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.
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.
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.
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.
ALS; beta-barrel dimer; metal binding; protein folding; thermodynamics and kinetics
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.
seeding; subcellular localization; SOD1; TDP-43; non-cell-autonomous motor neuron death; antibody
Cu,Zn SOD1 (superoxide dismutase 1) is implicated in FALS (familial amyotrophic lateral sclerosis) through the accumulation of misfolded proteins that are toxic to neuronal cells. Loop VI (residues 102–115) of the protein is at the dimer interface and could play a critical role in stability. The free cysteine residue, Cys111 in the loop, is readily oxidized and alkylated. We have found that modification of this Cys111 with 2-ME (2-mercaptoethanol; 2-ME-SOD1) stabilizes the protein and the mechanism may provide insights into destabilization and the formation of aggregated proteins. Here, we determined the crystal structure of 2-ME-SOD1 and find that the 2-ME moieties in both subunits interact asymmetrically at the dimer interface and that there is an asymmetric configuration of segment Gly108 to Cys111 in loop VI. One loop VI of the dimer forms a 310-helix (Gly108 to His110) within a unique β-bridge stabilized by a hydrogen bond between Ser105-NH and His110-CO, while the other forms a β-turn without the H-bond. The H-bond (H-type) and H-bond free (F-type) configurations are also seen in some wild-type and mutant human SOD1s in the Protein Data Bank suggesting that they are interconvertible and an intrinsic property of SOD1s. The two structures serve as a basis for classification of these proteins and hopefully a guide to their stability and role in pathophysiology.
superoxide dismutase 1 (SOD1); crystal structure; amyotrophic lateral sclerosis (ALS); asymmetric configuration; ALS, amyotrophic lateral sclerosis; FALS, familial amyotrophic lateral sclerosis; 2-ME, 2-mercaptoethanol; PDB, protein data bank; SOD1, superoxide dismutase 1; WT, wild-type
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.
Amyotrophic lateral sclerosis; Cu; Zn superoxide dismutase; Lou Gehrig’s disease; zinc-deficient superoxide dismutase; crystal structure
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.
Disulfide; redox potential; glutaredoxins; yeast; Cu,Zn superoxide dismutase; monothiol
Copper-zinc superoxide dismutase (SOD1) is a detoxifying enzyme localized in the cytosol, nucleus, peroxisomes, and mitochondria. The discovery that mutations in SOD1 gene cause a subset of familial amyotrophic lateral sclerosis (FALS) has attracted great attention, and studies to date have been mainly focused on discovering mutations in the coding region and investigation at protein level. Considering that changes in SOD1 mRNA levels have been associated with sporadic ALS (SALS), a molecular understanding of the processes involved in the regulation of SOD1 gene expression could not only unravel novel regulatory pathways that may govern cellular phenotypes and changes in diseases but also might reveal therapeutic targets and treatments. This review seeks to provide an overview of SOD1 gene structure and of the processes through which SOD1 transcription is controlled. Furthermore, we emphasize the importance to focus future researches on investigating posttranscriptional mechanisms and their relevance to ALS.
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.
Mutations in human copper-zinc superoxide dismutase (SOD1) cause an inherited form of amyotrophic lateral sclerosis (ALS, Lou Gehrig’s disease, motor neuron disease). Insoluble forms of mutant SOD1 accumulate in neural tissues of human ALS patients and in spinal cords of transgenic mice expressing these polypeptides, suggesting that SOD1-linked ALS is a protein misfolding disorder. Understanding the molecular basis for how the pathogenic mutations give rise to SOD1 folding intermediates, which may themselves be toxic, is therefore of keen interest. A critical step on the SOD1 folding pathway occurs when the copper chaperone for SOD1 (CCS) modifies the nascent SOD1 polypeptide by inserting the catalytic copper cofactor and oxidizing its intrasubunit disulfide bond. Recent studies reveal that pathogenic SOD1 proteins coming from cultured cells and from the spinal cords of transgenic mice tend to be metal-deficient and/or lacking the disulfide bond, raising the possibility that the disease-causing mutations may enhance levels of SOD1-folding intermediates by preventing or hindering CCS-mediated SOD1 maturation. This mini-review explores this hypothesis by highlighting the structural and biophysical properties of the pathogenic SOD1 mutants in the context of what is currently known about CCS structure and action. Other hypotheses as to the nature of toxicity inherent in pathogenic SOD1 proteins are not covered.
