Cu/Zn-superoxide dismutase and wild-type like fALS SOD1 mutants produce cytotoxic quantities of H2O2 via cysteine-dependent redox short-circuit

The Cu/Zn−superoxide dismutase (SOD1) is a ubiquitous enzyme that catalyzes the dismutation of superoxide radicals to oxygen and hydrogen peroxide. In addition to this principal reaction, the enzyme is known to catalyze, with various efficiencies, several redox side-reactions using alternative substrates, including biological thiols, all involving the catalytic copper in the enzyme’s active-site, which is relatively surface exposed. The accessibility and reactivity of the catalytic copper is known to increase upon SOD1 misfolding, structural alterations caused by a mutation or environmental stresses. These competing side-reactions can lead to the formation of particularly toxic ROS, which have been proposed to contribute to oxidative damage in amyotrophic lateral sclerosis (ALS), a neurodegenerative disease that affects motor neurons. Here, we demonstrated that metal-saturated SOD1WT (holo-SOD1WT) and a familial ALS (fALS) catalytically active SOD1 mutant, SOD1G93A, are capable, under defined metabolic circumstances, to generate cytotoxic quantities of H2O2 through cysteine (CSH)/glutathione (GSH) redox short-circuit. Such activity may drain GSH stores, therefore discharging cellular antioxidant potential. By analyzing the distribution of thiol compounds throughout the CNS, the location of potential hot-spots of ROS production can be deduced. These hot-spots may constitute the origin of oxidative damage to neurons in ALS.

In the nervous tissue, SOD1 is present at exceedingly high concentrations (~1% of total protein, i.e., 100-200 μM) 3 and is traditionally regarded as cytosolic protein 4 . However, in cell culture experiments, the SOD1 secretory pathways were shown to account for substantial quantities of extracellular SOD1, reaching ~ 20% of the intracellular SOD1 level 5 .

Results
Holo-SOD1 Wt and WTL SOD1 G93A are cytotoxic in the presence of thiol compounds. We tested the effect of CSH and other thiol compounds (namely, Hcy and GSH) on the viability of human neuroblastoma SH-SY5Y cells in the presence of extracellular holo-SOD1 WT . When applied separately, neither holo-SOD1 WT nor thiol compounds were cytotoxic at any of the tested concentrations (Fig. 1A,B). Conversely, their simultaneous application resulted in cytotoxicity, whose extent depended on the concentration of both holo-SOD1 WT and the thiol compounds. Thiol cytotoxicity was not observed in the presence of metal-depleted SOD1 WT (apo-SOD1 WT ) (Fig. 1B).
The fast kinetics of holo-SOD1 WT -induced cell death in the presence of thiol compounds, as demonstrated by Hcy (Fig. 1C), suggest that the origin of cytotoxicity is extracellular, rather than due to the internalization of holo-SOD1 WT by the cells 54,55 . Moreover, separating holo-SOD1 WT from the cells using a 3-kDa cutoff membrane did not abolish cytotoxicity, although the kinetics of cell death were significantly delayed ( Supplementary Fig. S1), demonstrating that cytotoxicity was facilitated by a diffusible low-molecular weight substance. The divalent metal ion chelator N,N,N' ,N'-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) abolished Hcy-induced holo-SOD1 WT cytotoxicity in a dose-dependent manner ( Fig. 2A), implying that metal ions are involved in the mechanism responsible for this cytotoxicity.
We also tested thiol-induced cytotoxicity in the presence of two fALS SOD1 mutants: WTL SOD1 G93A and metal binding-impaired SOD1 G85R , which were purified and reconstituted with metals using the same procedure H 2 o 2 is produced during the SOD1-catalyzed oxidation of thiol compounds. The toxicity of CSH and Hcy toward various cell types in the presence of free Cu 2+ (as CuCl 2 ) has been previously demonstrated and ascribed to the toxicity of H 2 O 2 and/or of hydroxyl radicals produced during Cu 2+ -catalyzed thiols oxidation [46][47][48][49][50] . Although free Cu 2+ was 5-fold more potent than holo-SOD1 WT in inducing cell death (Fig. 1B), the kinetics of the Hcy-dependent cell death were almost identical in both cases (Fig. 1C). Moreover, the cytotoxicity of Hcy was completely abolished by adding catalase at all examined concentrations of holo-SOD1 WT (Fig. 2B), indicating that this cytotoxicity was mediated by H 2 O 2 , and Cu 2+ ions are involved in the mechanism of its formation. The luck of cytotoxicity in apo-SOD1 WT and metal-deficient SOD1 G85R is in line with this conclusion.
