5 research outputs found
Atomic-resolution monitoring of protein maturation in live human cells by NMR
We use NMR directly in live human cells to describe the complete post-translational maturation process of human superoxide dismutase 1 (SOD1). We follow, at atomic resolution, zinc binding, homodimer formation and copper uptake, and discover that copper chaperone for SOD1 oxidizes the SOD1 intrasubunit disulfide bond through both copper-dependent and copper-independent mechanisms. Our approach represents a new strategy for structural investigation of endogenously expressed proteins in a physiological (cellular) environment
A random set scoring model for prioritization of disease candidate genes using protein complexes and data-mining of GeneRIF, OMIM and PubMed records
A molecular chaperone activity of CCS restores the maturation of SOD1 fALS mutants
Abstract Superoxide dismutase 1 (SOD1) is an important metalloprotein for cellular oxidative stress defence, that is mutated in familiar variants of Amyotrophic Lateral Sclerosis (fALS). Some mutations destabilize the apo protein, leading to the formation of misfolded, toxic species. The Copper Chaperone for SOD1 (CCS) transiently interacts with SOD1 and promotes its correct maturation by transferring copper and catalyzing disulfide bond formation. By in vitro and in-cell NMR, we investigated the role of the SOD-like domain of CCS (CCS-D2). 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. 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. Such molecular chaperone function of CCS-D2 is novel and its implications in SOD-linked fALS deserve further investigation
In-cell NMR reveals potential precursor of toxic species from SOD1 fALS mutants
Mutations in the superoxide dismutase 1 (SOD1) gene are related to familial cases of
amyotrophic lateral sclerosis (fALS). Here we exploit in-cell NMR to characterize the protein
folding and maturation of a series of fALS-linked SOD1 mutants in human cells and to obtain
insight into their behaviour in the cellular context, at the molecular level. The effect of various
mutations on SOD1 maturation are investigated by changing the availability of metal ions in
the cells, and by coexpressing the copper chaperone for SOD1, hCCS. We observe for most of
the mutants the occurrence of an unstructured SOD1 species, unable to bind zinc.
This species may be a common precursor of potentially toxic oligomeric species, that are
associated with fALS. Coexpression of hCCS in the presence of copper restores the correct
maturation of the SOD1 mutants and prevents the formation of the unstructured species,
confirming that hCCS also acts as a molecular chaperone