superoxide dismutase; SOD1; amyotrophic lateral sclerosis; motor neuron disease; protein misfolding; protein aggregation; protofibrils; amyloid
Dominant mutations in Cu,Zn-superoxide dismutase (SOD1) cause a familial form of amyotrophic lateral sclerosis (fALS). Misfolding and aggregation of mutant SOD1 proteins are a pathological hallmark of SOD1-related fALS cases; however, the molecular mechanism of SOD1 aggregation remains controversial. Here, I have used E. coli as a model organism and shown multiple distinct pathways of SOD1 aggregation that are dependent upon its thiol-disulfide status. Overexpression of fALS-mutant SOD1s in the cytoplasm of E. coli BL21 and SHuffleTM, where redox environment is reducing and oxidizing, respectively, resulted in the formation of insoluble aggregates with notable differences; a disulfide bond of SOD1 was completely reduced in BL21 or abnormally formed between SOD1 molecules in SHuffleTM. Depending upon intracellular redox environment, therefore, mutant SOD1 is considered to misfold/aggregate through distinct pathways, which would be relevant in description of the pathological heterogeneity of SOD1-related fALS cases.
SOD1; ALS; aggregation; disulfide bond
Mutations in superoxide dismutase 1 (SOD1), which are one cause of familial amyotrophic lateral sclerosis (fALS), induce misfolding and aggregation of the protein. Misfolding can be detected by the binding of antibodies raised against peptide epitopes that are normally buried in the native conformation, shifts in solubility in non-ionic detergents, and the formation of macromolecular inclusions. In the present study, we investigate the relationship between detergent-insoluble and sedimentable forms of mutant SOD1, forms of mutant SOD1 with aberrantly accessible epitopes, and mutant protein in inclusions with the goal of defining the spectrum of misfolded states that mutant SOD1 can adopt.
Using combined approaches in cultured cell models, we demonstrate that a substantial fraction of mutant SOD1 adopts a non-native conformation that remains soluble and freely mobile. We also show that mutant SOD1 can produce multimeric assemblies of which some are insoluble in detergent and large enough to sediment by ultracentrifugation and some are large enough to detect visually. Three conformationally restricted antibodies were found to be useful in discriminating mal-folded forms of mutant SOD1. An antibody termed C4F6 displays properties consistent with recognition of soluble, freely mobile, mal-folded mutant SOD1. An antibody termed SEDI, which recognizes C-terminal residues, detects larger inclusion structures as well as soluble misfolded entities. An antibody termed hSOD1, which recognizes aa 24-36, detects an epitope shared by soluble non-natively folded WT and mutant SOD1. This epitope becomes inaccessible in aggregates of mutant SOD1.
Our studies demonstrate how different methods of detecting misfolding and aggregation of mutant SOD1 reveal different forms of aberrantly folded protein. Immunological and biochemical methods can be used in combination to detect soluble and insoluble misfolded forms of mutant SOD1. Our findings support the view that mutant SOD1 can adopt multiple misfolded conformations with the potential that different structural variants mediate different aspects of fALS.
The rate-limiting step in the formation of the native dimeric state of human Cu, Zn superoxide dismutase (SOD1) is a very slow monomer folding reaction that governs the lifetime of its unfolded state. Mutations at dozens of sites in SOD1 are known to cause a fatal motor neuron disease, amyotrophic lateral sclerosis, and recent experiments implicate the unfolded state as a source of soluble oligomers and histologically observable aggregates thought to be responsible for toxicity. To determine the thermodynamic properties of the transition state ensemble (TSE) limiting the folding of this high contact order β-sandwich motif, a combined thermal/urea denaturation thermodynamic/kinetic analysis was performed. The barriers to folding and unfolding are dominated by the activation enthalpy at 298 K and neutral pH; the activation entropy is favorable and reduces the barrier height for both reactions. The absence of secondary structure formation or large-scale chain collapse prior to crossing the barrier for folding led to the conclusion that dehydration of nonpolar surfaces in the TSE is responsible for the large and positive activation enthalpy. Although the activation entropy favors the folding reaction, the transition from the unfolded state to the native state is entropically disfavored at 298 K. The opposing entropic contributions to the free energies of the TSE and the native state during folding provide insights into structural properties of the TSE. The results also imply a crucial role for water in governing the productive folding reaction and enhancing the propensity for the aggregation of SOD1.
