Paulo J. Martel

FOXO3 is integral in regulating numerous genes involved in critical cellular processes such as apoptosis, oxidative damage protection, cell growth, and cancer. Consequently, modulating FOXO3 activity holds significant potential for applications in cancer treatment and cellular aging. A promising approach involves identifying small-molecule modulators that can either enhance or inhibit FOXO3’s DNA-binding capability. This paper details a virtual screening protocol aimed at discovering such modulators. Utilizing the crystal structures of FOXO3 in both its free and DNA-bound forms, we pinpoint potential binding sites that may disrupt or facilitate the DNA–FOXO3 interaction. A comprehensive virtual screening of a small-molecule compound library is conducted using AutoDock Vina software. The highest-ranking hits for each site are carefully selected and analyzed to determine their binding modes. This protocol paves the way for identifying novel modulators of FOXO3, offering therapeutic avenues in cancer and aging-related research.

Aims: Protein disulfide isomerases (PDIs) are a family of chaperones resident in the endoplasmic reticulum (ER). In addition to holdase function, some members catalyze disulfide bond formation and isomerization, a crucial step for native folding and prevention of aggregation of misfolded proteins. PDIs are characterized by an arrangement of thioredoxin-like domains, with the canonical protein disulfide isomerase A1 (PDIA1) organized as four thioredoxin-like domains forming a horseshoe with two active sites, a and a ′, at the extremities. We aimed to clarify important aspects underlying the catalytic cycle of PDIA1 in the context of the full pathways of oxidative protein folding operating in the ER. Results: Using two fluorescent redox sensors, redox green fluorescent protein 2 (roGFP2) and HyPer (circularly permutated yellow fluorescent protein containing the regulatory domain of the H ₂ O ₂ -sensing protein OxyR), either unfolded or native, as client substrates, we identified the N-terminal a active site of PDIA1 as the main oxidant of thiols. From there, electrons can flow to the C-terminal a ′ active site, with the redox-dependent conformational flexibility of PDIA1 allowing the formation of an interdomain disulfide bond. The a ′ active site then acts as a crossing point to redirect electrons to ER downstream oxidases or back to client proteins to reduce scrambled disulfide bonds. Innovation and Conclusions: The two active sites of PDIA1 work cooperatively as an interdomain redox relay mechanism that explains PDIA1 oxidative activity to form native disulfides and PDIA1 reductase activity to resolve scrambled disulfides. This mechanism suggests a new rationale for shutting down oxidative protein folding under ER redox imbalance. Whether it applies to physiological substrates in cells remains to be shown.