Molecular characterisations and pathological implications of the antioxidant role of S-nitrosoglutathione reductase

Oxidative and nitrosative stress are harmful conditions for cellular homeostasis. To counteract reactive oxygen and nitrogen species (ROS and RNS, respectively), cells are equipped with a powerful antioxidant defense including enzymatic and non-enzymatic systems. However, ROS and RNS are also second messengers involved in contributing to signal transduction pathways underlying almost all cellular processes. ROS- and RNS -driven molecular information (redox signal) is transduced by means of reactive cysteine residues that can undergo Shydroxylation, upon reaction with ROS (i.e. H2O2) or S-nitrosylation, which is now emerging as the main posttranslational modification underlying nitric oxide (NO) bioactivity. The extent of S-nitrosylated proteins (PSNOs) depends on the balance between the rate of production, carried out by NO synthase (NOS), and their removal by denitrosylases, among which Snitrosoglutathione reductase (GSNOR) is the best-characterized example. GSNOR has a key role in the maintenance of nitrosothiols (SNOs) homeostasis and, as a consequence, in preventing nitrosative stress. Indeed, since it completely reduces the NO moiety of SNOs, it prevents the reaction between O2.- and any other still reactive NO-derived species, to generate one of the most oxidizing molecule, peroxynitrite (ONOO-). In this PhD thesis, I provide evidence of a strong GSNOR induction in in vitro cellular models of acute and chronic oxidative stress, the latter recapitulating pathological (neurodegenerative) conditions. I demonstrate that HEK293 cells, subjected to a single burst of H2O2, significantly upregulate GSNOR as protective factor to face nitroxidative-induced cell death. However, I observe that the increase in GSNOR protein levels is not associated with a matched transcript increase but relies on post transcriptional events. In order to investigate the molecular mechanism(s) underlying GSNOR enhanced translation, I provide evidence on the activation of a non-canonical oxidative stress-induced signaling cascade, which starts from the upstream protein kinase ATM, involves Chk2 and culminates in the activation of p53 as the final effector. I further demonstrate the importance of this signaling axis in mediating GSNOR upregulation by taking advantage of the p53-null HCT116 cell line, which did not show the ability to upregulate GSNOR after ROS increase, unless p53 was ectopically re-established. Nitroxidative stress is a hallmark of several diseases, such as neurodegenerations. I, therefore, investigate whether GSNOR was also upregulated in in vitro models of familiar amyotrophic lateral sclerosis (fALS). Namely I take advantage of neuroblastoma SH-SY5Y cells overexpressing the mutant form of SOD1G93A, which is one of the mutations found to be associated with fALS inducing mitochondrial impairment and ROS overproduction. I provide evidence that GSNOR is fundamental to protect the cells against SOD1G93A toxicity. Moreover, I demonstrate that clones stably expressing SOD1G93A fALS mutant (G93A clones) have an increased GSNOR expression as an essential adaptation factor selected to allow survival under chronic stress condition. Interestingly, and at variance with what previously demonstrated in HEK293, G93A clones upregulated GSNOR at the transcriptional level to ensure cell survival. In particular, I show that chronic and sustained ROS fluxes (e.g., those produced upon G93A expression or rotenone treatment respectively) induce the phosphoactivation of ATM-Chk2 signaling axis, which is associated with SOD1 translocation into the nuclei. In the G93A cell system, I also demonstrate that this event is associated with SOD1 interaction with Chk2 and its binding to GSNOR promoter, suggesting a new transcriptional role of SOD1 being operative in the antioxidant response of mammalian cells as recently proposed in yeast. In vivo studies performed on two distinct mouse models of fALS demonstrated that in spinal cord and skeletal muscles of fALS mice, which represent the tissues that more than other are affected in ALS, we were unable to appreciate any activation of GSNOR-inducing signaling pathways. This resulted in massive oxidative damage, i.e., increase of protein carbonylation and nitrosylation, eventually confirming that GSNOR has a crucial role in the antioxidant response, and that ectopic modulation of its expression could be a potential strategy in therapies against oxidative stress-based diseases, such as ALS.