A Drosophila model to study the redox regulation of FOXO in vivo

Abstract

Insulin/insulin-like growth factor signaling (IIS) is a key evolutionarily-conserved pathway associated with growth, metabolism and ageing from flies to humans. Forkhead box O (FOXO) transcription factors are the principal downstream effectors of the IIS pathway. FOXO is implicated in the regulation of many biological processes, such as energy metabolism, oxidative stress resistance and healthy ageing. FOXO proteins are known to be strongly post-translationally modified, however the contribution of redox regulation to the modulation of FOXO function is still underappreciated. Using Drosophila as a powerful in vivo model system, my goal is to dissect the redox regulation of the single fly orthologue, dFOXO, in the context of metabolic health, stress responses and survival. Redox signalling operates through reversible changes to cysteine residues on target proteins. Using differential labelling of cysteine residues, I have developed a mobility shift approach to assess the redox state dFOXO, and confirmed the presence of solvent-exposed cysteines implicated in redox signalling. I have optimised the redox proteomic analysis of dFOXO to uncover the redox state of all its nine cysteine residues. To gain mechanistic insight into FOXO redox regulation in vivo, I have generated a series of novel transgenic knock-in fly lines. These dFOXO redox-mutants each contain a single cysteine residue in an otherwise cysteine-less background. The methods developed and optimised in this study are applied to explore the redox regulation of dFOXO at the level of the cysteine residue, the protein and the whole fly. Studying the redox control of dFOXO in vivo will be important to understand how its key role in the regulation of metabolism, stress resistance and healthy lifespan is orchestrated.