Loss of Metal Ions, Disulfide Reduction and Mutations Related to Familial ALS Promote Formation of Amyloid-Like Aggregates from Superoxide Dismutase

Abstract

Mutations in the gene encoding Cu-Zn superoxide dismutase (SOD1) are one of the causes of familial amyotrophic lateral sclerosis (FALS). Fibrillar inclusions containing SOD1 and SOD1 inclusions that bind the amyloid-specific dye thioflavin S have been found in neurons of transgenic mice expressing mutant SOD1. Therefore, the formation of amyloid fibrils from human SOD1 was investigated. When agitated at acidic pH in the presence of low concentrations of guanidine or acetonitrile, metalated SOD1 formed fibrillar material which bound both thioflavin T and Congo red and had circular dichroism and infrared spectra characteristic of amyloid. While metalated SOD1 did not form amyloid-like aggregates at neutral pH, either removing metals from SOD1 with its intramolecular disulfide bond intact or reducing the intramolecular disulfide bond of metalated SOD1 was sufficient to promote formation of these aggregates. SOD1 formed amyloid-like aggregates both with and without intermolecular disulfide bonds, depending on the incubation conditions, and a mutant SOD1 lacking free sulfhydryl groups (AS-SOD1) formed amyloid-like aggregates at neutral pH under reducing conditions. ALS mutations enhanced the ability of disulfide-reduced SOD1 to form amyloid-like aggregates, and apo-AS-SOD1 formed amyloid-like aggregates at pH 7 only when an ALS mutation was also present. These results indicate that some mutations related to ALS promote formation of amyloid-like aggregates by facilitating the loss of metals and/or by making the intramolecular disulfide bond more susceptible to reduction, thus allowing the conversion of SOD1 to a form that aggregates to form resembling amyloid. Furthermore, the occurrence of amyloid-like aggregates per se does not depend on forming intermolecular disulfide bonds, and multiple forms of such aggregates can be produced from SOD1.

The Disulfide Bond, but Not Zinc or Dimerization, Controls Initiation and Seeded Growth in Amyotrophic Lateral Sclerosis-linked Cu,Zn Superoxide Dismutase (SOD1) Fibrillation

“…Both wild-type and ALS-mutant human SOD1 can be induced to aggregate in vitro using a variety of different treatments, including low concentrations of reducing agents such as DTT or glutathione (16 -19), low pH (18,20), structural perturbants such as trifluoroethanol and elevated temperature (21,22), oxidative modification induced by hydrogen peroxide (23), and by long term air exposure leading to cross-linking of nondisulfide cysteines (24). Some of these conditions, notably low pH, elevated temperatures, trifluoroethanol, and incubation in reducing agents, generate aggregates that bear a remarkable resemblance to amyloid fibrils.…”

Altered Thiol Chemistry in Human Amyotrophic Lateral Sclerosis-linked Mutants of Superoxide Dismutase 1

“…Disulfide isomerization may lead to disulfide cross-linked hSOD1 aggregates, which have been detected in spinal cords of model ALS mice (12). Indeed, these abnormal configurations have been implicated in the gain of toxic function of hSOD1 (13,14,47). In order to examine thiol/disulfide exchange reactions that are involved in the aggregation of hSOD1, there is a need to develop reliable assays that can probe directly the reactivity of the cysteines in hSOD1.…”

“…In this regard, it has been shown that mutation G93A leads to noticeable changes in the structure and dynamics of hSOD1 (53,54). In addition, the mutation may render Cys-111 more sensitive to oxidation (47,48), which would make Cys-111 unreactive. Indeed, equivalent loss of reactivity of Cys-111 can be reproduced in hSOD1 WT by preincubation with maleimide, an alkylating agent that targets free, accessible thiols (Fig.…”

Folding and Aggregation of Cu, Zn-Superoxide Dismutase

“…Temperature, pH, macromolecular crowding, agitation, and ionic strength are all variables that can influence aggregation (Chi et al 2003;Munishkina et al 2004;Mahler et al 2009;Sicorello et al 2009). A number of studies have used different solution conditions (increased temperature, decreased pH, increased ionic strength, sonication or agitation) to promote the formation of well-structured, fibrillar amyloid aggregates (see 1.2.3) by various forms of SOD1 (Stathopulos et al 2004;Chattopadhyay et al 2008;Chattopadhyay and Valentine 2009;Oztug Durer et al 2009). Other studies have demonstrated soluble oligomer and small aggregate formation by various forms of SOD1 in quiescent, physiologically relevant solution conditions (Vassall, 2011, Hwang, 2010, Banci, 2008.…”

“…It should be noted that ALS is not classified by pathologists as an amyloid disease (Kerman et al 2010). Recent studies have reported the formation of SOD1 aggregates in vitro that exhibit some features of amyloid (Banci et al 2008;Furukawa et al 2008;Oztug Durer et al 2009); however, the relevance of such studies to human disease is not known. Typically, there is considerable structural heterogeneity in amyloid (Platt and Radford 2009) and in other amorphous or ordered aggregate structures formed by many peptides and proteins (Fink 1998;Seshadri et al 2009) (see 1.2.4).…”