| Cu,Zn-superoxide dismutase (SOD1) provides a cellular defense system toward oxidative stress by catalyzing disproportionation of superoxide anion into hydrogen peroxide and dioxygen. SOD1 requires three post-translational modifications to be fully active: copper insertion, zinc insertion, and disulfide formation. Recent studies have revealed that an accessory protein called the copper chaperone for SOD1 (CCS) mediates copper insertion and disulfide formation. In this study, we show that CCS is able to transfer a zinc ion to SOD1 in vitro, and this function appears to be unique in human (or mammalian) CCS. The experimental data reveals that the zinc ion is directly transferred from human CCS (hCCS) domain II to human SOD1 (hSOD1) via a protein-protein interaction. Further biochemical assays implicate that this process is mediated by a cysteine cluster in hCCS domain II. This is the first example of an intracellular zinc chaperone protein, and provides a new paradigm for intracellular zinc trafficking processes.;Although CCS is known to transfer a copper ion to SOD1, quantitative analysis of the copper transfer reaction has never been reported. In vitro assays developed in examination of the zinc transfer process allowed for quantitative evaluation of copper transfer from hCCS to hSOD1. Surprisingly, hCCS transfers only 30-40 % of its bound copper ions, while copper transfer seems to saturate at less than 0.5 moles of copper ion per hSOD1 monomer, resulting in hSOD1 with high enzymatic activity. These observations imply substoichiometric copper binding in hSOD1 in vivo, which is consistent with previous studies reporting significant pool of Cu-free SOD1 in vivo.;Point mutations in the sod1 gene cause neurodegenerative disease, ALS. Based on the structural similarity and functional relevance, screening the ccs gene for mutations in patients with ALS has been conducted, and two mutations in ALS cases have been identified: R71W mutation in a sporadic ALS case and G222R mutation in a familial case. Biochemical and biophysical analysis demonstrates that ALS-associated CCS mutants present remarkable defects in protein folding/refolding and a significantly increased propensity to form fibrillar aggregates, which are general hallmark of neurodegenerative disease such as Alzheimer's disease, Parkinson's disease, and Bovine spongiform encephalopathy (prion disease). |