Protein folding; enthalpy; entropy; heat capacity; enthalpy-entropy compensation
Superoxide dismutases (SOD) are important anti-oxidant enzymes that guard against superoxide toxicity. Various SOD enzymes have been characterized that employ either a copper, manganese, iron or nickel co-factor to carry out the disproportionation of superoxide. This review focuses on the copper and manganese forms, with particular emphasis on how the metal is inserted in vivo into the active site of SOD. Copper and manganese SODs diverge greatly in sequence and also in the metal insertion process. The intracellular copper SODs of eukaryotes (SOD1) can obtain copper post-translationally, by way of interactions with the CCS copper chaperone. CCS also oxidizes an intrasubunit disulfide in SOD1. Adventitious oxidation of the disulfide can lead to gross misfolding of immature forms of SOD1, particularly with SOD1 mutants linked to amyotrophic lateral sclerosis. In the case of mitochondrial MnSOD of eukaryotes (SOD2), metal insertion cannot occur post-translationally, but requires new synthesis and mitochondrial import of the SOD2 polypeptide. SOD2 can also bind iron in vivo, but is inactive with iron. Such metal ion mis-incorporation with SOD2 can become prevalent upon disruption of mitochondrial metal homeostasis. Accurate and regulated metallation of copper and manganese SOD molecules is vital to cell survival in an oxygenated environment.
Copper; Manganese; Iron; Mitochondria; ALS; Superoxide dismutase; SOD; CCS; Copper chaperone; Posttranslational modification; Disulfide isomerase; SOD1; SOD2; EC-SOD
The copper-zinc superoxide dismutase-1 (SOD1) is a highly structured protein and, a priori, one of the least likely proteins to be involved in a misfolding disease. However, more than 140, mostly missense, mutations in the SOD1 gene cause aggregation of the affected protein in familial forms of amyotrophic lateral sclerosis (ALS). The remarkable diversity of the effects of these mutations on SOD1 properties has suggested that they promote aggregation by a variety of mechanisms. Experimental assessment of surface hydrophobicity using a sensitive fluorescent-based assay, revealed that diverse ALS-causing mutations provoke SOD1 aggregation by increasing their propensity to expose hydrophobic surfaces. These findings could not be anticipated from analysis of the amino acid sequence. Our results uncover the biochemical nature of the misfolded aggregation-prone intermediate and reconcile the seemingly diverse effects of ALS-causing mutations into a unifying mechanism. Furthermore, the method we describe here will be useful for investigating and interfering with aggregation of various proteins and thereby provide insight into the molecular mechanisms underlying many neurodegenerative diseases.
superoxide dismutase; amyotrophic lateral sclerosis; aggregation; neurodegeneration
We recently described a set of drug-like molecules obtained from an in silico screen that stabilize mutant superoxide dismutase-1 (SOD-1) linked to familial amyotrophic lateral sclerosis (ALS) against unfolding and aggregation but exhibited poor binding specificity towards SOD-1 in presence of blood plasma. A reasonable, but not a conclusive model for the binding of these molecules, was proposed based on restricted docking calculations and site-directed mutagenesis of key residues at the dimer interface. A set of hydrogen bonding constraints obtained from these experiments were used to guide docking calculations with compound library around the dimer interface. A series of chemically unrelated hits were predicted, which were experimentally tested for their ability to block aggregation. At least six of the new molecules exhibited high specificity of binding towards SOD-1 in presence of blood plasma. These molecules represent a new class of molecules for further development into clinical candidates.
The most common cause of amyotrophic lateral sclerosis (ALS) is mutations in superoxide dismutase-1 (SOD1). Since there is evidence for the involvement of non-neuronal cells in ALS, we searched for signs of SOD1 abnormalities focusing on glia. Spinal cords from nine ALS patients carrying SOD1 mutations, 51 patients with sporadic or familial ALS who lacked such mutations, and 46 controls were examined by immunohistochemistry. A set of anti-peptide antibodies with specificity for misfolded SOD1 species was used. Misfolded SOD1 in the form of granular aggregates was regularly detected in the nuclei of ventral horn astrocytes, microglia, and oligodendrocytes in ALS patients carrying or lacking SOD1 mutations. There was negligible staining in neurodegenerative and non-neurological controls. Misfolded SOD1 appeared occasionally also in nuclei of motoneurons of ALS patients. The results suggest that misfolded SOD1 present in glial and motoneuron nuclei may generally be involved in ALS pathogenesis.
Electronic supplementary material
The online version of this article (doi:10.1007/s00401-011-0805-3) contains supplementary material, which is available to authorized users.
Amyotrophic lateral sclerosis; Superoxide dismutase; Motoneurons; Intranuclear; Glia
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.
