Over 100 mutations in the gene encoding human copper-zinc superoxide dismutase (SOD1) cause an inherited form of the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS). Two pathogenic SOD1 mutations, His46Arg (H46R) and His48Gln (H48Q), affect residues that act as copper ligands in the wild type enzyme. Transgenic mice expressing a human SOD1 variant containing both mutations develop paralytic disease akin to ALS. Here we show that H46R/H48Q SOD1 possesses multiple characteristics that distinguish it from the wild type. These properties include: 1) an ablated copper-binding site; 2) a substantially weakened affinity for zinc; 3) a binding site for calcium ion; 4) the ability to form stable heterocomplexes with the Copper Chaperone for SOD1 (CCS); and 5) compromised CCS-mediated oxidation of the intrasubunit disulfide bond in vivo. The results presented here, together with data on pathogenic SOD1 proteins coming from cell culture and transgenic mice, suggest that incomplete posttranslational modification of nascent SOD1 polypeptides via CCS may be a characteristic shared by fALS SOD1 mutants, leading to a population of destabilized, off-pathway folding intermediates that are toxic to motor neurons.The homodimeric antioxidant enzyme copper-zinc superoxide dismutase (SOD1) has been studied for nearly four decades. In 1993, interest in the molecule intensified when mutations in the gene encoding SOD1 were linked to the lethal neurodegenerative disease amyotrophic lateral sclerosis (ALS) (1,2). Since then, ∼100 distinct pathogenic mutations have been documented [reviewed in (3)], with most resulting in single amino acid substitutions and a few in truncations in the C-terminal portion of the poypeptide. . Supporting Information Available: Supplementary Figure 1 shows additional details of the calcium binding site and its role in SOD1 crystal packing interactions. Supplementary Figure 2 shows additional details of H46R/H48Q SOD1 interactions with CCS in solution using analytical ultracentrifugation and analytical gel filtration. This material is available free of charge via the Internet at http://pubs.acs.org". NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 April 21. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptLandmark studies in transgenic mice established that pathogenic SOD1 proteins elicit motor neuron dysfunction through the acquisition of a deleterious property and not a loss of enzymatic function (4-6). SOD1-enriched inclusions are observed in cell culture model systems, ALS-SOD1 transgenic mice, and in fALS patients, suggesting that SOD1-linked ALS pathology is related to misfolding or aggregation [reviewed in (7-9)]. However, the precise molecular mechanism(s) underlying SOD1 toxicity to motor neurons is unknown, and it remains to be clarified whether the observed inclusions are causal or symptomatic of motor neuron dysfunction.Given the observations described above, ALS-mutant SOD1 proteins likely possess propert...

The CCS copper chaperone is critical for maturation of Cu, Zn-superoxide dismutase (SOD1) through insertion of the copper co-factor and oxidization of an intra-subunit disulfide. The disulfide helps stabilize the SOD1 polypeptide, which can be particularly important in cases of amyotrophic lateral sclerosis (ALS) linked to misfolding of mutant SOD1. Surprisingly, however, over-expressed CCS was recently shown to greatly accelerate disease in a G93A SOD1 mouse model for ALS. Herein we show that disease in these G93A/CCS mice correlates with incomplete oxidation of the SOD1 disulfide. In the brain and spinal cord, CCS over-expression failed to enhance oxidation of the G93A SOD1 disulfide and if anything, effected some accumulation of disulfide-reduced SOD1. This effect was mirrored in culture with a C244,246S mutant of CCS that has the capacity to interact with SOD1 but can neither insert copper nor oxidize the disulfide. In spite of disulfide effects, there was no evidence for increased SOD1 aggregation. If anything, CCS over-expression prevented SOD1 misfolding in culture as monitored by detergent insolubility. This protection against SOD1 misfolding does not require SOD1 enzyme activation as the same effect was obtained with the C244,246S allele of CCS. In the G93A SOD1 mouse, CCS over-expression was likewise associated with a lack of obvious SOD1 misfolding marked by detergent insolubility. CCS over-expression accelerates SOD1-linked disease without the hallmarks of misfolding and aggregation seen in other mutant SOD1 models. These studies are the first to indicate biological effects of CCS in the absence of SOD1 enzymatic activation.