Studies of the thioredoxin system in redox signaling and oxidative stress

Abstract: Reactive oxygen species exert reversible posttranslational modifications in proteins containing redox sensitive thiols, thereby affecting several cellular processes and protein functions. Reductive enzyme systems, such as the thioredoxin system, can reverse most of these cysteine modifications. Therefore, a tight control between oxidizing and reducing events is the central principle of redox signaling. The studies within this thesis have covered several aspects of the Trx system in the modulation of cellular signaling pathways. In Paper I, we evaluated how b-AP15, a small inhibitor of proteasome-associated DUB, exerts redox perturbations in tumor cells. It is known that b-AP15triggers an increase level of reactive oxygen species (ROS) and proteotoxic stress in can- cer cells. However, its efficacy inducing apoptosis diminished by antioxidants. To identify the precise mechanism by which b-AP15 induces redox perturbations, we generated cells deprived of mitochondrial DNA. We found that in cells lacking mitochondria, the oxidative stress generated by b-AP15 was completely abrogated. Furthermore, to exclude that the observed increase in the levels of oxidative stress were due to an inhibition of TrxR1, we evaluated a number of proteasome asso- ciated DUBs inhibitors that did not inhibit TrxR1. Similarly, to b-AP15 all the inhibitors tested induced oxidative stress and the expression of HO-1. In parallel, we observed mitochondrial dysfunction, measuring the levels of COX5b and TOMM34, in both cases their respective levels decreased in those cells treated with b-AP15. Based in all the results we could conclude that the source of ROS in cells treated with b-AP15 was of mitochondrial origin. In Paper II, we performed a drug-screen of compounds sharing a common enone motif with b-AP15 and many natural products with antineoplastic effect. Through biochemical and structural analyses, we could demonstrate the binding of the enone containing compounds to the proteasome-associated cysteine deubiquitinase, USP14, inhibiting its activity. Additionally, we further analyzed a subset of those compounds in a zebrafish embryo model where they showed antineoplastic activity. These findings suggest that DUB inhibition is a relatively common mode of action by cytotoxic compounds containing motifs and it helps to explain the antineoplastic effects of natural products containing such functional group. In Paper III, we identified HRI as a redox-regulated protein, which becomes oxidized when activated upon As(III) exposure. TrxR1 associates with HRI in cells and together with TRP14 and Trx1 reduces HRI in vitro. Moreover, several specific inhibitors of TrxR1 lead to HRI-dependent eIF2α phosphorylation, trans- lation suppression and stress granule formation. Based on our finding that HRI- mediated translation suppression is essential for cellular survival under conditions of high As(III), we revealed the Trx system as a regulator of the HRI dependent translational stress response. In Paper IV, we evaluated the role of TRP14 in the regulation of different redox- regulated transcriptional factors using our unique tool pTRAF (plasmid for tran- scription factor reporter activation based upon fluorescence). We discovered that using TRP14 knockdown HEK293 cells, NRF2 activation increased upon treat- ment with auranofin, we also uncovered that TRP14 is crucial for HIFactivation upon TNF-α stimulation in hypoxic conditions. Furthermore, endogenous TRP14 levels increased under hypoxia or TNF-α treatment, suggesting that TRP14 could itself be regulated by NFκB and HIF, which is compatible with the presence of the corresponding response elements in the proximal TXNDC17 promoter region. Surprisingly, using TRP14 knockout HEK293 cells we found that global protein translation was reduced, which could be reverted with methionine or N- acetylcysteine supplementation. TRP14 knockout cells were also, in contrast to controls, highly sensitive to PPG. We conclude that TRP14 has several roles in control of redox signaling pathways, and that TRP14 is the main intracellular reductase for liberation of cysteine from cystine. In Paper V, we de novo synthesized eight vinyl sulfone compounds and evalu- ated their capacity to activate NRF2, NFκB and HIF1 in comparison with DMF using our previously mention tool pTRAF. We selected a set of compounds that activate NRF2 more selectively than DMF and characterize their downstream effects using in vitro and in vivo models. Our selected compounds display a more selective oligodendrocyte associated effect which could be explore in the future as a regenerative drug in demyelinating disorders.