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1.  Glutathionylation at Cys-111 Induces Dissociation of Wild Type and FALS Mutant SOD1 Dimers 
Biochemistry  2011;50(32):7057-7066.
Mutation of the ubiquitous cytosolic enzyme Cu/Zn superoxide dismutase (SOD1) is hypothesized to cause familial amyotrophic lateral sclerosis (FALS) through structural destabilization leading to misfolding and aggregation. Considering the late onset of symptoms as well as the phenotypic variability among patients with identical SOD1 mutations, it is clear that nongenetic factor(s) impact ALS etiology and disease progression. Here we examine the effect of Cys-111 glutathionylation, a physiologically prevalent post-translational oxidative modification, on the stabilities of wild type SOD1 and two phenotypically diverse FALS mutants, A4V and I112T. Glutathionylation results in profound destabilization of SOD1WT dimers, increasing the equilibrium dissociation constant Kd to ~10−20 μM, comparable to that of the aggressive A4V mutant. SOD1A4V is further destabilized by glutathionylation, experiencing an ~30-fold increase in Kd. Dissociation kinetics of glutathionylated SOD1WT and SOD1A4V are unchanged, as measured by surface plasmon resonance, indicating that glutathionylation destabilizes these variants by decreasing association rate. In contrast, SOD1I112T has a modestly increased dissociation rate but no change in Kd when glutathionylated. Using computational structural modeling, we show that the distinct effects of glutathionylation on different SOD1 variants correspond to changes in composition of the dimer interface. Our experimental and computational results show that Cys-111 glutathionylation induces structural rearrangements that modulate stability of both wild type and FALS mutant SOD1. The distinct sensitivities of SOD1 variants to glutathionylation, a modification that acts in part as a coping mechanism for oxidative stress, suggest a novel mode by which redox regulation and aggregation propensity interact in ALS.
PMCID: PMC3281512  PMID: 21739997
2.  SOD1 oxidation and formation of soluble aggregates in yeast: Relevance to sporadic ALS development 
Redox Biology  2014;2:632-639.
Misfolding and aggregation of copper–zinc superoxide dismutase (Sod1) are observed in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Mutations in Sod1 lead to familial ALS (FALS), which is a late-onset disease. Since oxidative damage to proteins increases with age, it had been proposed that oxidation of Sod1 mutants may trigger their misfolding and aggregation in FALS. However, over 90% of ALS cases are sporadic (SALS) with no obvious genetic component. We hypothesized that oxidation could also trigger the misfolding and aggregation of wild-type Sod1 and sought to confirm this in a cellular environment. Using quiescent, stationary-phase yeast cells as a model for non-dividing motor neurons, we probed for post-translational modification (PTM) and aggregation of wild-type Sod1 extracted from these cells. By size-exclusion chromatography (SEC), we isolated two populations of Sod1 from yeast: a low-molecular weight (LMW) fraction that is catalytically active and a catalytically inactive, high-molecular weight (HMW) fraction. High-resolution mass spectrometric analysis revealed that LMW Sod1 displays no PTMs but HMW Sod1 is oxidized at Cys146 and His71, two critical residues for the stability and folding of the enzyme. HMW Sod1 is also oxidized at His120, a copper ligand, which will promote loss of this catalytic metal cofactor essential for SOD activity. Monitoring the fluorescence of a Sod1-green-fluorescent-protein fusion (Sod1-GFP) extracted from yeast chromosomally expressing this fusion, we find that HMW Sod1-GFP levels increase up to 40-fold in old cells. Thus, we speculate that increased misfolding and inclusion into soluble aggregates is a consequence of elevated oxidative modifications of wild-type Sod1 as cells age. Our observations argue that oxidative damage to wild-type Sod1 initiates the protein misfolding mechanisms that give rise to SALS.
•Key Sod1 catalytic and structure-stabilizing residues (Cys146, His120, His71) are oxidized in stationary-phase yeast.•Oxidized Sod1 is isolated in an inactive, high-molecular-weight, soluble aggregate.•Sod1 with native mass isolated from the same samples is not oxidized and is catalytically active.•Our results argue that oxidation triggers the formation of soluble Sod1-containing aggregates that may contribute to sporadic ALS development.
Graphical abstract
PMCID: PMC4052529  PMID: 24936435
Wild-type Sod1; Oxidative PTMs; Soluble aggregates; Sporadic ALS; Yeast
3.  An emerging role for misfolded wild-type SOD1 in sporadic ALS pathogenesis 
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder that targets motor neurons, leading to paralysis and death within a few years of disease onset. While several genes have been linked to the inheritable, or familial, form of ALS, much less is known about the cause(s) of sporadic ALS, which accounts for ~90% of ALS cases. Due to the clinical similarities between familial and sporadic ALS, it is plausible that both forms of the disease converge on a common pathway and, therefore, involve common factors. Recent evidence suggests the Cu,Zn-superoxide dismutase (SOD1) protein to be one such factor that is common to both sporadic and familial ALS. In 1993, mutations were uncovered in SOD1 that represent the first known genetic cause of familial ALS. While the exact mechanism of mutant-SOD1 toxicity is still not known today, most evidence points to a gain of toxic function that stems, at least in part, from the propensity of this protein to misfold. In the wild-type SOD1 protein, non-genetic perturbations such as metal depletion, disruption of the quaternary structure, and oxidation, can also induce SOD1 to misfold. In fact, these aforementioned post-translational modifications cause wild-type SOD1 to adopt a “toxic conformation” that is similar to familial ALS-linked SOD1 variants. These observations, together with the detection of misfolded wild-type SOD1 within human post-mortem sporadic ALS samples, have been used to support the controversial hypothesis that misfolded forms of wild-type SOD1 contribute to sporadic ALS pathogenesis. In this review, we present data from the literature that both support and contradict this hypothesis. We also discuss SOD1 as a potential therapeutic target for both familial and sporadic ALS.
