Dissociation of Human Copper-Zinc Superoxide Dismutase Dimers Using Chaotrope and Reductant

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145

Protein aggregation is a hallmark of many diseases, including amyotrophic lateral sclerosis (ALS), where aggregation of Cu/Zn superoxide dismutase (SOD1) is implicated in causing neurodegeneration. Recent studies have suggested that destabilization and aggregation of the most immature form of SOD1, the disulfide-reduced, unmetallated (apo) protein is particularly important in causing ALS. We report herein in depth analyses of the effects of chemically and structurally diverse ALS-associated mutations on the stability and aggregation of reduced apo SOD1. In contrast with previous studies, we find that various reduced apo SOD1 mutants undergo highly reversible thermal denaturation with little aggregation, enabling quantitative thermodynamic stability analyses. In the absence of ALS-associated mutations, reduced apo SOD1 is marginally stable but predominantly folded. Mutations generally result in slight decreases to substantial increases in the fraction of unfolded protein. Calorimetry, ultracentrifugation, and light scattering show that all mutations enhance aggregation propensity, with the effects varying widely, from subtle increases in most cases, to pronounced formation of 40–100 nm soluble aggregates by A4V, a mutation that is associated with particularly short disease duration. Interestingly, although there is a correlation between observed aggregation and stability, there is minimal to no correlation between observed aggregation, predicted aggregation propensity, and disease characteristics. These findings suggest that reduced apo SOD1 does not play a dominant role in modulating disease. Rather, additional and/or multiple forms of SOD1 and additional biophysical and biological factors are needed to account for the toxicity of mutant SOD1 in ALS.

Section: Dsc Reveals Complex Effects Of Als-associated Mutations On Thesupporting

confidence: 86%

Section: Resultsmentioning

confidence: 99%

Decreased stability and increased formation of soluble aggregates by immature superoxide dismutase do not account for disease severity in ALS

Vassall¹,

Stubbs²,

Primmer³

et al. 2011

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145

“…For example, the apoprotein is dimeric when the disulfide bond is intact, but it is monomeric when the disulfide bond is reduced. By contrast, the disulfide-reduced SOD1 protein is dimeric when zinc ions are bound (22). Our results, described here, demonstrate that the dimeric SOD1 apoprotein, even with its intrasubunit disulfide bonds intact, has a marked tendency to form soluble highmolecular-weight oligomers under conditions very close to physiological.…”

Section: Discussionmentioning

confidence: 56%

Metal-free superoxide dismutase forms soluble oligomers under physiological conditions: A possible general mechanism for familial ALS

Banci

Bertini

Durazo³

et al. 2007

Self Cite

225

262

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder selectively affecting motor neurons; 90% of the total cases are sporadic, but 2% are associated with mutations in the gene coding for the antioxidant enzyme copper-zinc superoxide dismutase (SOD1). The causes of motor neuron death in ALS are poorly understood in general, but for SOD1-linked familial ALS, aberrant oligomerization of SOD1 mutant proteins has been strongly implicated. In this work, we show that wild-type human SOD1, when lacking both its metal ions, forms large, stable, soluble protein oligomers with an average molecular mass of Ϸ650 kDa under physiological conditions, i.e., 37°C, pH 7.0, and 100 M protein concentration. It further is shown here that intermolecular disulfide bonds are formed during oligomerization and that Cys-6 and Cys-111 are implicated in this bonding. The formation of the soluble oligomers was monitored by their ability to enhance the fluorescence of thioflavin T, a benzothiazole dye that increases in fluorescence intensity upon binding to amyloid fibers, and by disruption of this binding upon addition of the chaotropic agent guanidine hydrochloride. Our results suggest a general, unifying picture of SOD1 aggregation that could operate when wild-type or mutant SOD1 proteins lack their metal ions. Although we cannot exclude other mechanisms in SOD1-linked familial ALS, the one proposed here has the strength of explaining how a large and diverse set of SOD1 mutant proteins all could lead to disease through the same mechanism.amyloid ͉ neurodegeneration ͉ protein aggregation ͉ amyotrophic lateral sclerosis ͉ protein misfolding P rotein oligomerization, aggregation, and formation of insoluble amyloid deposits commonly are observed in neurodegenerative diseases, but the factors initiating and modulating the abnormal protein-protein interactions that lead to oligomerization remain elusive (1, 2). Metal ions frequently have been implicated in these phenomena, but how exactly they are involved remains unclear (3). Over 114 different variants of human copper-zinc superoxide dismutase (Cu 2 Zn 2 SOD1) have been linked to the neurodegenerative disease familial ALS (FALS) by a gain-of-function mechanism (4-6). Although the exact cellular sites and mechanisms of toxicity are unknown, aberrant SOD1 protein oligomerization has been strongly implicated in disease causation (7,8). Several recent publications have presented compelling evidence that abnormal disulfide cross-linking of ALS-mutant SOD1 plays a role in this oligomerization, and disulfide-linked SOD1 multimers have been detected in neural tissues of SOD1-ALS transgenic mice that are presumed to be components of higher-molecular-weight species or intermediates in their formation (7, 9-11).Wild-type (WT) human SOD1 is an exceptionally stable protein in its holo form and, although some of the ALS-mutant SOD1 proteins are severely destabilized by their mutations, others largely retain the stability of WT SOD1 (4). In the fully demetallated (apo) states, some ...

