Metal deficiency increases aberrant hydrophobicity of mutant superoxide dismutases that cause amyotrophic lateral sclerosis.
The mechanisms by which mutant variants of Cu/Zn-superoxide dismutase (SOD1) cause familial amyotrophic lateral sclerosis are not clearly understood. Evidence to date suggests that altered conformations of amyotrophic lateral sclerosis mutant SOD1s trigger perturbations of cellular homeostasis that ultimately cause motor neuron degeneration. In this study we correlated the metal contents and disulfide bond status of purified wild-type (WT) and mutant SOD1 proteins to changes in electrophoretic mobility and surface hydrophobicity as detected by 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence. As-isolated WT and mutant SOD1s were copper-deficient and exhibited mobilities that correlated with their expected negative charge. However, upon disulfide reduction and demetallation at physiological pH, both WT and mutant SOD1s underwent a conformational change that produced a slower mobility indicative of partial unfolding. Furthermore, although ANS did not bind appreciably to the WT holoenzyme, incubation of metal-deficient WT or mutant SOD1s with ANS increased the ANS fluorescence and shifted its peak toward shorter wavelengths. This increased interaction with ANS was greater for the mutant SOD1s and could be reversed by the addition of metal ions, especially Cu(2+), even for SOD1 variants incapable of forming the disulfide bond. Overall, our findings support the notion that misfolding associated with metal deficiency may facilitate aberrant interactions of SOD1 with itself or with other cellular constituents and may thereby contribute to neuronal toxicity.
Tiwari
Ashutosh
A
Department of Neurology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA. tiwari@mtu.edu
Liba
Amir
A
Sohn
Se Hui
SH
Seetharaman
Sai V
SV
Bilsel
Osman
O
Matthews
C Robert
CR
Hart
P John
PJ
Valentine
Joan Selverstone
JS
Hayward
Lawrence J
LJ
D000690:Amyotrophic Lateral Sclerosis / Q000201:enzymology*; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D004586:Electrophoresis; D020035:Holoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D008670:Metals / Q000378:metabolism*; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011487:Protein Conformation; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000072105:Superoxide Dismutase-1; D017186:Titrimetry
D000817:Anilino Naphthalenesulfonates; D004220:Disulfides; D020035:Holoenzymes; D008670:Metals; D050505:Mutant Proteins; C000606290:SOD1 protein, human; C027132:1-anilino-8-naphthalenesulfonate; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1