PMCID: PMC3863749  PMID: 24379756
amyotrophic lateral sclerosis (ALS); sporadic amyotrophic lateral sclerosis; SOD1; protein misfolding; immunotherapy
4.  The Effects of Glutaredoxin and Copper Activation Pathways on the Disulfide and Stability of Cu,Zn Superoxide Dismutase*… 
The Journal of biological chemistry  2006;281(39):28648-28656.
Mutations in Cu,Zn superoxide dismutase (SOD1) can cause amyotrophic lateral sclerosis (ALS) through mechanisms proposed to involve SOD1 misfolding, but the intracellular factors that modulate folding and stability of SOD1 are largely unknown. By using yeast and mammalian expression systems, we demonstrate here that SOD1 stability is governed by post-translational modification factors that target the SOD1 disulfide. Oxidation of the human SOD1 disulfide in vivo was found to involve both the copper chaperone for SOD1 (CCS) and the CCS-independent pathway for copper activation. When both copper pathways were blocked, wild type SOD1 stably accumulated in yeast cells with a reduced disulfide, whereas ALS SOD1 mutants A4V, G93A, and G37R were degraded. We describe here an unprecedented role for the thiol oxidoreductase glutaredoxin in reducing the SOD1 disulfide and destabilizing ALS mutants. Specifically, the major cytosolic glutaredoxin of yeast was seen to reduce the intramolecular disulfide of ALS SOD1 mutant A4V SOD1 in vivo and in vitro. By comparison, glutaredoxin was less reactive toward the disulfide of wild type SOD1. The apo-form of A4V SOD1 was highly reactive with glutaredoxin but not SOD1 containing both copper and zinc. Glutaredoxin therefore preferentially targets the immature form of ALS mutant SOD1 lacking metal co-factors. Overall, these studies implicate a critical balance between cellular reductants such as glutaredoxin and copper activation pathways in controlling the disulfide and stability of SOD1 in vivo.
PMCID: PMC2757158  PMID: 16880213
5.  Zinc Binding Modulates the Entire Folding Free Energy Surface of Human Cu,Zn Superoxide Dismutase 
Journal of molecular biology  2008;384(2):540-555.
Over 100 amino acid replacements in human Cu, Zn superoxide dismutase (SOD) are known to cause amyotrophic lateral sclerosis, a gain-of-function neurodegenerative disease that destroys motor neurons. Supposing that aggregates of partially-folded states are primarily responsible for toxicity, the role of the structurally-important zinc ion in defining the folding free energy surface of dimeric SOD was determined by comparing the thermodynamic and kinetic folding properties of the zinc-free and zinc-bound forms of the protein. The presence of zinc was found to decrease the free energies of a peptide model of the unfolded monomer, a stable variant of the folded monomeric intermediate and the folded dimeric species. The unfolded state binds zinc weakly with a micromolar dissociation constant, and the folded monomeric intermediate and the native dimeric form both bind zinc tightly, with sub-nanomolar dissociation constants. Coupled with the strong driving force for the subunit association reaction, the shift in the populations towards more well-folded states in the presence of zinc decreases the steady-state populations of higher-energy states in SOD under expected in vivo zinc concentrations (∼nanomolar). The significant decrease in the population of partially-folded states is expected to diminish their potential for aggregation and account for the known protective effect of zinc. The ∼100-fold increase in the rate of folding of SOD in the presence of micromolar concentrations of zinc demonstrates a significant role for a pre-organized zinc-binding loop in the transition state ensemble for the rate-limiting monomer folding reaction in this β-barrel protein.
PMCID: PMC2756654  PMID: 18840448
ALS; beta-barrel dimer; metal binding; protein folding; thermodynamics and kinetics
6.  Glutathionylation potentiates benign superoxide dismutase 1 variants to the toxic forms associated with amyotrophic lateral sclerosis 
Scientific Reports  2013;3:3275.
Dissociation of superoxide dismutase 1 dimers is enhanced by glutathionylation, although the dissociation constants reported to date are imprecise. We have quantified the discreet dissociation constants for wild-type superoxide dismutase 1 and six naturally occurring sequence variants, in their unmodified and glutathionylated forms, at the ratios expressed. Unmodified superoxide dismutase 1 variants that shared similar dissociation constants with SOD1WT had disproportionately increased dissociation constants when glutathionylated. This defines a key role for glutathionylation in superoxide dismutase 1 associated familial amyotrophic lateral sclerosis.
PMCID: PMC3834562  PMID: 24253732
7.  Ligand binding and aggregation of pathogenic SOD1 
Nature Communications  2013;4:1758-.
Mutations in the gene encoding Cu/Zn superoxide dismutase-1 cause amyotrophic lateral sclerosis. Superoxide dismutase-1 mutations decrease protein stability and promote aggregation. The mutant monomer is thought to be an intermediate in the pathway from the superoxide dismutase-1 dimer to aggregate. Here we find that the monomeric copper-apo, zinc-holo protein is structurally perturbed and the apo-protein aggregates without reattainment of the monomer–dimer equilibrium. Intervention to stabilize the superoxide dismutase-1 dimer and inhibit aggregation is regarded as a potential therapeutic strategy. We describe protein–ligand interactions for two compounds, Isoproterenol and 5-fluorouridine, highlighted as superoxide dismutase-1 stabilizers. We find both compounds interact with superoxide dismutase-1 at a key region identified at the core of the superoxide dismutase-1 fibrillar aggregates, β-barrel loop II–strand 3, rather than the proposed dimer interface site. This illustrates the need for direct structural observations when developing compounds for protein-targeted therapeutics.
Mutations of the SOD1 gene are implicated in neurodegenerative diseases such as amyotrophic lateral sclerosis. Wright and colleagues find that SOD1 aggregation in cells is arrested by compounds that bind at the core of SOD1 aggregates, rather than at the dimer interface site.
