Conformational Disorder of the Most Immature Cu, Zn-Superoxide Dismutase Leading to Amyotrophic Lateral Sclerosis

Abstract

Misfolding of Cu,Zn-superoxide dismutase (SOD1) is a pathological change in the familial form of amyotrophic lateral sclerosis caused by mutations in the SOD1 gene. SOD1 is an enzyme that matures through the binding of copper and zinc ions and the formation of an intramolecular disulfide bond. Pathogenic mutations are proposed to retard the post-translational maturation, decrease the structural stability, and hence trigger the misfolding of SOD1 proteins. Despite this, a misfolded and potentially pathogenic conformation of immature SOD1 remains obscure. Here, we show significant and distinct conformational changes of apoSOD1 that occur only upon reduction of the intramolecular disulfide bond in solution. In particular, loop regions in SOD1 lose their restraint and become significantly disordered upon dissociation of metal ions and reduction of the disulfide bond. Such drastic changes in the solution structure of SOD1 may trigger misfolding and fibrillar aggregation observed as pathological changes in the familial form of amyotrophic lateral sclerosis.Mutations in Cu,Zn-superoxide dismutase (SOD1) 2 are linked to familial forms of amyotrophic lateral sclerosis (fALS) (1). A major pathological change observed in SOD1-related fALS is the abnormal accumulation of misfolded mutant SOD1 proteins in affected motor neurons (2). Actually, many in vivo as well as in vitro studies have supported that pathogenic mutations facilitate the misfolding of SOD1 proteins (3); however, the molecular mechanism triggering the misfolding of SOD1 remains controversial.SOD1 is known as one of the most stable proteins to the extent that its melting temperature (T m ) is Ͼ90°C (4); therefore, a misfolding event appears quite unlikely for SOD1. Nonetheless, SOD1 was found to have acquired such high stability through several post-translational processes including copper and zinc binding and disulfide bond formation (Fig. 1A). Actually, disulfide-reduced apoSOD1 exhibits significantly decreased stability (T m ϳ 42°C) and is susceptible to unfolding/ misfolding at physiological temperatures (5, 6). Intracellular deregulation of metal binding and/or disulfide formation will, hence, be a key event triggering the misfolding of SOD1.Notably, many pathogenic mutations are found to disturb the post-translational control of SOD1 maturation (7, 8) and thereby increase intracellular fractions of the apo-(9) and/or disulfide-reduced state (10). Only when both metal ions and disulfide bond are absent, SOD1 forms fibrillar aggregates (11). Given that SOD1 fibrillation is a pathological hallmark in SOD1-related fALS patients (12) as well as model mice (13), the most immature form of SOD1 will provide a clue to understand the molecular pathomechanism of this devastating disease. In a number of previous studies, the roles of metal binding and disulfide formation in the misfolding of SOD1 have been suggested by a variety of experimental methods (6, 8, 11, 14 -19); however, conformational information on SOD1 in solution lacking both metal ions and the disulfi...

A Phosphomimetic Mutation Stabilizes SOD1 and Rescues Cell Viability in the Context of an ALS-Associated Mutation

“…Furthermore, we find strong correlations within the zinc-binding loop (residues 49–60) only in A4V–SOD1, whereas in WT-, T2D-, and T2D/A4V–SOD1 these correlated motions are suppressed (Figure S6). The zinc-binding loop has been suggested to allosterically regulate the conformation of the C4F6 epitope (Bosco et al, 2010), which is directly linked to neural-toxicity of SOD1 in a primary microglia activation assay (Furukawa et al, 2015; Rotunno et al, 2014). We propose that in A4V–SOD1 the cross-correlated motions promote the exposure of the disease-related epitope, and that the T2D/A4V double mutant prevents this effect.…”

Immunochemical characterization on pathological oligomers of mutant Cu/Zn-superoxide dismutase in amyotrophic lateral sclerosis

“…Actually, we have shown that reduction of the disulfide bond drastically increases fluctuation of the loops IV and VII (Fig. 8b), temporarily “peels” those loops off from the β-barrel scaffold, and thus potentially exposes the epitope of anti-SOD1 int antibody [55]. As shown in Fig.…”

Tryptophan residue 32 in human Cu-Zn superoxide dismutase modulates prion-like propagation and strain selection

“…WT SOD1 is highly expressed in motor neurons, approaching supersaturation, which could enhance its probability of misfolding [43]. In vitro, purified WT SOD1 can be readily induced to aggregate into amyloid-like fibrillary structures by de-metalation (Cu) and disulfide reduction [44][45][46][47][48][49][50][51][52][53][54]. Within the amino acid sequence of SOD1 there are several small sequence motifs that are inherently amyloidogenic [55].…”