Destabilization of apoprotein is insufficient to explain Cu,Zn-superoxide dismutase-linked ALS pathogenesis

144

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: Als-associated Mutations Have Complex Effects On Stability Andsupporting

confidence: 77%

Section: Als-associated Mutations Have Complex Effects On Stability Andmentioning

confidence: 98%

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

144

“…That fact that regions of apo-SOD1 are highly dynamic has been recognized for years (25), but it is not clear whether the observed variables, such as NMR relaxation rates and amide hydrogen exchange with solvent deuterium (18,19,25,33), imply that SOD1 experiences a conformational continuum within a single basin in the energy landscape, large-scale unfolding of the affected regions, or exchange to a relatively well-defined excited state. Our present results provide evidence for the latter scenario, and further show that the excited state is a unifying feature of all SOD1 variants investigated herein, despite their significant variation in stability toward global unfolding.…”

Section: Biophysics and Computational Biologymentioning

confidence: 99%

Transient structural distortion of metal-free Cu/Zn superoxide dismutase triggers aberrant oligomerization

Teilum

Smith

Schulz

et al. 2009

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease linked to the misfolding of Cu/Zn superoxide dismutase (SOD1).ALS-related defects in SOD1 result in a gain of toxic function that coincides with aberrant oligomerization. The structural events triggering oligomerization have remained enigmatic, however, as is the case in other protein-misfolding diseases. Here, we target the critical conformational change that defines the earliest step toward aggregation. Using nuclear spin relaxation dispersion experiments, we identified a short-lived (0.4 ms) and weakly populated (0.7%) conformation of metal-depleted SOD1 that triggers aberrant oligomerization. This excited state emanates from the folded ground state and is suppressed by metal binding, but is present in both the disulfide-oxidized and disulfide-reduced forms of the protein. Our results pinpoint a perturbed region of the excited-state structure that forms intermolecular contacts in the earliest nonnative dimer/ oligomer. The conformational transition that triggers oligomerization is a common feature of WT SOD1 and ALS-associated mutants that have widely different physicochemical properties. But compared with WT SOD1, the mutants have enhanced structural distortions in their excited states, and in some cases slightly higher excited-state populations and lower kinetic barriers, implying increased susceptibility to oligomerization. Our results provide a unified picture that highlights both (i) a common denominator among different SOD1 variants that may explain why diverse mutations cause the same disease, and (ii) a structural basis that may aid in understanding how different mutations affect disease propensity and progression.amyotrophic lateral sclerosis ͉ nuclear magnetic resonance relaxation ͉ protein misfolding

“…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)(5)(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).…”

mentioning

confidence: 99%

“…In the fully demetallated (apo) states, some of the ALS-mutant SOD1 proteins actually are more stable than apo WT SOD1 (4). WT human SOD1 contains four cysteines, Cys-6, Cys-57, Cys-111, and Cys-146.…”

mentioning

confidence: 99%

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

220

261

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 ...