**4. Molecular link between copper‐mediated chemistry and cysteine oxidation in mutant SOD1**

Oxidative reactivity and modification of Cys111, such as glutathionylation [28, 29] and peroxidation [30], is documented with human or chick wild‐type SOD1. Because Cys111 is located on the edge of the dimer interface of each subunit, the modification of Cys111 can interrupt the dimer contact at the interface stereochemically and cause the dissociation of SOD1. Molecular dynamic simulations of SOD1 imply that the region including Cys111 is important for the residue interaction network in the protein and is likely to affect the dimer interface through the network and may disrupt their coupled motions [31]. Indeed, it was noted *in vitro* that the Cys111 modification caused wild‐type SOD1 liable to monomerize and decrease its enzymatic activity [32]. On the other hand, substitution of Cys111 to serine (C111S) is known to increase the structural stability and resistance to heat inactivation of wild‐type SOD1 [33], also implying that the mode of Cys111 may regulate the conformational state of

Changes in the redox state of cysteine residues have been reported in ALS‐linked mutant SOD1. Mutant SOD1 exhibits aberrant vulnerability to mild reducing conditions, which cleave the intra‐subunit Cys57‐Cys146 disulfide bond to destabilize the SOD1 dimer [34]. The dimer dissociation results in the exposure of the hydrophobic region of the SOD1 subunit and promotes aggregation of the protein [35]. Alternatively, insoluble mutant SOD1 oligomers can be formed by crosslinking via inter‐subunit disulfide bonds at Cys57 and Cys146 [26] or by disulfide scrambling of all four cysteine residues [36]. Such insoluble SOD1 oligomers were also detected in the spinal cord of mutant SOD1 transgenic mice in parallel to the disease onset [37]. These oligomers were mostly reversed by a reducing reagent, supposing that disulfide‐ mediated crosslinking at cysteine residues is a major factor for mutant SOD1 to form aggre‐ gates and ALS phenotype. Conversely, replacement of cysteine residues, especially of Cys6 and Cys111, decreased disulfide‐crosslinked mutant SOD1 oligomers and aggregate forma‐ tion, and improved cell viability in cultured cells [38, 39]. Glutaredoxins, which specifically catalyze the reduction of protein‐SSG‐mixed disulfides, significantly increased the solubility of mutant SOD1 and protected neuronal cells [39, 40]. On the other hand, the intermolecular disulfide binding at cysteines is shown to have a limited effect on the aggregation of mutant

With regard to Cys111, posttranslational modifications of Cys111 per se are also known in mutant SOD1. The change of the protein structure, which would affect the hindrance of Cys111 near the dimer interface, can enhance oxidative modification of Cys111 at the sulfhydryl moiety by substrates in mutant SOD1. Mutant SOD1 is commonly glutathionylated at Cys111 [42], and Cys111‐peroxidized SOD1 is detected in the inclusion bodies of spinal motor neurons in G93A mutant SOD1 transgenic mice [30]. Those indicate the pathogenic significance of Cys111‐oxidized SOD1 for misfolding and aggregation to acquire neuronal toxicity. Moreover, even in the spinal cord of sporadic ALS patients without SOD1 mutation, misfolded SOD1 deposits have been detected and the SOD1 species are peroxidized at Cys111, indicating that misfolding and aggregation of wild‐type SOD1 may also be a factor in the pathogenesis of sporadic ALS [43]. However, in the vast majority of sporadic ALS, an RNA‐binding protein TDP‐43 is well known to mislocalize from the nucleus and deposit in the cytoplasm [44, 45]. SOD1 does not interact or co‐localize with TDP‐43 in general in such cases [46], which is

SOD1.

90 Update on Amyotrophic Lateral Sclerosis

SOD1 [41].

Then, what is the molecular mechanism by which mutant SOD1 causes the copper‐mediated oxidative stress in relation to misfolding of the protein? In case that mutant SOD1 enhances an aberrant side reaction through the discoordinated copper due to the abnormality of the protein structure, we can develop treatment strategies by identifying the responsible site and its conformational state in the protein. To clarify a possible aberrant interaction of mutant SOD1 with copper, we fractionated cell lysates from the spinal cord of SOD1 transgenic mice and SOD1 expressing cultured cells by immobilized metal affinity chromatography (IMAC), a method that separates proteins based on their affinities with an immobilized metal such as copper [47]. Mutant SOD1 was eluted commonly in an aberrant fraction with high affinity for copper, in addition to that with low affinity for copper seen in wild‐type SOD1 as well. Considering that mutant SOD1 is separated into two distinct fractions and the interaction of proteins on IMAC is determined by topology of metal‐coordinating residues on solvent‐facing surfaces [48], conformational transition from the native to non‐native state is implicated in the high‐affinity fraction for the copper of mutant SOD1.

Therefore, we further analyzed mutant SOD1 in the high‐affinity fraction for copper to know its biochemical characteristics compared to that in the low‐affinity fraction. Existence of Cys111 was critical to the appearance of the high‐affinity fraction species, and mutant SOD1 was in a monomer state and oxidatively modified at Cys111 in this fraction [49]. Peroxidation of wild‐ type SOD1 forced by oxidants such as hydrogen peroxide made it to monomerize and generate the high‐affinity fraction species by copper IMAC. Furthermore, these mutant SOD1 and Cys111‐peroxidized wild‐type SOD1 showed higher thiol oxidase activity, an adverse side activity reported in SOD1 [50], than untreated wild‐type SOD1. These results indicate that mutant SOD1 is labile to be monomerized by peroxidaton of Cys111 near the dimer interface, which will expose the copper of the protein and raise its reactivity to cause oxidative stress, and eventually forms intracellular aggregates or inclusion bodies to cause neurodegeneration (**Figure 1**).

**Figure 1.** Proposed model of mutant SOD1 toxicity.

Oxidation of Cys111 leads mutant SOD1 dimers to dissociate into monomers, which causes oxidative stress through the copper and aggregate formation of the monomers.
