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.
Highlights
More than sixty human diseases are accompanied by the formation of protein aggregates called amyloid [1]
The AS mutant, C6A/C111S, is a ‘‘pseudo-WT’’ SOD1 in which both free cysteines have been removed by mutation, with the buried cys6 mutated to alanine and the surface cys111 changed to serine
The results presented here lead to three principal conclusions: 1) Metal-free SOD1S-S and metalated SOD12SH self-assemble into amyloid fibrils at physiological temperature, pH and ionic strength
Summary
More than sixty human diseases are accompanied by the formation of protein aggregates called amyloid [1]. These include a number of neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and Creutzfeldt-Jakob (prion) disease. Amyloid deposits may be involved in the neurodegenerative disease amyotrophic lateral sclerosis (ALS), commonly known as. 2% of ALS cases are caused by mutations in the gene encoding the anti-oxidant enzyme copperzinc superoxide dismutase (SOD1). These mutations represent one of the few known causes of ALS and underlie the most well-studied mouse models of this devastating disease. Electron microscopy has revealed a fibrillar morphology of the SOD1 aggregates found in motor neurons of FALS patients [6], in COS cells expressing mutant but not wild-type (WT) SOD1 [7], in neuroblastoma cells expressing ALS mutant SOD1 which were subjected to endoplasmic reticulum stress [8], and in transgenic mice expressing ALS mutant SOD1 [6,9,10]
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