CRITICAL ROLE OF THE THIOREDOXIN AND THE GLUTATHIONE SYSTEMS IN MITOCHONDRIAL PATHOPHYSIOLOGY

The thioredoxin and the glutathione systems are important thiol redox regulating networks. The mitochondrial thioredoxin system is composed by NADPH, thioredoxin reductase 2 (TrxR2) and thioredoxin 2 (Trx2) that, in turn, can reduce peroxiredoxin 3 (Prx3) which has a hydroperoxide scavenging activity. Cyclophilin D (CypD) is a small protein of the mitochondrial matrix having a crucial role in the control of the mitochondrial membrane permeability transition. CypD activity and redox state were found to be subjected to thioredoxin system-mediated reduction in both isolated rat heart mitochondria and in human cell lines. Furthermore, CypD interaction with Trx2 and Prx3 was observed with both the co-immunoprecipitation technique and with a molecular docking prediction. Thus, CypD can be redox modulated by the mitochondrial thioredoxin system. The thiol redox regulating systems, especially TrxR2, are often overexpressed in cancer cells to counteract the increased ROS level due to cancer progression. Therefore, the search for specific TrxR2 inhibitors is a possible new anticancer strategy. Several novel compounds, obtained in the frame of different international collaborations, were studied. In particular, a new Au(III) complex bearing a bidentate N-donor ligand, various cyclometalated 2,6-diphenylpyridine Au(III) complexes and a series of mono and bis N-heterocyclic carbene Au(I) complexes were synthesized and were found to inhibit selectively thioredoxin reductase, disrupting the overall cellular redox homeostasis in human ovarian cancer cell lines. Afterwards, a class of non-gold based metallodrugs, derived from tamoxifen and called tamoxifen-like metallocifens (TLMs), were studied. Interestingly, TLMs act as pro-drugs. In fact, upon enzymatic oxidation, they can be transformed into new oxidized derivatives endowed with remarkable TrxR2 inhibitory properties. In the lymphoblastoid cell line Jurkat, TLMs-mediated TrxR2 inhibition stimulated Trx2 oxidation, ROS production and intrinsic apoptotic pathway activation. The effects of TrxR2 genetic depletion was also investigated in different cancer cell lines utilizing the Crispr/Cas9 method. Notably, an inverse correlation between TrxR2 protein level and cellular ROS production was observed, indicating the strong pro-oxidizing condition derived from TrxR2 depletion. Then, the research was focused on glutaredoxin 2 (Grx2). Grx2 was reported to link the thioredoxin and the glutathione systems, but its specific role in redox signaling events is unclear. Grx2 catalyzes protein de-glutathionylations and can also coordinate an iron-sulfur cluster, forming dimers. Grx2 monomeric and dimeric state was analyzed upon HeLa cells treatment with different oxidizing conditions. Grx2 stayed principally as an inactive dimer, while it dissociated, and its activity was stimulated specifically in the mitochondrial compartment, only upon the combined hindering of both the glutathione and the thioredoxin systems. A large increase of free iron ions in the mitochondrial matrix, induction of lipid peroxidation and decrease of the mitochondrial membrane potential were also observed, indicating that Grx2 monomerization implied the release of the iron-sulfur cluster. In collaboration with Prof. A. Holmgren’s group at the Karolinska Institutet, the role of Grx2 in mitochondria has been further studied in a murine model knockout for Grx2 in mitochondria (mGrx2 KO). The overall redox state of mitochondria isolated from different organs of WT or mGrx2 KO mice at three months of age was assessed and interestingly it was not affected from Grx2 deletion. However, a significant increase of mitochondrial ROS production was noted in the liver associated to a decrease of the mitochondrial respiratory capacity, to a reduction of the mitochondrial membrane potential and to an increased sensitivity to calcium ions. Altogether, these results suggest that Grx2 deletion in mouse mitochondria affects mainly the mitochondrial functioning in the liver