Metal Deficiency Increases Aberrant Hydrophobicity of Mutant Superoxide Dismutases That Cause Amyotrophic Lateral Sclerosis

Osman Bilsel,Lawrence J Hayward,C.Robert Matthews,Amir Liba,Sai V Seetharaman,Joan Selverstone Valentine,Se Hui Sohn,Ashutosh Tiwari,P.John Hart

doi:10.1074/jbc.m109.043729

Abstract

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.

Highlights

Familial forms of amyotrophic lateral sclerosis (ALS) is unknown
Our group and others subsequently showed that the mutant SOD1 proteins share a susceptibility to increased hydrophobicity under conditions that reduce disulfide bonds and/or chelate metal ions [5] and that similar hydrophobic species exist in tissue lysates from mutant SOD1 transgenic mice (4 – 6)
One consequence of such hydrophobic exposure could be the facilitation of abnormal interactions between the mutant enzymes and other cellular constituents, which might influence pathways leading to motor neuron death (15, 16, 24 –27)

Summary

EXPERIMENTAL PROCEDURES

Materials—EDTA was from Invitrogen; sodium phosphate (monobasic and dibasic), potassium phosphate (monobasic and dibasic), KCl, NaCl, glycine, and Tris base were from J. Titration of ANS into the buffer without protein yielded a fluorescence peak at 518 Ϯ 1.5 nm for free ANS that was independent of the ANS concentration, and these curves provided a constraint for subsequent fitting. ANS fluorescence values at emission wavelengths from 400 – 600 nm were fitted simultaneously with the Kd as a global variable parameter. The bound ANS fluorescence intensity at each wavelength (for 201 values using 1-nm increments) was treated as a local variable parameter to be optimized during the fit. 203 parameters (201 local and 2 global) were simultaneously fitted to 201 binding curves This analysis yielded a Kd value for ANS binding and allowed us to deconvolute the spectra for bound versus free ANS fluorescence. After the addition and mixing of metal ions, the protein solutions were centrifuged at 20,000 ϫ g for 5 min at 4 °C before spectral observations and were maintained at 4 °C between successive scans

RESULTS

Raw Data Fit Curve Bound ANS Free ANS

Iodoacetamide tagging followed by

DISCUSSION