“…Aggregation propensity and loss of stability of SOD1 are synergistic risk factors for fALS patient disease severity (23), and it has been suggested that a common property of fALS variants in vitro and in vivo is their propensity to aggregate (24,25). A prevailing hypothesis for the mechanism of the toxicity of fALS-SOD1 variants involves dimer destabilization and dissociation into monomers, which then nucleate the formation of higher-order aggregates (13,(26)(27)(28). Indeed, variant proteins, such as the G85R SOD1 used in this study, are found as monomers in vivo (29)(30)(31).…”

mentioning

confidence: 99%

Strategies for stabilizing superoxide dismutase (SOD1), the protein destabilized in the most common form of familial amyotrophic lateral sclerosis

Auclair

Boggio

Petsko

et al. 2010

Amyotrophic lateral sclerosis (ALS) is a disorder characterized by the death of both upper and lower motor neurons and by 3-to 5-yr median survival postdiagnosis. The only US Food and Drug Administration-approved drug for the treatment of ALS, Riluzole, has at best, moderate effect on patient survival and quality of life; therefore innovative approaches are needed to combat neurodegenerative disease. Some familial forms of ALS (fALS) have been linked to mutations in the Cu/Zn superoxide dismutase (SOD1). The dominant inheritance of mutant SOD1 and lack of symptoms in knockout mice suggest a "gain of toxic function" as opposed to a loss of function. A prevailing hypothesis for the mechanism of the toxicity of fALS-SOD1 variants, or the gain of toxic function, involves dimer destabilization and dissociation as an early step in SOD1 aggregation. Therefore, stabilizing the SOD1 dimer, thus preventing aggregation, is a potential therapeutic strategy. Here, we report a strategy in which we chemically cross-link the SOD1 dimer using two adjacent cysteine residues on each respective monomer (Cys111). Stabilization, measured as an increase in melting temperature, of ∼20°C and ∼45°C was observed for two mutants, G93A and G85R, respectively. This stabilization is the largest for SOD1, and to the best of our knowledge, for any disease-related protein. In addition, chemical cross-linking conferred activity upon G85R, an otherwise inactive mutant. These results demonstrate that targeting these cysteine residues is an important new strategy for development of ALS therapies.mass spectrometry | thiol-disulfide I nnovative approaches are needed to combat neurodegenerative disease, among the most serious of which is amyotrophic lateral sclerosis (ALS), a disorder characterized by the death of both upper and lower motor neurons and by 3-to -5-yr median survival postdiagnosis. The only US Food and Drug Administrationapproved drug for the treatment of ALS, Riluzole, has at best, moderate effect on patient survival and quality of life (1-3). Although the causes of sporadic neurodegenerative diseases remain a mystery, mutations causing familial forms of many of these diseases (e.g., Alzheimer's, Parkinson, and ALS) are known. For example, mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1) are responsible for ∼20% of the familial ALS cases (fALS) and 2% of all ALS (4, 5). Two such mutations are G93A, which maintains wild-type-like enzymatic activity, and the metal-deficient G85R, which is essentially inactive. Posttranslational modifications of proteins involved in familial diseases have been invoked in the etiology of the corresponding sporadic diseases, for example, alpha-synuclein (6) and Parkin (7) modification in Parkinson, Abeta (8) and tau (9) modification in Alzheimer's, and TDP43 (10) and SOD1 (11-14) modification in ALS. The hope, therefore, is that strategies for treating familial diseases may translate to at least a subset of sporadic diseases.Both dominant inheritance of mutant SOD1 (15) and lack of symptoms i...