Extracellular H 2 O 2 is an effective and potent neurotoxin, which can induce either apoptosis or necrosis in neurons [56][57][58] . Such cell death is associated with a burst-like increase in intracellular Ca 2+ after a brief (several minutes) exposure to H 2 O 2 , as a result of the influx of Ca 2+ from the extracellular space via redox-sensitive Ca 2+ channels, such as TRPM2, which are expressed in neurons 59 . In agreement with this mechanism of neuronal death, a brief (<15 min) exposure of SH-SY5Y cells to extracellular holo-SOD1 WT in the presence of Hcy irreversibly damaged the cells (Fig. 2C).
Since the plasma membrane is permeable to H 2 O 2 60 , extracellularly produced H 2 O 2 may also aggravate existing oxidative stress conditions. Cells with an impaired ability to scavenge ROS are expected to be less resistant to an oxidative insult, including one of extracellular origin. Consistent with this idea, the cytotoxicity of  The viability of SH-SY5Y cells was determined after 6 h of incubation with or without 500 U/ml catalase in the presence of 0.5 mM Hcy and the indicated concentrations of holo-SOD1 WT . (C) SH-SY5Y cells were incubated with 50 μM holo-SOD1 WT in the presence of 0.5 mM Hcy for the indicated periods of time, followed by further incubation in a fresh medium (without SOD1 or Hcy), and the viability was determined after 12 h of incubation in total. The viability of untreated cells was set to 100%. Results represent normalized means ± SD and are representative of at least three independent experiments performed in triplicates.
We then directly quantified the formation of H 2 O 2 in the mixtures of SOD1 and thiol compounds using a phenol red/HRP H 2 O 2 assay 62 . In cell experiments, all tested thiol compounds were toxic in the presence of holo-SOD1 WT , with GSH being the most potent (Fig. 1A); in contrast, in a reconstituted reaction mixture, different rates of H 2 O 2 formation were observed for different thiols ( Fig. 5A and Supplementary Fig. S2). The fastest rates of oxidation were obtained with CSH, whereas Hcy was inferior and GSH failed to produce any significant amount of H 2 O 2 . Similar (but not identical) patterns of H 2 O 2 production were observed in the presence of free Cu 2+ (CuCl 2 ) as a catalyst ( Supplementary Fig. S2). In particular, the oxidation of Hcy in the presence of free Cu 2+ exhibited a lag phase followed by a sigmoidal increase in the rate of H 2 O 2 formation, whereas a monotonic asymptotic increase without lag phase was obtained in the presence of holo-SOD1 WT ( Supplementary Fig. S2C,D).
The apparent discrepancy between the cytotoxic potencies of the thiol compounds and their respective rates of H 2 O 2 formation in the reconstituted reaction mixture (in the presence of catalytically active SOD1) was reconciled when thiol oxidation was performed in the presence of cystine (oxidized cysteine dimer, CS-SC, 200 μM) -a component of the standard DMEM cell growth medium used in our cytotoxicity experiments. In the presence of cystine, all thiol compounds, including GSH, demonstrated similar rates of holo-SOD1 WT -catalyzed H 2 O 2 production (Fig. 5B), which were consistent with their cytotoxic potencies (Fig. 1A). Comparable rates of H 2 O 2 formation were also observed when the measurements were performed directly in the DMEM cell growth medium ( Supplementary Fig. S3).
We also monitored the kinetics of CSH substrate consumption in the course of H 2 O 2 formation by using Ellman's reagent (DTNB). The oxidation of CSH went to a completion in the presence of the holo-SOD1 WT , while  www.nature.com/scientificreports www.nature.com/scientificreports/ no significant thiol oxidation was observed with the metal-depleted apo-SOD1 WT , Fig. 6A. As expected, the rate of CSH oxidation decreased under the conditions of limiting oxygen, and the reaction came to a halt when the remaining O 2 was consumed.
