ALS-causing SOD1 mutations promote production of copper-deficient misfolded species |
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Authors: | Ip Philbert Mulligan Vikram Khipple Chakrabartty Avijit |
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Institution: | 1 Campbell Family Institute for Cancer Research, Ontario Cancer Institute/University Health Network, Department of Biochemistry, University of Toronto, TMDT 4-305, 101 College Street, Toronto, Ontario, Canada M5G 1L72 Campbell Family Institute for Cancer Research, Ontario Cancer Institute/University Health Network, Department of Medical Biophysics, University of Toronto, TMDT 4-307, 101 College Street, Toronto, Ontario, Canada M5G 1L7 |
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Abstract: | Point mutations scattered throughout the sequence of Cu,Zn superoxide dismutase (SOD1) cause a subset of amyotrophic lateral sclerosis (ALS) cases. SOD1 is a homodimer in which each subunit binds one copper atom and one zinc atom. Inclusions containing misfolded SOD1 are seen in motor neurons of SOD1-associated ALS cases. The mechanism by which these diverse mutations cause misfolding and converge on the same disease is still not well understood. Previously, we developed several time-resolved techniques to monitor structural changes in SOD1 as it unfolds in guanidine hydrochloride. By measuring the rates of Cu and Zn release using an absorbance-based assay, dimer dissociation through chemical cross-linking, and β-barrel conformation changes by tryptophan fluorescence, we established that wild-type SOD1 unfolds by a branched pathway involving a Zn-deficient monomer as the dominant intermediate of the major pathway, and with various metal-loaded and Cu-deficient dimers populated along the minor pathway. We have now compared the unfolding pathway of wild-type SOD1 with those of A4V, G37R, G85R, G93A, and I113T ALS-associated mutant SOD1. The kinetics of unfolding of the mutants were generally much faster than those of wild type. However, all of the mutants utilize the minority pathway to a greater extent than the wild-type protein, leading to greater populations of Cu-deficient intermediates and decreases in Zn-deficient intermediates relative to the wild-type protein. The greater propensity of the mutants to populate Cu-deficient states potentially implicates these species as a pathogenic form of SOD1 in SOD1-associated ALS and provides a novel target for therapeutic intervention. |
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Keywords: | SOD1 Cu Zn superoxide dismutase ALS amyotrophic lateral sclerosis fALS familial ALS PAR 4-(2-pyridylazo)resorcinol TCEP Tris(2-carboxyethyl)phosphine hydrochloride |
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