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

Melissa L Kemp,Shreya Shukla,Linda E Kippner,Nnenna A Finn

doi:10.1186/1752-0509-5-164

Abstract

BackgroundCellular 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.ResultsStable 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.ConclusionsOur 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.

Highlights

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
The most efficient short hairpin RNA (shRNA) for each gene of interest showed greater than 50% perturbation of mRNA levels as measured by qRT PCR (Figure 1A) and protein levels were further confirmed by western blot (Figure 1B)
The fold changes in glutaredoxin 1 (Grx1), thioredoxin 1 (Trx1), and glucose6-phosphate dehydrogenase (G6PD) levels were normalized relative to a housekeeping gene, b-actin, and compared to mRNA levels in the empty lentivirus cells

Summary

Introduction

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. The cellular redox environment is determined by numerous electron couples, including glutathione/glutathione disulfide (GSH/GSSG), reduced thioredoxin/ oxidized thioredoxin, NAD(P)H/NAD(P)+, and cysteine/ cystine, that transfer electrons during changes in intracellular oxidation state. These redox couples are in turn maintained out of equilibrium by a network of protein thiol/disulfide oxidation to the removal of hydrogen peroxide from the intracellular environment. This model was specific to the Jurkat T-lymphocyte cell line, yet through the adjustment of initial enzyme, glutathione, and NAD(P)H concentrations this model could in principle simulate the oxidative protection mechanisms of other cell types. Numerous studies have examined the cDNA changes that occur across the genome in response to alterations in the oxidative environment (e.g. HIV infection, hypoxia, age); these studies primarily report the significant up- or down-regulated gene hits from the conditions assayed without consideration of the subtle changes that could occur across the redox network

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