**6. Role of cysteine-oxidized mutant SOD1 in familial ALS**

What is the role of Cys111 modification on the neuronal toxicity by mutant SOD1? In the spinal cords of familial ALS patients and mutant SOD1 transgenic mice, degenerating motor neurons contain SOD1-positive inclusion bodies, suggesting that mutant SOD1 is conformationally misfolded and subject to aggregate (Chattopadhyay & Valentine, 2009). As seen in other neurodegenerative diseases, abnormal protein accumulation in neurons can impair their cellular functions such as axonal transport (Tateno et al., 2009), oxidative phosphorylation in mitochondria and protein degradation machinery.

Various factors can cause conformational rearrangement or misfolding of mutant SOD1, including decreased metallation (Hayward et al., 2002), hydrophobicity (Tiwari et al., 2005) and reduction of repulsive charge (Sandelin et al., 2007). Modification of amino acid residues, especially by oxidative stress, can be a critical factor to enhance the misfolding of mutant SOD1 (Rakhit & Chakrabartty, 2006). 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 inter-molecular disulfide bond, which have important roles to maintain or disrupt physiological conformation of proteins. Oxidative reactivity and modification of Cys111, such as glutathionylation (Kajihara et al., 1988; Schinina et al., 1996) and peroxidation (Fujiwara et al., 2007), was documented with human or chick wild type SOD1, although the effect of which on the enzymatic activity or dimer stability had not been determined. Because Cys111 is located on the edge of the dimer interface of each subunit, the modification of Cys111, especially when a large molecule such as glutathione is adducted to the residue, can interrupt the dimer contact at the interface stereochemically and cause the dissociation of SOD1. Molecular dynamics simulations of SOD1 imply that the region including Cys111 is important for the residue interaction network in the protein, and likely to affect the dimer interface through the network and may disrupt their coupled motions (Khare & Dokholyan, 2006). In fact, glutathionylation of Cys111 has been confirmed with native human SOD1 in erythrocytes (Nakanishi et al., 1998; Wilcox et al., 2009), and it was noted that the modification caused SOD1 liable to monomerize and decrease its enzymatic activity (Wilcox et al., 2009). The SOD1 monomer is prone to form aggregates that might be the origin of intracellular inclusions found in motor neurons with SOD1-linked familial ALS. Supporting that, Cys111-peroxidized SOD1 was detected in the neuronal inclusions of mutant SOD1 mice (Fujiwara et al., 2007).

Oxidative modification of cysteine residues, including Cys111, is also possible to be involved in the aggregation process of mutant SOD1. High molecular weight dimers and multimers of mutant SOD1 can be detected in the spinal cords of transgenic mice in parallel to the disease onset (Deng et al., 2006; Furukawa et al., 2006). They are detergentinsoluble and reversed by reductants, supposing that disulfide-mediated crosslinking at cysteine residues is a major factor for mutant SOD1 to form aggregates and ALS phenotype. Cysteines forming the intramolecular disulfide bond (Cys57 and Cys146) are possibly involved in the crosslinking, since the disulfide bond between the residues is labile to be reduced (Tiwari & Hayward, 2003) and cause aberrant oxidation in mutant SOD1. The disulfide-reduced mutant SOD1 is actually enriched in the spinal cord of transgenic mice (Jonsson et al., 2006). The reduced form of mutant SOD1 can also translocate into the intermembrane space of mitochondria cooperated by CCS (Field et

Oxidative Modifications of Cu, Zn-Superoxide

can be the first-line treatment strategy of this incurable disease.

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**8. Conclusion** 

**9. References** 

Dismutase (SOD1) – The Relevance to Amyotrophic Lateral Sclerosis (ALS) 307

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al., 2003), which may be components of aggregates in mitochondria (Deng et al., 2006; Ferri et al., 2006) and harmful to the mitochondrial function. However, intermolecular disulfide bonds mediated at free cysteines (Cys6 and Cys111) can also be components of the detergent-insoluble SOD1 aggregates (Banci et al., 2007; Niwa et al., 2007). In either case, apo SOD1 is more prone to the disulfide-linked oxidative aggregation than holo SOD1 (Banci et al., 2007; Furukawa & O'Halloran, 2005). That is in concert with the notion that immature SOD1 is the pathogenic species in familial ALS (Seetharaman et al., 2009).

It is still controversial whether the cysteine-mediated misfolding or aggregation of mutant SOD1 is the origin of the protein's toxicity. Removal of free cysteines, especially of Cys111, strongly reduced the ability of mutant SOD1 to form disulfide crosslinking and aggregates, and improved cell viability in cultured cells (Cozzolino et al., 2008; Niwa et al., 2007). Moreover, glutaredoxins, which specifically catalyze the reduction of protein-SSG-mixed disulfides, significantly increased the solubility of mutant SOD1 and protected neuronal cells (Cozzolino et al., 2008; Ferri et al., 2010). On the other hand, the intermolecular disulfide binding at cysteines is shown to have a limited effect on the aggregation of mutant SOD1 (Karch & Borchelt, 2008). Even in this case, Cys111-modified mutant SOD1 may cause neuronal toxicity independently of the aggregation, by oxidative stress such as thiol oxidase activity we have shown (Kishigami et al., 2010).