In agreement with their respective cytotoxic potencies (Fig. 3), the WTL SOD1 G93A was by 40% less efficient in catalyzing H 2 O 2 formation in the presence of CSH as compared to the holo-SOD1 WT , whereas metal-deficient SOD1 G85R failed to produce any significant amounts of H 2 O 2 (Fig. 5C). We suggest that the efficiency of thiol oxidation catalyzed by SOD1 is a function of its Cu 2+ content, which in turn reflected the efficiency, with which Cu 2+ was incorporation into the tested SOD1 variants upon the reconstitution (see Methods).
Although GSH cannot be oxidized directly by SOD1 to produce H 2 O 2 , it is apparently capable of reducing cystine to regenerate free CSH: 2GSH + CS-SC → GS-SG + 2CSH 53,63 . The CSH that is thereby produced enters another cycle of oxidation. Similarly to GSH, Hcy is capable of regenerating CSH from cystine 53,63 (Fig. 5A,B). The total amount of H 2 O 2 produced by a given amount of CSH or cystine was proportional to the amount of GSH present in the system (Fig. 7). The dependence of H 2 O 2 accumulation on GSH concentrations, however, was bell-shaped, demonstrating that H 2 O 2 buildup was inhibited at high GSH concentrations (Fig. 7C).
To distinguish between the possibilities that the inhibitory effect of high GSH concentrations was caused by the ability of the thiol compounds (including GSH) to effectively scavenge H 2 O 2 in a non-enzymatic Cu 2+ -independent process (2GSH + H 2 O 2 → GS-SG + 2H 2 O) 64 , or alternatively, by the inhibition of H 2 O 2 formation, we monitored the kinetics of free thiol oxidation in the CSH/GSH mixtures in the presence of holo-SOD1 WT , Fig. 6B. Although GSH alone was resistant − at any of the tested concentrations − to the holo-SOD1 WT -catalyzed oxidation (not shown), in the presence of small quantities of CSH (100 μM), GSH was rapidly and completely  www.nature.com/scientificreports www.nature.com/scientificreports/ oxidized, demonstrating a marked ability of the redox short-circuit thus established to deplete GSH stores. At the GSH concentrations higher than 2 mM, however, no significant GSH oxidation was observed, consistent with the lack of H 2 O 2 formation at high GSH concentrations (Fig. 7). The mechanism responsible for the inhibitory effect of high GSH concentrations on the rate of H 2 O 2 formation is unclear, but it may involve a competitive inhibition of CSH binding to the SOD1 active-site by GSH 65 ; this competition, however does not prevent much smaller substrates, such as superoxide, from accessing the catalytic site, therefore enabling the enzyme's action at the physiological GSH concentrations.
Thiol compounds increase the accessibility of Cu 2+ to external chelators. As high-affinity binding of metals to SOD1 is characterized by fast association-dissociation dynamics, strong chelators may interfere with the equilibrium 2 . To test whether thiol compounds increase the accessibility of holo-SOD1 WT metals to external chelators, we used as chelator the colorimetric divalent metal sensor 4-(2-pyridylazo)resorcinol (PAR) 36 . The incubation of holo-SOD1 WT with PAR in the presence of thiol compounds resulted in an increased formation of PAR-copper complexes (Fig. 8), while virtually no effect of thiol compounds was observed on the rate of PAR-Zn 2+ complex formation (not shown).
The mechanism by which the thiol compounds increase the accessibility of PAR to SOD1 copper is unknown. It is possible that thiol compounds reduce Cu 2+ to Cu + directly in the active site of SOD1 53 , thus changing its coordination geometry to non-tetrahedral and facilitating ligand replacement (PAR binding) 52 . In addition, the formation of a binary thiol ̶ Cu + complex could increase copper accessibility to PAR. The latter possibility, however, appears unlikely, since in line with the assumption that the thiol oxidation is catalyzed by the  www.nature.com/scientificreports www.nature.com/scientificreports/ enzyme-bound Cu 2+ , CSH (similarly to other thiols) was incapable of extracting metal ions (Cu 2+ or Zn 2+ ) from the active site of holo-SOD1 WT (Fig. 9). The filtrate of the reaction mixture containing 100 μM holo-SOD1 WT and 0.5 mM CSH was analyzed for the metal presence using PAR. No labile metal ions were detected in the filtrate after the complete oxidation of the remaining thiols, indicating that, during the catalysis, no soluble binary complexes between the thiol compound and SOD1-derived Cu 2+ were formed.
