D2 alone is sufficient to interact with the immature, metastable state of fALS SOD1 mutants, and effectively prevents the accumulation of unstructured species. We showed that the SOD-like domain of CCS, D2, acts as a molecular chaperone in the human cytoplasm. We characterized by in-cell NMR the interaction between CCS and SOD1, both WT and the WT-L fALS mutants A4V, T54R, G93A, I113T, in the cytoplasm of living human cells. Here, we investigated further how CCS exerts this protective role. Importantly, this mechanism can be reverted by co-expressing CCS, indicating that the copper chaperone is able to pull the nascent SOD1 molecules towards the correct folding and maturation pathway. It has been shown that some WT-L fALS SOD1 mutants fail to bind zinc when they are overexpressed in the human cytoplasm, and accumulate as unstructured species that may act as precursors in the pathogenic aggregation pathway 28. Recently, the maturation steps of SOD1 have been observed in living human cells 25 through in-cell NMR 26, 27. CCS acts as a metallochaperone through its N-terminal Atx1-like domain (D1) and as oxidoreductase through its C-terminal domain (D3), whereas the second, SOD-like domain (D2) is responsible for the interaction with SOD1 15– 24. Copper binding and disulfide bond formation are mediated by a specific partner, the Copper Chaperone for SOD1 (CCS) 15, 16. Disulfide-reduced, monomeric apo-SOD1 binds zinc spontaneously, and forms a stable homodimer 13, 14. The molecular events leading the mature, enzymatically active form of SOD1 have been extensively studied in vitro. These mutations however are known to further destabilize the structure of the apo protein, increasing the fraction of unfolded protein and eventually leading to misfolding and to the formation of the cytotoxic species 7, 8, 11, 12. Some fALS mutations, termed wild type-like (WT-L), do not affect the enzymatic activity of the mature protein, as they do not perturb the geometry of the SOD1 metal binding sites, nor the disulfide bond 9, 10. All these post-translational events stabilize the fold of SOD1, which in the apo state is intrinsically unstable and prone to unfolding and aggregation 7, 8. In order to reach its active conformation, SOD1 has to undergo several post-translational maturation events in the cytoplasm, which include zinc binding, dimerization, copper binding and the formation of an intramolecular disulfide bond. These mutations have been linked to the formation of amorphous aggregates enriched in SOD1 in the spinal cord of fALS patients and in transgenic mice 3– 6. Several missense mutations in the sod1 gene scattered throughout the polypeptide of SOD1 have been correlated with the onset of fALS 2. It has been shown that SOD1 is implicated in a sizable fraction of the familial variants of ALS (fALS) 1. Human copper, zinc superoxide dismutase (SOD1) is a conserved intracellular metalloprotein that protects the cell from oxidative damage. Such molecular chaperone function of CCS-D2 is novel and its implications in SOD-linked fALS deserve further investigation.Īmyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by the death of motor neurons in the brain and spinal cord. We further showed that CCS-D2 interacts with apo-SOD1 in vitro, suggesting that in cells CCS stabilizes mutant apo-SOD1 prior to zinc binding. We showed that CCS-D2 forms a stable complex with zinc-bound SOD1 in human cells, that has a twofold stabilizing effect: it both prevents the accumulation of unstructured mutant SOD1 and promotes zinc binding. By in vitro and in-cell NMR, we investigated the role of the SOD-like domain of CCS (CCS-D2). The Copper Chaperone for SOD1 (CCS) transiently interacts with SOD1 and promotes its correct maturation by transferring copper and catalyzing disulfide bond formation. Some mutations destabilize the apo protein, leading to the formation of misfolded, toxic species. Superoxide dismutase 1 (SOD1) is an important metalloprotein for cellular oxidative stress defence, that is mutated in familiar variants of Amyotrophic Lateral Sclerosis (fALS).
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