PMCID: PMC3644087  PMID: 23612299
8.  Prognostic role of “prion-like propagation” in SOD1-linked familial ALS: an alternative view 
“Prion-like propagation” has recently been proposed for disease spread in Cu/Zn superoxide dismutase 1 (SOD1)-linked familial amyotrophic lateral sclerosis (ALS). Pathological SOD1 conformers are presumed to propagate via cell-to-cell transmission. In this model, the risk-based kinetics of neuronal cell loss over time appears to be represented by a sigmoidal function that reflects the kinetics of intercellular transmission. Here, we describe an alternative view of prion-like propagation in SOD1-linked ALS – its relation to disease prognosis under the protective-aggregation hypothesis. Nucleation-dependent polymerization has been widely accepted as the molecular mechanism of prion propagation. If toxic species of misfolded SOD1, as soluble oligomers, are formed as on-pathway intermediates of nucleation-dependent polymerization, further fibril extension via sequential addition of monomeric mutant SOD1 would be protective against neurodegeneration. This is because the concentration of unfolded mutant SOD1 monomers, which serve as precursor of nucleation and toxic species of mutant SOD1, would decline in proportion to the extent of aggregation. The nucleation process requires that native conformers exist in an unfolded state that may result from escaping the cellular protein quality control machinery. However, prion-like propagation-SOD1 aggregated form self-propagates by imposing its altered conformation on normal SOD1-appears to antagonize the protective role of aggregate growth. The cross-seeding reaction with normal SOD1 would lead to a failure to reduce the concentration of unfolded mutant SOD1 monomers, resulting in continuous nucleation and subsequent generation of toxic species, and influence disease prognosis. In this alternative view, the kinetics of neuronal loss appears to be represented by an exponential function, with decreasing risk reflecting the protective role of aggregate and the potential for cross-seeding reactions between mutant SOD1 and normal SOD1.
PMCID: PMC4215625  PMID: 25400549
aggregate; amyotrophic lateral sclerosis; mutation; nucleation; prion; SOD1; kinetic model
9.  Post-translational modification of Cu/Zn superoxide dismutase under anaerobic conditions† 
Biochemistry  2012;51(2):677-685.
In eukaryotic organisms, the largely cytosolic copper and zinc containing superoxide dismutase enzyme (Cu/Zn SOD) represents a key defense against reactive oxygen toxicity. Although much is known about the biology of this enzyme under aerobic conditions, less is understood regarding the effects of low oxygen on Cu/Zn SOD from diverse organisms. We show here that like bakers’ yeast (Saccharomyces cerevisiae), adaptation of the multicellular Caenorhabditis elegans to growth in low oxygen involves strong down-regulation of its Cu/Zn SOD. Much of this regulation occurs at the post-translational level where CCS-independent activation of Cu/Zn SOD is inhibited. Hypoxia inactivates the endogenous Cu/Zn SOD of C. elegans Cu/Zn SOD as well as a P144 mutant of S. cerevisiae Cu/Zn SOD (herein denoted as Sod1p) that is independent of CCS. In our studies of S. cerevisiae Sod1p we noted a post-translational modification to the inactive enzyme during hypoxia. Analysis of this modification by mass spectrometry revealed phosphorylation on serine 38. Serine 38 represents a putative proline-directed kinase target site located on a solvent exposed loop that is positioned at one end of the Sod1p beta-barrel, a region immediately adjacent to residues previously shown to influence CCS-dependent activation. Although phosphorylation of serine 38 is minimal when the Sod1p is abundantly active (e.g., high oxygen), up to 50% of Sod1p can be phosphorylated when CCS-activation of the enzyme is blocked, e.g., by hypoxia or low copper conditions. Serine 38 phosphorylation can be a marker for inactive pools of Sod1p.
PMCID: PMC3264780  PMID: 22148750
10.  Pathological Roles of Wild-Type Cu, Zn-Superoxide Dismutase in Amyotrophic Lateral Sclerosis 
Dominant mutations in a Cu, Zn-superoxide dismutase (SOD1) gene cause a familial form of amyotrophic lateral sclerosis (ALS). While it remains controversial how SOD1 mutations lead to onset and progression of the disease, many in vitro and in vivo studies have supported a gain-of-toxicity mechanism where pathogenic mutations contribute to destabilizing a native structure of SOD1 and thus facilitate misfolding and aggregation. Indeed, abnormal accumulation of SOD1-positive inclusions in spinal motor neurons is a pathological hallmark in SOD1-related familial ALS. Furthermore, similarities in clinical phenotypes and neuropathology of ALS cases with and without mutations in sod1 gene have implied a disease mechanism involving SOD1 common to all ALS cases. Although pathogenic roles of wild-type SOD1 in sporadic ALS remain controversial, recent developments of novel SOD1 antibodies have made it possible to characterize wild-type SOD1 under pathological conditions of ALS. Here, I have briefly reviewed recent progress on biochemical and immunohistochemical characterization of wild-type SOD1 in sporadic ALS cases and discussed possible involvement of wild-type SOD1 in a pathomechanism of ALS.
PMCID: PMC3395178  PMID: 22830015
11.  Colocalization of 14-3-3 Proteins with SOD1 in Lewy Body-Like Hyaline Inclusions in Familial Amyotrophic Lateral Sclerosis Cases and the Animal Model 
PLoS ONE  2011;6(5):e20427.
Background and Purpose
Cu/Zn superoxide dismutase (SOD1) is a major component of Lewy body-like hyaline inclusion (LBHI) found in the postmortem tissue of SOD1-linked familial amyotrophic lateral sclerosis (FALS) patients. In our recent studies, 14-3-3 proteins have been found in the ubiquitinated inclusions inside the anterior horn cells of spinal cords with sporadic amyotrophic lateral sclerosis (ALS). To further investigate the role of 14-3-3 proteins in ALS, we performed immunohistochemical analysis of 14-3-3 proteins and compared their distributions with those of SOD1 in FALS patients and SOD1-overexpressing mice.
We examined the postmortem brains and the spinal cords of three FALS cases (A4V SOD1 mutant). Transgenic mice expressing the G93A mutant human SOD1 (mutant SOD1-Tg mice), transgenic mice expressing the wild-type human SOD1 (wild-type SOD1-Tg mice), and non-Tg wild-type mice were also subjected to the immunohistochemical analysis.