Thiol oxidation promotes the formation of the intramolecular disulfide in holo-SOD1 Wt . The H 2 O 2 produced during holo-SOD1 WT -catalyzed thiol oxidation would increase the oxidizing potential of the protein's immediate environment, potentially affecting redox-sensitive surface-exposed groups of the protein. The SOD1 contains a highly conserved intramolecular disulfide bond (Cys 57 -Cys 146 ), which is required for the long-term stability and full catalytic activity of SOD1 66 . The formation of this bond in newly synthesized SOD1 is facilitated by CCS1 Cu-chaperone and is coupled to copper insertion 66,67 . The process is initiated by a copper-mediated (and CCS1-independent) oxidative step, in which one of the SOD1 cysteine residues becomes sulfenylated. The sulfenylation is subsequently resolved, in the presence of CCS1, to form a stable disulfide bond 66 . This disulfide, which is surface-exposed, is rather unusual feature for the protein found predominantly in the highly reducing cytosolic environment [66][67][68] . We tested the effect of holo-SOD1 WT -catalyzed CSH oxidation on the redox status of the SOD1 intramolecular disulfide bond (Fig. 10). The holo-SOD1 WT or apo-SOD1 WT were initially reduced by a high concentration of DTT (5 mM) and then exposed to the increasing concentrations of CSH (0-1 mM). After blocking free cysteine groups with iodoacetamide to prevent disulfide bond scrambling, the disulfide status of the proteins was analyzed using a non-reducing SDS-PAGE 69,70 . Counterintuitively, considering the cysteine's status as cellular reductant, increasing concentrations of CSH resulted in a progressively increasing proportion of holo-SOD1 WT , but not apo-SOD1 WT , containing oxidized disulfide bond (Fig. 10). We therefore concluded that the disulfide bond formation in the metallated SOD1 was facilitated, in the absence of CCS1, by ROS generated via the active-site Cu 2+ -catalyzed CSH oxidation. Understanding the physiological significance of such ability of metallated SOD1 WT to 'self-repair' its disulfide bond in the presence of ubiquitous biological thiols, and how this ability is affected by fALS mutations, requires further investigation. It is tempting to speculate, however, that the ROS-producing reaction may contribute to the long-term stability of SOD1 by preserving the oxidized status of its conserved disulfide in a highly reducing atmosphere of cellular interior.  Fig. 3). The reaction mixture was separated from the protein by ultrafiltration, and the filtrate was analyzed for the presence of labile metal ions after the complete oxidation of the remaining thiols (10 h at 37 °C) by adding 100 μM PAR. Metal standards (0-10 μM) were added to separate reaction mixtures to calibrate the system. The concentration of metals was determined as described in the Methods. Figure 10. Cysteine promotes disulfide bond formation in metallated SOD1 WT . Holo-SOD1 WT or apo-SOD1 WT (50 μM) were fully reduced by DTT (5 mM) and then exposed to the indicated concentrations of CSH for 30 min at 37 °C. After blocking free cysteine groups with iodoacetamide, the protein was separated by a nonreducing 12% SDS-PAGE. Data are representative of three independent experiments. Full-length gels are presented in Supplementary Figure S4. www.nature.com/scientificreports www.nature.com/scientificreports/

Discussion
The active-site Cu 2+ of holo-SOD1 WT and fALS WTL SOD1 G93A mutant is capable of catalyzing the oxidation of various thiol compounds, including CSH and Hcy, with a concomitant production of H 2 O 2 . Conversely, the ubiquitous cellular thiol antioxidant GSH is resistant to the SOD1-catalyzed oxidation. In the presence of small quantities of CSH or cystine, however, GSH becomes potent pro-oxidant that fuels the CSH-dependent H 2 O 2 formation by reducing cystine back to CSH. The GSH/CSH mixtures, therefore, may constitute a potent redox short-circuit that, under certain pathophysiological metabolic circumstances, could drain − in the presence of metallated SOD1 − GSH stores and, thereby, discharge the antioxidant potential of the cell.
