinoblastoma phosphorylation, the amyloid conformation abolishes this inhibition, and reduction restores both structure and function. Expression of drP18 in zebrafish embryos yields Congo red-positive, oxidation-dependent aggregates in vivo. Together, our findings show that a regulatory cysteine controls an executioner cysteine to induce reversible, functional amyloid formation, revealing that proteins can encode sophisticated mechanisms to control amyloid assembly.
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BibTeX
@misc{sethi2026hierarchical,
title = {{Hierarchical cysteine oxidation controls reversible amyloid formation in an ankyrin repeat protein}},
author = {A. Sethi and H. Darroch and N. J. Magon and G. Greene and K. M. O. Connor and A. D. Botha and S. G. Gray and P. d. Cordovez and R. Coldicott and J. Horsfield and V. K. Morris and C. Goebl},
howpublished = {bioRxiv},
year = {2026},
month = aug,
doi = {10.64898/2026.08.30.748053},
url = {https://www.biorxiv.org/content/10.64898/2026.08.30.748053v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Hierarchical cysteine oxidation controls reversible amyloid formation in an ankyrin repeat protein
AU - A. Sethi
AU - H. Darroch
AU - N. J. Magon
AU - G. Greene
AU - K. M. O. Connor
AU - A. D. Botha
AU - S. G. Gray
AU - P. d. Cordovez
AU - R. Coldicott
AU - J. Horsfield
AU - V. K. Morris
AU - C. Goebl
PY - 2026
DA - 2026/08/30
PB - bioRxiv
DO - 10.64898/2026.08.30.748053
UR - https://www.biorxiv.org/content/10.64898/2026.08.30.748053v1
AB - The formation of amyloids, including functional amyloids, is observed for an increasing number of proteins but the molecular mechanisms that control this structural transition remain poorly understood. Here we report that the kinase inhibitor protein P18 (drP18) from Danio rerio (zebrafish), which contains two cysteine residues, undergoes a complex and hierarchical redox switch that strictly governs reversible amyloid formation. We identify cysteine 50 (C50) acting as a regulatory residue. Upon oxidation, C50 forms an intramolecular disulfide bond with the executioner cysteine 128 (C128), thereby blocking it. C50 can become S-glutathionylated, and upon oxidation, C128 then forms intermolecular disulfides that lead to rapid transition into amyloid fibrils. S-glutathionylation of C50 therefore enables amyloid formation of drP18 and the outcome is oxidant-dependent with diamide, hydrogen peroxide, peroxymonocarbonate and hypothiocyanous acid each leading to amyloid assembly with distinct kinetics and morphologies. These amyloids are fully reversible, where disulfide reduction is leading to disassembly. Whereas monomeric drP18 inhibits CDK4-mediated retinoblastoma phosphorylation, the amyloid conformation abolishes this inhibition, and reduction restores both structure and function. Expression of drP18 in zebrafish embryos yields Congo red-positive, oxidation-dependent aggregates in vivo. Together, our findings show that a regulatory cysteine controls an executioner cysteine to induce reversible, functional amyloid formation, revealing that proteins can encode sophisticated mechanisms to control amyloid assembly.
ER -