In all the FALS patients, LBHIs were observed in the cytoplasm of the anterior horn cells, and these inclusions were immunopositive intensely for pan 14-3-3, 14-3-3β, and 14-3-3γ. In the mutant SOD1-Tg mice, a high degree of immunoreactivity for misfolded SOD1 (C4F6) was observed in the cytoplasm, with an even greater degree of immunoreactivity present in the cytoplasmic aggregates of the anterior horn cells in the lumbar spinal cord. Furthermore, we have found increased 14-3-3β and 14-3-3γ immunoreactivities in the mutant SOD1-Tg mice. Double immunofluorescent staining showed that C4F6 and 14-3-3 proteins were partially co-localized in the spinal cord with FALS and the mutant SOD1-Tg mice. In comparison, the wild-type SOD1-Tg and non-Tg wild-type mice showed no or faint immunoreactivity for C4F6 and 14-3-3 proteins (pan 14-3-3, 14-3-3β, and 14-3-3γ) in any neuronal compartments.
These results suggest that 14-3-3 proteins may be associated with the formation of SOD1-containing inclusions, in FALS patients and the mutant SOD1-Tg mice.
PMCID: PMC3105059  PMID: 21655264
12.  Redox Properties of the Disulfide Bond of Human Cu,Zn Superoxide Dismutase and the Effects of Human Glutaredoxin 1 
The Biochemical journal  2012;446(1):59-67.
The intramolecular disulfide bond in human Cu,Zn superoxide dismutase 1 (hSOD1) plays a key role in maintaining the protein’s stability and quaternary structure. In mutant forms of SOD1 that cause familial amyotrophic lateral sclerosis (ALS), this disulfide bond is more susceptible to chemical reduction, which may lead to destabilization of the dimer and aggregation. During hSOD1 maturation, disulfide formation is catalyzed by the copper chaperone CCS1. Previous studies in yeast demonstrate that the yeast glutathione (GSH)/glutaredoxin redox system promotes reduction of the hSOD1 disulfide in the absence of CCS1. Herein, we further probe the interaction between hSOD1, GSH, and glutaredoxins to provide mechanistic insight into the redox kinetics and thermodynamics of the hSOD1 disulfide. We demonstrate that human glutaredoxin 1 (hGrx1) uses a monothiol mechanism to reduce the hSOD1 disulfide, and the GSH/hGrx1 system reduces ALS mutant SOD1 at a faster rate than WT hSOD1. However, redox potential measurements demonstrate that the thermodynamic stability of the disulfide is not consistently lower in ALS mutants compared to WT hSOD1. Furthermore, the presence of the metal cofactors does not influence the disulfide redox potential. Overall, these studies suggest that differences in the GSH/hGrx1 reaction rate with WT vs. ALS mutant hSOD1 and not the inherent thermodynamic stability of the hSOD1 disulfide bond may contribute to the greater pathogenicity of ALS mutant hSOD1.
PMCID: PMC3533437  PMID: 22651090
Disulfide; redox potential; glutaredoxins; yeast; Cu,Zn superoxide dismutase; monothiol
13.  Protein Misdirection Inside and Outside Motor Neurons in Amyotrophic Lateral Sclerosis (ALS): A Possible Clue for Therapeutic Strategies 
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by progressive muscle wasting and weakness with no effective cure. Emerging evidence supports the notion that the abnormal conformations of ALS-linked proteins play a central role in triggering the motor neuron degeneration. In particular, mutant types of superoxide dismutase 1 (SOD1) and TAR DNA binding protein 43kDa (TDP-43) are key molecules involved in the pathogenesis of familial and sporadic ALS, respectively. The commonalities of the two proteins include a propensity to aggregate and acquire detrimental conformations through oligomerization, fragmentation, or post-translational modification that may drive abnormal subcellular localizations. Although SOD1 is a major cytosolic protein, mutated SOD1 has been localized to mitochondria, endoplasmic reticulum, and even the extracellular space. The nuclear exclusion of TDP-43 is a pathological hallmark for ALS, although the pathogenic priority remains elusive. Nevertheless, these abnormal behaviors based on the protein misfolding are believed to induce diverse intracellular and extracellular events that may be tightly linked to non-cell-autonomous motor neuron death. The generation of mutant- or misfolded protein-specific antibodies would help to uncover the distribution and propagation of the ALS-linked proteins, and to design a therapeutic strategy to clear such species. Herein we review the literature regarding the mislocalization of ALS-linked proteins, especially mutant SOD1 and TDP-43 species, and discuss the rationale of molecular targeting strategies including immunotherapy.
PMCID: PMC3211022  PMID: 22072931
seeding; subcellular localization; SOD1; TDP-43; non-cell-autonomous motor neuron death; antibody
14.  Early steps in thermal unfolding of superoxide dismutase 1 are similar to the conformational changes associated with the ALS-associated A4V mutation 
There are over 100 mutations in Cu/Zn superoxide dismutase (SOD1) that result in a subset of familial amyotrophic lateral sclerosis (fALS) cases. The hypothesis that dissociation of the dimer, misfolding of the monomer and subsequent aggregation of mutant SOD1 leads to fALS has been gaining support as an explanation for how these disparate missense mutations cause the same disease. These forms are only responsible for a fraction of the ALS cases; however, the rest are sporadic. Starting with a folded apo monomer, the species considered most likely to be involved in misfolding, we used high-temperature all-atom molecular dynamics simulations to explore the events of the wild-type protein unfolding through the denatured state. All simulations showed early loss of structure along the β5–β6 edge of the β-sandwich, supporting earlier findings of instability in this region. Transition state structures identified from the simulations are in good agreement with experiment, providing detailed, validated molecular models for this elusive state. Furthermore, we compare the process of thermal unfolding investigated here to that of the lethal A4V mutant-induced unfolding at physiological temperature and find that the pathways are very similar.