Further investigation is required to elucidate the significance of the SOD1-catalyzed thiol oxidation with the concomitant H 2 O 2 production to ALS pathogenesis. The reaction involves catalytic copper and as such it characterizes both holo-SOD1 WT and WTL fALS SOD1 mutants. Conversely, metal-deficient fALS mutants lack this ability, which raises the possibility that the described reaction is a peculiar catalytic ability of metallated SOD1 and it may not be related to ALS. Alternatively, one may hypothesize that ALS is facilitated by a synergistic interplay between a fALS SOD1 mutant and SOD1 WT . According to this scenario, a catalytically inactive SOD1 mutant may act as a prion-like transmitter of the misfolding signal to efficiently spread the pathology, whereas the neurotoxic effect per se is caused by the ubiquitous SOD1 WT , the structural and catalytic properties of which compromised by its interaction with the misfolded SOD1 mutant.
Under the assumption that the described mechanism of H 2 O 2 production by thiol oxidation contributes to ALS pathogenesis, two important questions need to be addressed. The first one is whether the metabolic conditions required for the CSH-dependent short-circuit of GSH oxidation exist in CNS. A significant fraction of the cellular SOD1 may reach the extracellular space 5 . Although the cytotoxicity of extracellular SOD1 may represent a plausible mechanism of pathogenesis in disorders characterized by high concentrations of extracellular thiols (e.g., in homocystinuria, where plasma Hcy may reach 500 μM 71 ), it is unlikely the pathogenic mechanism in ALS. This is because the extracellular, as demonstrated by CSF, concentrations of both GSH and CSH (or cystine) are very low [72][73][74][75][76][77] . Although the intracellular concentration of GSH in neurons is high [77][78][79] , the CSH content is very low (under the detection limit 77 ) and, therefore, an efficient CSH-dependent GSH oxidation is unlikely in these cells. In astrocytes, by contrast, the intracellular content of GSH is one of the highest among mammalian cells (8-10 mM) [77][78][79] , and the level of CSH is substantial 80 . Supplemented with the ubiquitously expressed SOD1 81 , this combination renders astrocytes a possible locale for the CSH-dependent short-circuiting of GSH oxidation to produce high quantities of H 2 O 2 .
The second question is as to how misfolding may affect the SOD1 catalytic properties to facilitate disease. H 2 O 2 is considered a relatively benign ROS due to the presence of H 2 O 2 -detoxifying enzymes, such as catalase and GSH peroxidases (although the performance of the latter is compromised by low GSH concentrations). It was previously demonstrated that SOD1 can utilize H 2 O 2 as a sole substrate to produce hydroxyl radical 51,92,93 , a highly potent ROS that targets lipids, sugars, DNA bases, amino acids, and organic acids 38,94 , and against which no enzymatic defense exists. In this process, a reduction of SOD1 Cu 2+ by H 2 O 2 (Reaction 3) is followed by a Cu + oxidation by another H 2 O 2 in a Fenton-type reaction to generate a hydroxyl radical (Reaction 4) 51,92,93 : The hydroxyl radical formation is markedly accelerated in the fALS SOD1 mutants, as compared with the native SOD1 WT , a feature attributed to structural instability of the former manifested in the increased openness and accessibility of the active-site Cu 2+ to substrates other than superoxide 35,51,95 . However, due to the low affinity of H 2 O 2 to the SOD1 active site, non-physiologically high concentrations of H 2 O 2 (10-20 mM) were required to produce substantial amounts of HO• 92 , questioning the significance of this reaction to ALS pathogenesis. It was proposed that the HO• formation in the presence of H 2 O 2 could, in principle, be facilitated by a cellular reductant other than H 2 O 2 (whose identity remains unknown) capable of activating the active-site Cu 2+ 35,51 , therefore decreasing the amount of H 2 O 2 required to produce HO• 57,59 . We speculate that ubiquitous thiol compounds may play the role of such activating substance. Moreover, during thiol oxidation, H 2 O 2 substrate of the second step (Reaction 4) is generated by SOD1 itself, thus the effective local concentration of H 2 O 2 near the enzyme's active-site is high. It could be especially true when considering the abnormal tendency of misfolded SOD1 to accumulate on particular locations in the cell 96,97 , hence further increasing local H 2 O 2 concentration and accelerating the reaction by mass action.
