Study of the involvement of glutaredoxin 1 in copper homeostasis in neuroblastoma cells

Copper (Cu) is a redox transition metal existing in two ionic forms, Cu+ and Cu++. In biological systems the electrochemical properties of copper ions make it an important cofactor for many enzymes that catalyze a wide range of biochemical processes such as oxidative phosphorylation, antioxidant defense, catecholamine synthesis, iron homeostasis and connective tissue cross-linking. However, the same properties if uncontrolled, may damage biological systems through the formation of reactive oxygen species that can adversely modify proteins, lipid and nucleic acids thus affecting the overall cell physiology including the function of organelles like mitochondria (Jomova and Valko, 2011). To maintain the control on copper homeostasis, biological systems have evolved a class of proteins called copper chaperones characterized by the presence of one or more copper binding domains (CXXC) (Palumaa, 2013). Copper enters the cells by the high affinity Copper transporter 1 (Ctr1); it is a plasmamembrane pore, responsible for the uptake of at least 80% of copper. Copper itself modulates Ctr1 abundance: elevated intracellular copper levels stimulate endocytosis and degradation of Ctr1; moreover copper regulates Ctr1 expression through Sp1 (Specific protein 1). This mechanism may prevent the accumulation of potentially toxic levels of copper (Wang et al, 2011). Intracellular copper levels are also controlled by the activity of ATP7A pump, copper chaperone devoted to the metallation of Cu-dependent enzymes of the secretory pathway and to the efflux of excess Cu from the cell (Palumaa, 2013). It was shown that ATP7A activity is affected by glutaredoxin 1 (Grx1) a protein belonging to the thioredoxin family, which catalyzes protein deglutathionilation by interacting with the Cu-binding domains in the presence of the metal thus promoting their function (Lim et al., 2006; Singleton et al., 2010). Grx1 has a mitochondrial homologue, glutaredoxin 2 (Grx2), which is the first [Fe-S] Grx discovered; it has a modification in the active site (Ser for Pro) that allows the enzyme to complex a redox inactive [2Fe-2S]2+ cluster that bridge two Grx2 molecules to form a dimeric holo Grx2 complex. In this condition the enzyme is inactive; the increase of intracellular oxidative stress determines the disruption of the complex and the subsequent activation of Grx2. The present study is aimed at further investigating the effect of Grx1 on copper metabolism, in an experimental model represented by SH-SY5Y human neuroblastoma cell line constitutively over-expressing Grx1 (SHGrx1 cells). In the first part of my work, I focused my attention on the intracellular Cu content of SH-SY5Y and SH-Grx1 cells, under basal conditions and after CuSO4 treatment (50 or 150 μM, for 24 hours). The SH-Grx1 cells show a higher copper content than control cells, particularly after copper overload. In order to understand this issue, expression level of Ctr1 has been measured, showing that it is higher in SH-Grx1 cells. This effect is accompanied by a higher expression of Sp1 transcription factor. However, SH-Grx1 cells seem to be more resistant to copper induced cell toxicity. CCS (the cytosolic copper chaperone), which is expected to be degraded in copper loaded cells (Bertinato and L’Abbè, 2003), decreases more in SHGrx1 than in SH-SY5Y cells. SOD1 activity doesn’t change between the two cell lines. It has been demonstrated that upon copper exposure, mitochondrial copper overload occurs, accompanied by mitochondrial damage (Arciello et al., 2005). By comparing the two cell lines, under basal conditions, I observed that the mitochondrial copper content is comparable; however, after copper exposure, it increases more in the SH-SY5Y cells than in SHGrx1, suggesting that Grx1 restrains copper translocation to mitochondria, possibly sequestering the metal in the cytosol, thus protecting cells from copper-induced toxicity. To investigate this issue, I used ImmobilizedMetal-Affinity-Chromatography to select Cu-binding proteins from SHGrx1 total cell lysates and by Western blot analysis I identified Grx1, in the fraction eluting at 40 mM imidazole. Eventually, I measured Grx1 activity in SH-Grx1 cells, after treatment with 150 μM CuSO4 for 24 hours, and I found that Grx1 activity seems to be lower than under basal conditions. Given these results, I hypothesize that copper binds to the cysteines localized in the catalytic domain of Grx1, affecting its activity. In the second part of my work, I analyzed the effect of Grx2 overexpression on copper metabolism in SH-SY5Y cells, with particular focus to mitochondrial functionality. These cells seem to be more susceptible to copper induced toxicity and accumulate more copper than control cells, but at a lesser extent than SH-Grx1 cells. In fact, the increase of intracellular copper content observed in SH-Grx2 cells treated with CuSO4 (150 µM) is not sufficient to induce CCS degradation. Despite SH-Grx2 cells are more sensible to copper overload, they accumulate less copper in mitochondria than control cells when exposed to exogenous copper. This data needs to be further investigated in order to understand the mechanism underlyng this phenomenon. I observed that the protein level of COX17, a mitochondrial copper chaperone involved in copper transfer to cytochrome c oxidase (COX), is lower in mitochondria of SH-Grx2 cells; this is accompanied by a reduction of COX enzyme activity and protein content. In fact, it is known from literature, that COX17 downregulation affects COX assembly and so its enzyme activity (Oswald et al., 2009). However, this aspect must be examine in depth in order to clarify how Grx2 may modulate COX17 protein level and COX activity. The last part of my work focused to study the effect of sodium selenite, used in cancer theraphy, on SH-SY5Y neuroblastoma cells. It was demonstrated that sodium selenite induces Grx1 transcription, translation and activity (Wallenberg et al, 2010). Interestingly, selenite was shown to revert the acquired resistance to cisplatin, another well-known chemotherapic agent, used against a wide variety of solid tumors (Siddik et al, 2006). Cisplatin is known to enter the cells via Ctr1 (Ishida et al, 2002) and lower Ctr1 levels have been reported to be associated with innate or acquired cisplatin resistance (Lin et al., 2002; Holzer et al., 2004b, 2006a; Larson et al., 2009). Therefore, experiments were planned in order to understand whether sodium selenite treatment of SH-SY5Y cells, by increasing the level of Grx1; may lead to the Sp1-mediated induction of Ctr1 level. The results obtained support this hypothesis and may thus represent an important base for further investigations.