PMCID: PMC3711394  PMID: 23784844
ALS; misfolding; molecular dynamics; SOD1; unfolding
Journal of neurochemistry  2008;108(4):1009-1018.
Mutations in superoxide dismutase 1 (SOD1, EC cause familial amyotrophic lateral sclerosis (fALS); with aggregated forms of mutant protein accumulating in spinal cord tissues of transgenic mouse models and human patients. Mice over-expressing wild-type human SOD1 (WT hSOD1) do not develop ALS-like disease, but co-expression of WT enzyme at high levels with mutant SOD1 accelerates the onset of motor neuron disease compared to mice expressing mutant hSOD1 alone. Spinal cords of mice expressing both proteins contain aggregated forms of mutant protein and, in some cases, evidence of co-aggregation of WT hSOD1 enzyme. In the present study, we used a cell culture model of mutant SOD1 aggregation to examine how the presence of WT SOD1 affects mutant protein aggregation, finding that co-expression of WT SOD1, human (hSOD1) or mouse (mSOD1), delayed the formation of mutant hSOD1 aggregates; in essence appearing to slow the aggregation rate. In some combinations of WT and mutant hSOD1 co-expression, the aggregates that did eventually form appeared to contain WT hSOD1 protein. However, WT mSOD1 did not co-aggregate with mutant hSOD1 despite displaying a similar ability to slow mutant hSOD1 aggregation. Together, these studies indicate that WT SOD1 (human or mouse), when expressed at levels equivalent to the mutant protein, modulates aggregation of FALS-mutant hSOD1.
PMCID: PMC2801375  PMID: 19077113
superoxide; dismutase; aggregation; amyotrophic lateral sclerosis
16.  Different regulation of wild-type and mutant Cu,Zn superoxide dismutase localization in mammalian mitochondria 
Human Molecular Genetics  2008;17(21):3303-3317.
The antioxidant enzyme Cu,Zn superoxide dismutase (SOD1) is predominantly localized in the cytosol, but it is also found in mitochondria. Studies in yeast suggest that apoSOD1 is imported into mitochondria and trapped inside by folding and maturation, which is facilitated by its copper chaperone for SOD1 (CCS). Here, we show that in mammalian cells, SOD1 mitochondrial localization is dictated by its folding state, which is modulated by several interconnected factors. First, the intracellular distribution of CCS determines SOD1 partitioning in cytosol and mitochondria: CCS localization in the cytosol prevents SOD1 mitochondrial import, whereas CCS in mitochondria increases it. Second, the Mia40/Erv1 pathway for import of small intermembrane space proteins participates in CCS mitochondrial import in a respiratory chain-dependent manner. Third, CCS mitochondrial import is regulated by oxygen concentration: high (20%) oxygen prevents import, whereas physiological (6%) oxygen promotes it. Therefore, SOD1 localization responds to changes in environmental conditions following redistribution of CCS, which operates as an oxygen sensor. Fourth, all of the cysteine residues in human SOD1 are critical for its retention in mitochondria due to their involvement in intramolecular disulfide bonds and in the interaction with CCS. Mutations in SOD1 are associated with autosomal dominant familial amyotrophic lateral sclerosis. Like the wild-type protein, mutant SOD1 localizes to mitochondria, where it induces bioenergetic defects. We find that the physiological regulation of mitochondrial localization is either inefficient or absent in SOD1 pathogenic mutants. We propose misfolding and aggregation of these mutants that trap them inside mitochondria.
PMCID: PMC2566526  PMID: 18703498
17.  Metal-deficient aggregates and diminished copper found in cells expressing SOD1 mutations that cause ALS 
Disruptions in metal ion homeostasis have been described in association with amyotrophic lateral sclerosis (ALS) for a number of years but the precise mechanism of involvement is poorly understood. Metal ions are especially important to familial ALS cases caused by mutations in the metalloenzyme copper-zinc superoxide dismutase (SOD1). To investigate the role of metals in aggregation of mutant SOD1, we have examined the localization of metal ions in a cell culture model of overexpression. Chinese hamster ovary cells (CHO-K1) were transfected to overexpress SOD1 fused to yellow fluorescent protein (YFP) to readily identify the transfected cells and the intracellular aggregates that develop in the cells expressing mutant or wild-type (WT) SOD1. The concentration and distribution of iron, copper, and zinc were determined for four SOD1 mutants (A4V, G37R, H80R, and D125H) as well as a WT SOD1 using X-ray fluorescence microscopy (XFM). Results demonstrated that the SOD1 aggregates were metal-deficient within the cells, which is consistent with recent in vitro studies. In addition, all SOD1 mutants showed significantly decreased copper content compared to the WT SOD1 cells, regardless of the mutant’s ability to bind copper. These results suggest that SOD1 overexpression creates an unmet demand on the cell for copper. This is particularly true for the SOD1 mutants where copper delivery may also be impaired. Hence, the SOD1 mutants are less stable than WT SOD1 and if copper is limited, aggregate formation of the metal-deficient, mutant SOD1 protein occurs.
PMCID: PMC4059277  PMID: 24982630
amyotrophic lateral sclerosis; superoxide dismutase; X-ray fluorescence microscopy; synchrotron
18.  Posttranslational Modifications in Cu,Zn-Superoxide Dismutase and Mutations Associated with Amyotrophic Lateral Sclerosis 
Antioxidants & redox signaling  2006;8(5-6):847-867.
Activation of the enzyme Cu,Zn-superoxide dismutase (SOD1) involves several posttranslational modifications including copper and zinc binding, as well as formation of the intramolecular disulfide bond. The copper chaperone for SOD1, CCS, is responsible for intracellular copper loading in SOD1 under most physiological conditions. Recent in vitro and in vivo assays reveal that CCS not only delivers copper to SOD1 under stringent copper limitation, but it also facilitates the stepwise conversion of the disulfide-reduced immature SOD1 to the active disulfide-containing enzyme. The two new functions attributed to CCS, (i.e., O2-dependent sulfhydryl oxidase- and disulfide isomerase-like activities) indicate that this protein has attributes of the larger class of molecular chaperones. The CCS-dependent activation of SOD1 is dependent upon oxygen availability, suggesting that the cell only loads copper and activates this enzyme when O2-based oxidative stress is present. Thiol/disulfide status as well as metallation state of SOD1 significantly affects its structure and protein aggregation, which are relevant in pathologies of a neurodegenerative disease, amyotrophic lateral sclerosis (ALS). The authors review here a mechanism for posttranslational activation of SOD1 and discuss models for ALS in which the most immature forms of the SOD1 polypeptide exhibits propensity to form toxic aggregates.