Since the thiol oxidation by SOD1 is insensitive to the presence of Mn-SOD, it was concluded that superoxide is not formed as intermediate in this reaction 53 . Such resistance to the Mn-SOD presence, however, might results from the differences in the kinetics of the competing reactions or because peroxide may not be released as a free intermediate 35 . If superoxide is generated during the catalysis, it may react with NO to produce peroxynitrite, a powerful and much more (10,000 folds) stable oxidant than the hydroxyl radical 38,98-100 . Peroxynitrite exhibits a broad range of tissue-damaging effects, including lipid peroxidation, enzyme and ion channel inactivation, and inhibition of mitochondrial respiration, 98 and the decomposition of peroxynitrite produces hydroxyl radicals 101 . www.nature.com/scientificreports www.nature.com/scientificreports/ It has been demonstrated that, in the absence of structural Zn 2+ , the coordination of Cu 2+ in the active site of SOD1 changes, rendering Cu 2+ a much more potent oxidant 35,36 . The Zn 2+ -deficient/Cu 2+ -SOD1 WT and fALS WTL SOD1 mutants were shown to efficiently oxidize ascorbate, with a subsequent reoxidation of SOD1-Cu + by oxygen to produce O 2 • − , which combines with NO afterward to produce peroxynitrite 35 . We speculate that the peroxynitrite production by misfolded metallated SOD1 may benefit, similarly to the HO• formation, by the ability of SOD1 enzyme to accept ubiquitous biological thiols as an alternative substrate for cooper activation.

Methods
Purification of recombinant SOD1 Wt , SOD1 G93A and SOD1 G85R proteins and their reconstitution with metals. The human SOD1 WT , SOD1 G93A and SOD1 G85R were produced in E. coli BL-21 grown without Zn 2+ and Cu 2+ supplements and purified to homogeneity under non-denaturing conditions, as described previously 102 .
The MALDI-TOF mass spectrometry analysis of SOD1 WT (Autoflex ™ speed MALDI TOF/TOF mass spectrometer, Bruker, Germany) ( Supplementary Fig. S5) and the purified protein's low enzymatic activity 3.1 ± 0.2 U/mg, determined by a superoxide dismutase assay kit (Cayman Chemical, Ann Arbor, MI), indicated that this protein is demetallated (apo-SOD1 WT ) or metallated only partially. As shown previously, the recombinant SOD1 WT produced in E. coli and reconstituted with metals by a dialysis after purification, regains its full catalytic activity and could be regarded as holo-SOD1  . Indeed, we showed that saturation with Zn 2+ and Cu 2+ by the dialysis increased the catalytic activity of the recombinant SOD1 WT by three order of magnitude (1200 ± 40 U/mg) to approach that of the SOD1 standard provided with the SOD1 activity assay kit (3700 ± 200 U/mg). The MALDI-TOF analysis revealed that the average molecular mass difference between the metallated and apo-SOD1 WT is consistent, within the instrument's sensitivity limit, with the addition of two metal ions per SOD1 monomer: 108 Da difference for the monomer peak (expected 129 Da) and 280 Da difference for the dimer peak (expected 258 Da), Supplementary Fig. S5.
Specifically, the purified SOD1 proteins were reconstituted with metals by an overnight dialysis against buffer A (50 mM Tris-HCl, pH 7.5, 0.1 M NaCl, and 10% glycerol) supplemented with ZnSO 4 and CuSO 4 (1 mM each). The dialyzed proteins were incubated for 1 h at 4 °C in the presence of 3 mM EDTA to chelate unbound and weakly bound metals, and then dialyzed overnight against buffer A. The dialyzed proteins were further buffer-exchanged using five 1:4 dilution (buffer A) and ultrafiltration (10 kDa cutoff, Amicon, Millipore, Burlington, MA) steps to remove residual metals and EDTA. The protein was then centrifuged at 110,000 × g for 1 h at 4 °C using an ultracentrifuge (Sorvall M120, Discovery, Thermo Fisher Scientific, UK) and stored, at the concentration 30 mg/ml, at −20 °C under argon until used. Protein concentration was measured by the Bradford method, using bovine serum albumin (fatty acid free, Sigma-Aldrich, Israel) as standard, and spectroscopically using a monomeric molar extinction coefficient at 280 nm of 5500 M −1 ·cm −1 (the two methods produced similar estimations).
