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.
Highlights
Lou Gehrig’s disease is accompanied by misfolding and aggregation of proteins
In human superoxide dismutase 1 (SOD1), a conserved disulfide bond and two free cysteine residues can engage in anomalous thiol/disulfide exchange resulting in non-native disulfides, a hallmark of Amyotrophic Lateral Sclerosis (ALS) that is related to protein misfolding and aggregation
Disulfide 57–146 Is Buried in the Folded State of human SOD1 (hSOD1)— Thiol/disulfide exchange reactions involving disulfide 57–146 and the free cysteines of hSOD1 have been proposed to contribute to aggregation and misfolding in ALS [15, 16, 26, 37,38,39]
Summary
Lou Gehrig’s disease is accompanied by misfolding and aggregation of proteins. Results: The intramolecular reactivity of cysteines of superoxide dismutase 1 results in new disulfide bonds in unusual positions. 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. Non-native disulfide formation contributes to form and/or stabilize SOD1 aggregates that accompany progression of the disease [12,13,14,15]. Cysteines in hSOD1 are exposed to the solution as the protein is translocated into the intermembrane space of mitochondria, resulting in a much enhanced reactivity that could lead to formation of non-native disulfides. We use recently developed single-molecule atomic force microscopy (AFM) techniques [23] to monitor directly the reactivity of Cys-111 toward the conserved disulfide in hSOD1, one of the reactions that triggers disulfide rearrangement associated with ALS. Aberrant thiol/disulfide chemistry in hSOD1 may influence the onset and progression of ALS
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