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Cellular toxicity of mutant SOD1 protein is linked to an easily soluble, non-aggregated form in vitro.

ABSTRACT: Mutations in superoxide dismutase 1 (SOD1) are found in approximately 20% of patients with familial amyotrophic lateral sclerosis. The propensity of mutant SOD1 to form aggregates in pathologically affected cells (i.e. motor neurons) has implicated these poorly soluble protein aggregates and/or their misfolded soluble precursors as being instrumental to the disease process. We investigated the relative solubility and toxicity of four different mutant SOD1 proteins in a cell-based model system and demonstrate that the mutant, misfolded SOD1 proteins that are the most soluble are also the most toxic. This toxicity was ameliorated by upregulating heat-shock protein chaperones in order to refold the soluble, misfolded protein, regardless of the presence of poorly soluble SOD1. We further demonstrate that increasing the solubility of a SOD1 mutant protein that is both poorly soluble and non-toxic, as compared to other mutant proteins, resulted in remarkably increased toxicity of the mutant SOD1. Again, this increased toxicity was attenuated by upregulating heat-shock protein chaperones in order to refold the soluble, misfolded proteins. These findings implicate easily soluble, misfolded SOD1 as being toxic to the cell and support the hypothesis that reducing solubility of mutant SOD1 proteins through aggregation may occur as a self-protective response in the cell.

SUBMITTER: Brotherton TE

PROVIDER: S-EPMC3549044 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Cellular toxicity of mutant SOD1 protein is linked to an easily soluble, non-aggregated form in vitro.

Brotherton Terrell E TE Li Yingjie Y Glass Jonathan D JD

Neurobiology of disease 20120825

Mutations in superoxide dismutase 1 (SOD1) are found in approximately 20% of patients with familial amyotrophic lateral sclerosis. The propensity of mutant SOD1 to form aggregates in pathologically affected cells (i.e. motor neurons) has implicated these poorly soluble protein aggregates and/or their misfolded soluble precursors as being instrumental to the disease process. We investigated the relative solubility and toxicity of four different mutant SOD1 proteins in a cell-based model system an ...[more]

PMID: 22926189

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Project description:Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the rapid and progressive degeneration of upper and lower motor neurons in the spinal cord, brain stem and motor cortex. The first gene linked to ALS was the gene encoding the free radical scavenging enzyme superoxide dismutase-1 (SOD1) that currently has over 180, mostly missense, ALS-associated mutations identified. SOD1-associated fALS patients show remarkably broad mean survival times (<1 year to ~17 years death post-diagnosis) that are mutation dependent. A hallmark of SOD1-associated ALS is the deposition of SOD1 into large insoluble aggregates in motor neurons. This is thought to be a consequence of mutation induced structural destabilization and/or oxidative damage leading to the misfolding and aggregation of SOD1 into a neurotoxic species. Here we aim to understand the relationship between SOD1 variant toxicity, structural stability, and aggregation propensity using a combination of cell culture and purified protein assays. Cell based assays indicated that aggregation of SOD1 variants correlate closely to cellular toxicity. However, the relationship between cellular toxicity and disease severity was less clear. We next utilized mass spectrometry to interrogate the structural consequences of metal loss and disulfide reduction on fALS-associated SOD1 variant structure. All variants showed evidence of unfolded, intermediate, and compact conformations, with SOD1G37R, SOD1G93A and SOD1V148G having the greatest abundance of intermediate and unfolded SOD1. SOD1G37R was an informative outlier as it had a high propensity to unfold and form oligomeric aggregates, but it did not aggregate to the same extent as SOD1G93A and SOD1V148G in in vitro aggregation assays. Furthermore, seeding the aggregation of DTT/EDTA-treated SOD1G37R with preformed SOD1G93A fibrils elicited minimal aggregation response, suggesting that the arginine substitution at position-37 blocks the templating of SOD1 onto preformed fibrils. We propose that this difference may be explained by multiple strains of SOD1 aggregate and this may also help explain the slow disease progression observed in patients with SOD1G37R.

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Cellular toxicity of mutant SOD1 protein is linked to an easily soluble, non-aggregated form in vitro.

Publications

Cellular toxicity of mutant SOD1 protein is linked to an easily soluble, non-aggregated form in vitro.

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