Cell viability assay. SH-SY5Y cells were seeded in 96-well plates at a starting density of 1.5 × 10 4 cells/ well in a high glucose Dulbecco's modified Eagle medium (DMEM) supplemented with 1% L-glutamine, 1% penicillin-streptomycin, and 10% (vol/vol) fetal bovine serum (FBS), and incubated for 10 h prior experiments. The experiments were performed in the medium containing 2% FBS. At the end of the experiment, the cells were washed once with Hank's balanced salt solution (HBSS) and cell viability was assessed by adding 20 µL of the CellTiter 96 AQueous One Solution reagent (Promega, Fitchburg, WI) to 100 μl HBSS, followed by incubation for 60 min at 37 °C, and the absorbance was measured at 490 nm using the Infinite 200 PRO plate-reader (Tekan, Switzerland). We found that mycoplasma infection decreased the cells susceptibility to SOD1 cytotoxicity; therefore, the cells were treated for two weeks prior the experiments with 25 μg/ml Plasmocin (Invitrogen, Sweden). We also found that the magnitude of the cytotoxic effect of SOD1 was inversely proportional to the cell density; therefore, the cell seeding conditions were adjusted to perform all the experiments at the final confluency of 70%.
In the compartmentalization experiments, SOD1 was separated from the cells cultured in a 24-well plate using a tightly fit dialysis insert equipped with a 3.5-kDa cutoff membrane (Thermo Fisher Scientific, UK).
Colorimetric metal assay. The holo-SOD1 WT (50 μM, monomer based) was incubated for 20 h at 37 °C in 50 mM Hepes buffer, 50 mM NaCl, pH 7.4, in the presence of the indicated concentrations of thiol compounds and 100 μM divalent metal dye 4-(2-pyridylazo)resorcinol (PAR), in 90 μl total volume in 384-well plates covered with a transparent adhesive film (to prevent evaporation). The absorbance was measured at 490 nm and 520 nm using the Infinite 200 PRO plate-reader (Tekan, Switzerland) and the concentrations of dye-complexed copper and zinc ions were determined using the method described by Mulligan et al. 106 .
To determine the concentration of metals present in solution, 100 μM holo-SOD1 WT were incubated for 15 min at 37 °C in 10 mM glycyl-glycine buffer, pH 7.5, 50 mM NaCl, in the absence or presence of 0.5 mM CSH. At the end of incubation, the protein was separated from the reaction mixture by ultra-filtration (5 kDa cutoff, Vivaspin 500, Sartorius, Germany) and the filtrate was analyzed for the presence of metals after a complete oxidation of the remaining thiols (10 h at 37 °C) by adding 100 μM PAR, as described above.
H 2 o 2 assay. H 2 O 2 was determined according to the method described by Pick et al. 107 with some modifications. Briefly, a reagent solution containing 0.56 mM phenol red (Alfa Aesar, UK) and 17 U/ml horseradish peroxidase (Type II, Sigma-Aldrich, Israel) in 10 mM glycyl-glycine buffer, pH 7.5, 50 mM NaCl, was added at a 1:1 volumetric ratio to the analyzed sample, and the mixture was incubated at room temperature for 2 min (5 min produced similar results), followed by the addition of NaOH to the final concentration of 50 mM to reach pH 12.5. The absorbance was measured at 610 nm using the Infinite 200 PRO plate-reader (Tekan, Switzerland).
Free thiol assay. The analyzed samples were incubated for 15 min at room temperature in 20 mM Tris-HCl buffer, pH 7.5, NaCl 100 mM, 1 mM EDTA, 5 % DMSO in the presence of 0.5 mM Ellman's Reagent (DTNB,