**3. Conformational change and oxidation of cysteine residues in mutant SOD1**

The subunit structure and dimer formation of SOD1 is critically affected by the binding state of copper and zinc, as well as the redox state of cysteine residues in the protein [24]. Human SOD1 has four cysteine residues (Cys6, Cys57, Cys111, and Cys146) in a subunit. Cys57 and Cys146 form an intra‐subunit disulfide bond that maintains the rigid structure and enzymatic activity of SOD1 protein, whereas Cys6 and Cys111 are present in a reduced state having free sulfhydryl groups. Cys6 is deeply buried in the core of the subunit and less accessible by other molecules, while Cys111 is located on the protein surface. The intra‐subunit Cys57‐Cys146 disulfide bond of SOD1 is physiologically formed by copper chaperone for SOD1 (CCS) coupled with copper incorporation into the enzymatic active site, meaning that the metal coordination and disulfide formation are mechanistically related to each other for the confor‐ mation of SOD1 protein [25]. Reduction of the Cys57‐Cys146 disulfide bond and/or deprivation of metals make human wild‐type SOD1 liable to misfold, resulting in monomerization [26].

Modification of amino acid residues, especially by oxidative stress, can be a critical factor to enhance the misfolding of proteins [27]. Cysteine is in particular susceptible to oxidative modification, since its sulfhydryl moiety is readily attacked by redox‐active substrates such as glutathione or peroxides to form S‐S or S‐O covalent modification. Sulfhydryl groups also crosslink each other to form intra‐ or intermolecular disulfide bond, which have important roles to maintain or disrupt physiological conformation of proteins.

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 SOD1.

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 SOD1 [41].

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 inconsistent with the hypothesis mentioned above. Although the involvement of Cys111‐ mediated misfolded SOD1 may be limited in sporadic ALS, the theory is attractive and further investigation will be needed.
