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J Biol Chem. 2014 Sep 26;289(39):26722-32. doi: 10.1074/jbc.M114.565333. Epub 2014 Aug 4.

Altered thiol chemistry in human amyotrophic lateral sclerosis-linked mutants of superoxide dismutase 1.

Author information

1
From the Laboratory of Cellular and Molecular Neurobiology, Department of Pathology and Experimental Therapeutics, Faculty of Medicine-Campus Bellvitge, University of Barcelona, Feixa Llarga s/n. Hospitalet de Llobregat, 08907 Barcelona, Spain, the Bellvitge Biomedical Research Institute (IDIBELL), Gran Via de l'Hospitalet, 199-203, L'Hospitalet de Llobregat, Barcelona, 08908 Barcelona, Spain, solsona@ub.edu.
2
the Department of Biochemistry and Molecular Biophysics, Columbia University Medical Center, New York, New York 10032, the Department of Biological Sciences, Columbia University, New York, New York 10027, and.
3
the Department of Biological Sciences, Columbia University, New York, New York 10027, and.
4
From the Laboratory of Cellular and Molecular Neurobiology, Department of Pathology and Experimental Therapeutics, Faculty of Medicine-Campus Bellvitge, University of Barcelona, Feixa Llarga s/n. Hospitalet de Llobregat, 08907 Barcelona, Spain, the Bellvitge Biomedical Research Institute (IDIBELL), Gran Via de l'Hospitalet, 199-203, L'Hospitalet de Llobregat, Barcelona, 08908 Barcelona, Spain.
5
the Department of Biological Sciences, Columbia University, New York, New York 10027, and the Vascular Biology and Inflammation Department, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Cl. Melchor Fernández Almagro 3, 28029 Madrid, Spain.

Abstract

Neurodegenerative diseases share a common characteristic, the presence of intracellular or extracellular deposits of protein aggregates in nervous tissues. Amyotrophic Lateral Sclerosis (ALS) is a severe and fatal neurodegenerative disorder, which affects preferentially motoneurons. Changes in the redox state of superoxide dismutase 1 (SOD1) are associated with the onset and development of familial forms of ALS. In human SOD1 (hSOD1), a conserved disulfide bond and two free cysteine residues can engage in anomalous thiol/disulfide exchange resulting in non-native disulfides, a hallmark of ALS that is related to protein misfolding and aggregation. Because of the many competing reaction pathways, traditional bulk techniques fall short at quantifying individual thiol/disulfide exchange reactions. Here, we adapt recently developed single-bond chemistry techniques to study individual disulfide isomerization reactions in hSOD1. Mechanical unfolding of hSOD1 leads to the formation of a polypeptide loop held by the disulfide. This loop behaves as a molecular jump rope that brings reactive Cys-111 close to the disulfide. Using force-clamp spectroscopy, we monitor nucleophilic attack of Cys-111 at either sulfur of the disulfide and determine the selectivity of the reaction. Disease-causing mutations G93A and A4V show greatly altered reactivity patterns, which may contribute to the progression of familial ALS.

KEYWORDS:

Amyotrophic Lateral Sclerosis (ALS) (Lou Gehrig disease); Atomic Force Microscopy (AFM); Atomic Force Spectroscopy; Cysteine-mediated Cross-linking; Disulfide; Neurodegenerative Disease; Protein Misfolding; Superoxide Dismutase (SOD)

PMID:
25096579
PMCID:
PMC4175315
DOI:
10.1074/jbc.M114.565333
[Indexed for MEDLINE]
Free PMC Article

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