PMCID: PMC1633719  PMID: 16771675
19.  Monoaminergic control of spinal locomotor networks in SOD1G93A newborn mice 
Mutations in the gene that encodes Cu/Zn-superoxide dismutase (SOD1) are the cause of approximately 20% of familial forms of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease characterized by the progressive loss of motor neurons. While ALS symptoms appear in adulthood, spinal motoneurons exhibit functional alterations as early as the embryonic and postnatal stages in the murine model of ALS, the SOD1 mice. Monoaminergic – i.e., dopaminergic (DA), serotoninergic (5-HT), and noradrenergic (NA) – pathways powerfully control spinal networks and contribute significantly to their embryonic and postnatal maturation. Alterations in monoaminergic neuromodulation during development could therefore lead to impairments in the motoneuronal physiology. In this study, we sought to determine whether the monoaminergic spinal systems are modified in the early stages of development in SOD1 mice. Using a post-mortem analysis by high performance liquid chromatography (HPLC), monoaminergic neuromodulators and their metabolites were quantified in the lumbar spinal cord of SOD1 and wild-type (WT) mice aged one postnatal day (P1) and P10. This analysis underscores an increased content of DA in the SOD1 lumbar spinal cord compared to that of WT mice but failed to reveal any modification of the other monoaminergic contents. In a next step, we compared the efficiency of the monoaminergic compounds in triggering and modulating fictive locomotion in WT and SOD1 mice. This study was performed in P1–P3 SOD1 mice and age-matched control littermates using extracellular recordings from the lumbar ventral roots in the in vitro isolated spinal cord preparation. This analysis revealed that the spinal networks of SOD1G93A mice could generate normal locomotor activity in the presence of NMA-5-HT. Interestingly, we also observed that SOD1 spinal networks have an increased sensitivity to NA compared to WT spinal circuits but exhibited similar DA responses.
PMCID: PMC4081764  PMID: 25071458
serotonin; dopamine; noradrenaline; spinal cord; ALS; fictive locomotion; HPLC; extracellular recordings
20.  Destabilizing Protein Polymorphisms in the Genetic Background Direct Phenotypic Expression of Mutant SOD1 Toxicity 
PLoS Genetics  2009;5(3):e1000399.
Genetic background exerts a strong modulatory effect on the toxicity of aggregation-prone proteins in conformational diseases. In addition to influencing the misfolding and aggregation behavior of the mutant proteins, polymorphisms in putative modifier genes may affect the molecular processes leading to the disease phenotype. Mutations in SOD1 in a subset of familial amyotrophic lateral sclerosis (ALS) cases confer dominant but clinically variable toxicity, thought to be mediated by misfolding and aggregation of mutant SOD1 protein. While the mechanism of toxicity remains unknown, both the nature of the SOD1 mutation and the genetic background in which it is expressed appear important. To address this, we established a Caenorhabditis elegans model to systematically examine the aggregation behavior and genetic interactions of mutant forms of SOD1. Expression of three structurally distinct SOD1 mutants in C. elegans muscle cells resulted in the appearance of heterogeneous populations of aggregates and was associated with only mild cellular dysfunction. However, introduction of destabilizing temperature-sensitive mutations into the genetic background strongly enhanced the toxicity of SOD1 mutants, resulting in exposure of several deleterious phenotypes at permissive conditions in a manner dependent on the specific SOD1 mutation. The nature of the observed phenotype was dependent on the temperature-sensitive mutation present, while its penetrance reflected the specific combination of temperature-sensitive and SOD1 mutations. Thus, the specific toxic phenotypes of conformational disease may not be simply due to misfolding/aggregation toxicity of the causative mutant proteins, but may be defined by their genetic interactions with cellular pathways harboring mildly destabilizing missense alleles.
Author Summary
Correct folding and stability are essential for protein function. In cells, a network of molecular chaperones and degradative enzymes facilitate folding, prevent aggregation and ensure degradation of the misfolded proteins, thus maintaining protein homeostasis. In many diseases, including Amyotrophic Lateral Sclerosis (ALS), expression of a single mutant protein that misfolds and aggregates causes cellular toxicity that is strongly dependent on the genetic background. To address the influence of genetic background on the toxicity of aggregation-prone proteins, we established a C. elegans model of misfolding and aggregation of several distinct ALS-related mutants of superoxide dismutase 1 (SOD1). In one wild type genetic background (N2), these proteins exhibited only mild cellular toxicity despite strong, mutant-specific aggregation phenotypes. However, when SOD1 mutants were expressed in the background of mildly destabilized protein polymorphisms, their toxicity was enhanced and a number of distinct phenotypes were exposed. These synthetic phenotypes reflected the loss-of-function of the destabilized polymorphic proteins. Furthermore, the degree to which each of these phenotypes was exposed depended on the nature of the SOD1 mutation. These data suggest that the presence of mildly destabilizing polymorphisms in the genetic background may modulate and direct the specific toxic phenotypes in protein aggregation diseases.
PMCID: PMC2642731  PMID: 19266020
21.  Molecular Mechanisms and Clinical Implications of Reversible Protein S-Glutathionylation 
Antioxidants & Redox Signaling  2008;10(11):1941-1988.
