Systemic remodeling of the redox regulatory network due to RNAi perturbations of glutaredoxin 1, thioredoxin 1, and glucose-6-phosphate dehydrogenase.

Background: Cellular clearance of reactive oxygen species is dependent on a network of tightly coupled redox enzymes; this network rapidly adapts to oxidative conditions such as aging, viral entry, or inflammation. Current widespread use of shRNA as a means to perturb specific redox couples may be misinterpreted if the targeted effects are not monitored in the context of potential global remodeling of the redox enzyme network.

Results: Stable cell lines containing shRNA targets for glutaredoxin 1, thioredoxin 1, or glucose-6-phosphate dehydrogenase were generated in order to examine the changes in expression associated with altering cytosolic redox couples. A qRT PCR array revealed systemic off-target effects of altered antioxidant capacity and reactive oxygen species formation. Empty lentiviral particles generated numerous enzyme expression changes in comparison to uninfected cells, indicating an alteration in antioxidant capacity irrespective of a shRNA target. Of the three redox couples perturbed, glutaredoxin 1, attenuation produced the most numerous off-target effects with 10/28 genes assayed showing statistically significant changes. A multivariate analysis extracted strong co-variance between glutaredoxin 1 and peroxiredoxin 2 which was subsequently experimentally verified. Computational modeling of the peroxide clearance dynamics associated with the remodeling of the redox network indicated that the compromised antioxidant capacity compared across the knockdown cell lines was unequally affected by the changes in expression of off-target proteins.

Conclusions: Our results suggest that targeted reduction of redox enzyme expression leads to widespread changes in off-target protein expression, changes that are well-insulated between sub-cellular compartments, but compensatory in both the production of and protection against intracellular reactive oxygen species. Our observations suggest that the use of lentivirus can in itself have off-target effects on dynamic responses to oxidative stress due to the changes in species concentrations.