Disulfide bond; Zn binding; protein folding; aggregation
Mutations in superoxide dismutase 1 (SOD1) are associated with familial cases of amyotrophic lateral sclerosis (fALS). Studies in transgenic mice have suggested that wild-type (WT) SOD1 can modulate the toxicity of mutant SOD1. In the present study, we demonstrate that the effects of WT SOD1 on the age at which transgenic mice expressing mutant human SOD1 (hSOD1) develop paralysis are influenced by the nature of the ALS mutation and the expression levels of WT hSOD1. We show that regardless of whether WT SOD1 changes the course of disease, both WT and mutant hSOD1 accumulate as detergent-insoluble aggregates in symptomatic mice expressing both proteins. However, using a panel of fluorescently tagged variants of SOD1 in a cell model of mutant SOD1 aggregation, we demonstrate that the interactions between mutant and WT SOD1 in aggregate formation are not simply a co-assembly of mutant and WT proteins. Overall, these data demonstrate that the product of the normal SOD1 allele in fALS has potential to influence the toxicity of mutant SOD1 and that complex interactions with the mutant protein may influence the formation of aggregates and inclusion bodies generated by mutant SOD1.
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.
Amyotrophic lateral sclerosis (ALS) is a disorder that involves the degeneration of motor neurons, muscle atrophy, and paralysis. In a few familiar forms of ALS, mutations in the superoxide dismutase-1 (SOD1) gene have been held responsible for the degeneration of motor neurons. Nevertheless, after the discovery of the SOD1 mutations no consensus has emerged as to which cells, tissues and pathways are primarily implicated in the pathogenic events that lead to ALS. Ubiquitous overexpression of mutant SOD1 in transgenic animals recapitulates the pathological features of ALS. However, the toxicity of mutant SOD1 is not necessarily limited to the central nervous system. Views about ALS pathogenesis are now enriched by the recent discovery of mutations in a pair of DNA/RNA-binding proteins called TDP-43 and FUS/TLS as causes of familial and sporadic forms of ALS. Although the steps that lead to the pathological state are well defined, several fundamental issues are still controversial: are the motor neurons the first direct targets of ALS; and what is the contribution of non-neuronal cells, if any, to the pathogenesis of ALS? The state of the art of ALS pathogenesis and the open questions are discussed in this review.
Amyotrophic lateral sclerosis; Neurodegenerative disease; Muscle wasting; Oxidative stress; Excitotoxicity; Protein aggregation; Mitochondrial dysfunction; Insulin-like growth factor 1
Mutations in human copper-zinc superoxide dismutase (SOD1) cause an inherited form of amyotrophic lateral sclerosis (ALS). Inclusions enriched in pathogenic SOD1 accumulate in the spinal cords of transgenic mice expressing these proteins, but endogenous mouse SOD1 is not found as a component of these aggregates. In the accompanying paper, Karch and colleagues analyze aggregation propensities of human/mouse SOD1 chimeras in cell culture and identify two sequence elements in the human enzyme that seem to enhance its aggregation relative to the mouse enzyme. Here, we report the first structure of mouse SOD1 along with those of SOD1 chimeras in which residues 1-80 come from human SOD1 and residues 81-153 come from mouse SOD1 and vice versa. Taken together, the structural and cell-based data suggest a model in which residues Q42 and Q123 in mouse SOD1 modulate nonnative SOD1-SOD1 intermolecular interactions at edge strands in the SOD1 Greek key β-barrel.
Copper-zinc superoxide dismutase; amyotrophic lateral sclerosis; X-ray crystallography; protein aggregation
The oxidative damage hypothesis proposed for the function gain of copper, zinc superoxide dismutase (SOD1) maintains that both mutant and wild-type (WT) SOD1 catalyze reactions with abnormal substrates that damage cellular components critical for viability of the affected cells. However, whether the oxidative damage of SOD1 is involved in the formation of aggregates rich in SOD1 or not remains elusive. Here, we sought to explore the oxidative aggregation of WT SOD1 exposed to environments containing both ascorbate (Asc) and DNA under neutral conditions. The results showed that the WT SOD1 protein was oxidized in the presence of Asc. The oxidation results in the higher affinity of the modified protein for DNA than that of the unmodified protein. The oxidized SOD1 was observed to be more prone to aggregation than the WT SOD1, and the addition of DNA can significantly accelerate the oxidative aggregation. Moreover, a reasonable relationship can be found between the oxidation, increased hydrophobicity, and aggregation of SOD1 in the presence of DNA. The crucial step in aggregation is neutralization of the positive charges on some SOD1 surfaces by DNA binding. This study might be crucial for understanding molecular forces driving the protein aggregation.
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.