Sulfhydryl chemistry plays a vital role in normal biology and in defense of cells against oxidants, free radicals, and electrophiles. Modification of critical cysteine residues is an important mechanism of signal transduction, and perturbation of thiol–disulfide homeostasis is an important consequence of many diseases. A prevalent form of cysteine modification is reversible formation of protein mixed disulfides (protein–SSG) with glutathione (GSH). The abundance of GSH in cells and the ready conversion of sulfenic acids and S-nitroso derivatives to S-glutathione mixed disulfides suggests that reversible S-glutathionylation may be a common feature of redox signal transduction and regulation of the activities of redox sensitive thiol-proteins. The glutaredoxin enzyme has served as a focal point and important tool for evolution of this regulatory mechanism, because it is a specific and efficient catalyst of protein–SSG deglutathionylation. However, mechanisms of control of intracellular Grx activity in response to various stimuli are not well understood, and delineation of specific mechanisms and enzyme(s) involved in formation of protein–SSG intermediates requires further attention. A large number of proteins have been identified as potentially regulated by reversible S-glutathionylation, but only a few studies have documented glutathionylation-dependent changes in activity of specific proteins in a physiological context. Oxidative stress is a hallmark of many diseases which may interrupt or divert normal redox signaling and perturb protein–thiol homeostasis. Examples involving changes in S-glutathionylation of specific proteins are discussed in the context of diabetes, cardiovascular and lung diseases, cancer, and neurodegenerative diseases. Antioxid. Redox Signal, 10, 1941–1988.
Potential Mechanisms of Protein–SSG Formation
Thiol-disulfide exchange
Sulfenic acid intermediates
Sulfenylamide intermediates
Thiyl radical intermediates
Thiosulfinate intermediates
S-Nitrosylated intermediates
Potential Catalysis of Protein Glutathionylation
Flavoprotein sulfhydryl oxidease (QSOX)
Other potential mechanisms of catalysis/control of protein S-glutathionylation
Proteomics of Discovery of Potential Protein–SSG Intermediates
Deglutathionylation (Reversal) of Protein–SSG: Properties of the Glutaredoxin Enzymes
Glutaredoxin Mechanism of Action
Modualtion of Grx Expression
Diabetes and Implications of Changes in S-Glutathionylation Status
Mechanism of hyperglycemic damage and ROS
Insulin-glucose dynamics and diabetes complications
Glucose metabolism: aldose reductase–SSG (Fig. 3, step 1a)
K+ channels: Grx regulated (Fig. 3, step 2a)
ATP-sensitive potassium channels
Voltage-gated potassium channels
Ca2+ channels: SERCA-SSG and Grx-reversible RyR-SSG (Fig. 3, step 3a)
Insulin exocytosis: Grx regulated (Fig. 3 step 6a)
Insulin receptor: Grx-reversible PTP1B-SSG (Fig. 3, step 6b)
Signal transduction [Fig. 3, Ras-SSG (step 7b), MEKK-SSG (step 8b), c-Jun-SSG (step 9b), Akt-SSG (step 10b), IKK-SSG (step 11b), NF-κB(p50)-SSG (steps 5a and 12b), and PKC-SSG (step 4a)]
Summary and discussion: Grx as a therapeutic target in diabetic complications
Cardiovascular Diseases and Alterations in Protein-S-Glutathionylation Status
Myocardial infarction
Protein kinase C (PKC)
Protein kinase A (PKA)
Nuclear factor κB (NF-κB)
Nonspecific oxidative injury
Cardiac hypertrophy
Implications of Protein S-Glutathionylation in Lung Disease
Tobacco exposure
Hyperoxic injury
Fibrotic and granulomatous diseases
Chronic obstructuve pulmonary disease (COPD)
Implications of Reversible Protein S-Glutathionylation in Cancer
Thiol oxidation and cancer
S-Glutathionylation and signal transduction in cancer
S-Glutathionylation and modulation of kinase/phosphatase signaling pathways
Protein kinase C (PKC)
I3 kinase and Akt
Protein tryosine phosphatase
c-Jun N-terminal kinase (JKN)
S-Glutathionylation and modulation of the proteasome pathway
S-Glutathionylation and modulation of transcription factors (c-Jun, NF-κB, p53, AP-1)
AP-1, c-Jun
Modulation of S-glutathionylation as a chemotherapeutic strategy for cancer
Implications of Protein S-Glutathionylation in Neurodegenerative Diseases
Oxidative stress and neurodegeneration
Sources of reactive oxygen and nitrogen species in brain
Alzheimer's disease
Parkinson's disease
Huntington's disease
Amyotrophic lateral sclerosis
Freidreich's ataxia
Glutaredoxin and neurodegeneration
Proteins associated with neurodegeneration that are redox regulated through S-glutathionylation
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
Mitochondrial NADP+-dependent isocitrate dehydrogenase (IDPm)
Tyrosine hydroxylase
Cytosolic calcium regulators
Proteasome degradation pathway
α-Ketoglutarate dehydrogenase
Summary and Conclusions
Frontier Areas of Investigation
PMCID: PMC2774718  PMID: 18774901
22.  Disulfide-Reduced ALS Variants of Cu, Zn Superoxide Dismutase Exhibit Increased Populations of Unfolded Species 
Journal of molecular biology  2010;398(2):320-331.
Cu, Zn superoxide dismutase (SOD1) is a dimeric metal binding enzyme responsible for the dismutation of toxic superoxide to hydrogen peroxide and oxygen in cells. Mutations at dozens of sites in SOD1 induce amyotrophic lateral sclerosis (ALS), a fatal gain-of-function neurodegenerative disease whose molecular basis is unknown. To obtain insights into effects of the mutations on the folded and unfolded populations of immature monomeric forms whose aggregation or self-association may be responsible for ALS, the thermodynamic and kinetic folding properties of a set of disulfide-reduced and disulfide-oxidized Zn-free and Zn-bound stable monomeric SOD1 variants were compared to the wild-type (WT) protein. The most striking effect of the mutations on the monomer stability was observed for the disulfide-reduced metal-free variants. Whereas the WT and S134N monomers are >95% folded at neutral pH and 37 °C, A4V, L38V, G93A, and L106V ranged from 50% to ∼90% unfolded. The reduction of the disulfide-bond was also found to reduce the apparent Zn affinity of the WT monomer by 750-fold, into the nanomolar range where it may be unable to compete for free Zn in the cell. With the exception of the S134N metal-binding variant, the Zn affinity of disulfide-oxidized SOD1 monomers showed little sensitivity to amino acid replacements. These results suggest a model for SOD1 aggregation where the constant synthesis of ALS-variants of SOD1 on ribosomes provides a pool of species in which the increased population of the unfolded state may favor aggregation over productive folding to the stable native dimeric state.
