sodium-dodecyl-sulfate and Amyotrophic-Lateral-Sclerosis

Accumulation of misfolded Cu/Zn superoxide dismutase (SOD1) occurs in patients with a subgroup of familial amyotrophic lateral sclerosis (fALS). To identify the conversion of SOD1 from a normally soluble form to insoluble aggregates, we investigated the change of SOD1 solubility with aging in fALS-linked H46R SOD1 transgenic mice. Mutant SOD1 specifically altered to insoluble forms, which were sequentially separated into Triton X-100-insoluble/sodium dodecyl sulfate (SDS)-soluble and SDS-insoluble/formic acid-soluble species. In spinal cords, the levels of SDS-dissociable soluble SOD1 monomers and SDS-stable soluble dimers were significantly elevated before motor dysfunction onset. In COS-7 cells expressing H46R SOD1, treatment with proteasome inhibitors recapitulated the alteration of SOD1 solubility in transgenic mice. In contrast, overexpression of Hsp70 reduced accumulation of mutant-specific insoluble SOD1. SDS-soluble low molecular weight species of H46R SOD1 may appear as early misfolded intermediates when their concentration exceeds the capacity of the proteasome and molecular chaperones.

Topics: Age Factors; Aging; Amyotrophic Lateral Sclerosis; Animals; Chlorocebus aethiops; COS Cells; Disease Progression; HSP40 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Humans; Mice; Mice, Transgenic; Mutation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Folding; Sodium Dodecyl Sulfate; Solubility; Superoxide Dismutase; Superoxide Dismutase-1

Although more than 100 mutations have been identified in the copper/zinc superoxide dismutase (Cu/Zn-SOD) in familial amyotrophic lateral sclerosis (FALS), the mechanism responsible for FALS remains unclear. The finding of the present study shows that FALS-causing mutant Cu/Zn-SOD proteins (FALS mutant SODs), but not wild-type SOD, are barely detected by three monoclonal antibodies (mAbs) in Western blot analyses. The enzyme-linked immunosorbent assay for denatured FALS mutant SODs by dithiothreitol, SDS, or heat treatment also showed a lowered immunoreactivity against the mAbs compared with wild-type SOD. Because all the epitopes of these mAbs are mapped within the Greek key loop (residues 102-115 in human Cu/Zn-SOD), these data suggest that different conformational changes occur in the loop between wild-type and FALS mutant SODs during the unfolding process. Circular dichroism measurements revealed that the FALS mutant SODs are sensitive to denaturation by dithiothreitol, SDS, or heat treatment, but these results do not completely explain the different recognition by the mAbs between wild-type and FALS mutant SODs under the denatured conditions. The study on the conformational changes in local areas monitoring with mAbs may provide a new insight into the etiology of FALS.

Topics: Amino Acid Sequence; Amyotrophic Lateral Sclerosis; Animals; Antibodies, Monoclonal; Blotting, Western; Cell Line; Circular Dichroism; Dithiothreitol; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Humans; Mice; Models, Molecular; Molecular Sequence Data; Mutagenesis; Mutation; Peptides; Sodium Dodecyl Sulfate; Superoxide Dismutase; Ultraviolet Rays