PMCID: PMC3075925  PMID: 20184893
Disulfide bond; Zn binding; protein folding; aggregation
23.  Structural Characterization of Zinc-deficient Human Superoxide Dismutase and Implications for ALS 
Journal of molecular biology  2007;373(4):877-890.
Over 130 mutations to copper, zinc superoxide dismutase (SOD) are implicated in the selective death of motor neurons found in 25% of familial amyotrophic lateral sclerosis (ALS) patients. Despite their widespread distribution, ALS mutations appear positioned to cause structural and misfolding defects. Such defects decrease SOD’s affinity for zinc, and loss of zinc from SOD is sufficient to induce apoptosis in motor neurons in vitro. To examine the importance of the zinc site in the structure and pathogenesis of human SOD, we determined the 2.0 Å resolution crystal structure of a designed zinc-deficient human SOD, in which two zinc-binding ligands have been mutated to hydrogen-bonding serine residues. This structure revealed a 9° twist of the subunits, which opens the SOD dimer interface and represents the largest inter-subunit rotational shift observed for a human SOD variant. Furthermore, the electrostatic loop and zinc-binding sub-loop were partly disordered, the catalytically important Arg143 was rotated away from the active site, and the normally rigid intramolecular Cys57-Cys146 disulfide bridge assumed two conformations. Together, these changes allow small molecules greater access to the catalytic copper, consistent with the observed increased redox activity of zinc-deficient SOD. Moreover, the dimer interface is weakened and the Cys57-Cys146 disulfide is more labile, as demonstrated by the increased aggregation of zinc-deficient SOD in the presence of a thiol reductant. However, equimolar Cu,Zn SOD rapidly forms heterodimers with zinc-deficient SOD (t1/2 ≈ 15 min) and prevents aggregation. The stabilization of zinc-deficient SOD as a heterodimer with Cu,Zn SOD may thus be a contributing factor to the dominant inheritance of ALS mutations. These results have general implications for the importance of framework stability on normal metalloenzyme function and specific implications for the role of zinc ion in the fatal neuropathology associated with SOD mutations.
PMCID: PMC2175016  PMID: 17888947
Amyotrophic lateral sclerosis; Cu; Zn superoxide dismutase; Lou Gehrig’s disease; zinc-deficient superoxide dismutase; crystal structure
24.  Muscle cells and motoneurons differentially remove mutant SOD1 causing familial amyotrophic lateral sclerosis 
Journal of Neurochemistry  2011;118(2):266-280.
Amyotrophic lateral sclerosis (ALS) is a fatal motoneuronal disease which occurs in sporadic or familial forms, clinically indistinguishable. About 15% of familial ALS cases are linked to mutations of the superoxide dismutase 1 (SOD1) gene that may induce misfolding in the coded protein, exerting neurotoxicity to motoneurons. However, other cell types might be target of SOD1 toxicity, because muscle-restricted expression of mutant SOD1 correlates with muscle atrophy and motoneurons death. We analysed the molecular behaviour of mutant SOD1 in motoneuronal NSC34 and muscle C2C12 cells. We found that misfolded mutant SOD1 clearance is much more efficient in muscle C2C12 than in motoneuronal NSC34 cells. Mutant SOD1 forms aggregates and impairs the proteasome only in motoneuronal NSC34 cells. Interestingly, NSC34 cells expressing mutant SOD1 are more sensitive to a superoxide-induced oxidative stress. Moreover, in muscle C2C12 cells mutant SOD1 remains soluble even when proteasome is inhibited with MG132. The higher mutant SOD1 clearance in muscle cells correlates with a more efficient proteasome activity, combined with a robust autophagy activation. Therefore, muscle cells seem to better manage misfolded SOD1 species, not because of an intrinsic property of the mutant protein, but in function of the cell environment, indicating also that the SOD1 toxicity at muscle level may not directly depend on its aggregation rate.
PMCID: PMC3206220  PMID: 21554318
amyotrophic lateral sclerosis; autophagy; motoneuron diseases; muscle cells; proteasome; SOD1
25.  Single chain variable fragment antibodies block aggregation and toxicity induced by familial ALS-linked mutant forms of SOD1 
Neurobiology of disease  2013;56:74-78.
Approximately 10% of amyotrophic lateral sclerosis (ALS) cases are familial (known as FALS) with an autosomal dominant inheritance pattern, and ~25% of FALS cases are caused by mutations in Cu/Zn superoxide dismutase (SOD1). There is convincing evidence that mutant SOD1 (mtSOD1) kills motor neurons (MNs) because of a gain-of-function toxicity, most likely related to aggregation of mtSOD1. A number of recent reports have suggested that antibodies can be used to treat mtSOD1-induced FALS. To follow up on the use of antibodies as potential therapeutics, we generated single chain fragments of variable region antibodies (scFvs) against SOD1, and then expressed them as ‘intrabodies’ within a motor neuron cell line. In the present study, we describe isolation of human scFvs that interfere with mtSOD1 in vitro aggregation and toxicity. These scFvs may have therapeutic potential in sporadic ALS, as well as FALS, given that sporadic ALS may also involve abnormalities in the SOD1 protein or activity.
PMCID: PMC3725968  PMID: 23607939
familial amyotrophic lateral sclerosis; mutant superoxide dismutase type 1; amyotrophic lateral sclerosis; ALS; single chain fragments of variable region antibodies; scFvs; motor neuron disease

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