Loss of in vitro metal ion binding specificity in mutant copper-zinc superoxide dismutases associated with familial amyotrophic lateral sclerosis

The presence of the copper ion at the active site of human wild type copper-zinc superoxide dismutase (CuZnSOD) is essential to its ability to catalyze the disproportionation of superoxide into dioxygen and hydrogen peroxide. Wild type CuZnSOD and several of the mutants associated with familial amyotrophic lateral sclerosis (FALS) (Ala(4) --> Val, Gly(93) --> Ala, and Leu(38) --> Val) were expressed in Saccharomyces cerevisiae. Purified metal-free (apoproteins) and various remetallated derivatives were analyzed by metal titrations monitored by UV-visible spectroscopy, histidine modification studies using diethylpyrocarbonate, and enzymatic activity measurements using pulse radiolysis. From these studies it was concluded that the FALS mutant CuZnSOD apoproteins, in direct contrast to the human wild type apoprotein, have lost their ability to partition and bind copper and zinc ions in their proper locations in vitro. Similar studies of the wild type and FALS mutant CuZnSOD holoenzymes in the "as isolated" metallation state showed abnormally low copper-to-zinc ratios, although all of the copper acquired was located at the native copper binding sites. Thus, the copper ions are properly directed to their native binding sites in vivo, presumably as a result of the action of the yeast copper chaperone Lys7p (yeast CCS). The loss of metal ion binding specificity of FALS mutant CuZnSODs in vitro may be related to their role in ALS.     

Transduction of familial amyotrophic lateral sclerosis-related mutant PEP-1-SOD proteins into neuronal cells

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by the selective death of motor neurons. Mutations in the SOD1 gene are responsible for a familial form of ALS (FALS). Although many studies suggest that mutant SOD1 proteins are cytotoxic, the mechanism is not fully understood. To investigate the role of mutant SOD1 in FALS, human SOD1 genes were fused with a PEP-1 peptide in a bacterial expression vector to produce in-frame PEP-1-SOD fusion proteins (wild type and mutants). The expressed and purified PEP-1-SOD fusion proteins were efficiently transduced into neuronal cells. Neurones harboring the A4V, G93A, G85R, and D90A mutants of PEP-1-SOD were more vulnerable to oxidative stress induced by paraquat than those harboring wild-type proteins. Moreover, neurones harboring the mutant SOD proteins had lower heat shock protein (Hsp) expression levels than those harboring wild-type SOD. The effects of the transduced SOD1 fusion proteins may provide an explanation for the association of SOD1 with FALS, and Hsps could be candidate agents for the treatment of ALS.     

The effects of glutaredoxin and copper activation pathways on the disulfide and stability of Cu,Zn superoxide dismutase

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.     

Effect of CCS on the accumulation of FALS SOD1 mutant-containing aggregates and on mitochondrial translocation of SOD1 mutants: implication of a free radical hypothesis

Missense mutations of SOD1 are linked to familial amyotrophic lateral sclerosis (FALS) through a yet-to-be identified toxic-gain-of-function. One of the proposed mechanisms involves enhanced aggregate formation. However, a recent study showed that dual transgenic mice overexpressing both G93A and CCS copper chaperone (G93A/CCS) exhibit no SOD1-positive aggregates yet show accelerated FALS symptoms with enhanced mitochondrial pathology compared to G93A mice. Using a dicistronic mRNA to simultaneously generate hSOD1 mutants, G93A, A4V and G85R, and hCCS in AAV293 cells, we revealed: (i) CCS is degraded primarily via a macroautophagy pathway. It forms a stable heterodimer with inactive G85R, and via its novel copper chaperone-independent molecular chaperone activity facilitates G85R degradation via a macroautophagy-mediated pathway. For active G93A and A4V, CCS catalyzes their maturation to form active and soluble homodimers. (ii) CCS reduces, under non-oxidative conditions, yet facilitates in the presence of H₂O₂, mitochondrial translocation of inactive SOD1 mutants. These results, together with previous reports showing FALS SOD1 mutants enhanced free radical-generating activity, provide a mechanistic explanation for the observations with G93A/CCS dual transgenic mice and suggest that free radical generation by FALS SOD1, enhanced by CCS, may, in part, be responsible for the FALS SOD1 mutant-linked aggregation, mitochondrial translocation, and degradation.     

Familial amyotrophic lateral sclerosis-associated mutations decrease the thermal stability of distinctly metallated species of human copper/zinc superoxide dismutase

We report the thermal stability of wild type (WT) and 14 different variants of human copper/zinc superoxide dismutase (SOD1) associated with familial amyotrophic lateral sclerosis (FALS). Multiple endothermic unfolding transitions were observed by differential scanning calorimetry for partially metallated SOD1 enzymes isolated from a baculovirus system. We correlated the metal ion contents of SOD1 variants with the occurrence of distinct melting transitions. Altered thermal stability upon reduction of copper with dithionite identified transitions resulting from the unfolding of copper-containing SOD1 species. We demonstrated that copper or zinc binding to a subset of "WT-like" FALS mutants (A4V, L38V, G41S, G72S, D76Y, D90A, G93A, and E133Delta) conferred a similar degree of incremental stabilization as did metal ion binding to WT SOD1. However, these mutants were all destabilized by approximately 1-6 degrees C compared with the corresponding WT SOD1 species. Most of the "metal binding region" FALS mutants (H46R, G85R, D124V, D125H, and S134N) exhibited transitions that probably resulted from unfolding of metal-free species at approximately 4-12 degrees C below the observed melting of the least stable WT species. We conclude that decreased conformational stability shared by all of these mutant SOD1s may contribute to SOD1 toxicity in FALS.     

A novel protocol to identify and quantify all spin trapped free radicals from in vitro/in vivo interaction of HO(.-) and DMSO: LC/ESR, LC/MS, and dual spin trapping combinations

When dimethyl sulfoxide (DMSO) is oxidized via hydroxyl radical (HO(.-)), it forms methyl radicals ((.-)CH(3)) that can be spin trapped and detected by electron spin resonance (ESR). This ESR spin trapping technique has been widely used in many biological systems to indicate in vivo HO(.-) formation. However, we recently reported that (.-)CH(3) might not be the only carbon-centered radical that was trapped and detected by ESR from in vivo DMSO oxidation. In the present study, newly developed combination techniques consisting of dual spin trapping (free radicals trapped by both regular and deuterated alpha-[4-pyridyl 1]-N-tert-butyl nitrone, d(0)/d(9)-POBN) followed by LC/ESR and LC/MS were used to characterize and quantify all POBN-trapped free radicals from the interaction of HO(.-) and DMSO. In addition to identifying the two well-known free radicals, (.-)CH(3) and (.-)OCH(3), from this interaction, we also characterized two additional free radicals, (.-)CH(2)OH and (.-)CH(2)S(O)CH(3). Unlike ESR, which can measure POBN adducts only in their radical forms, LC/MS identified and quantified all three redox forms, including the ESR-active radical adduct and two ESR-silent forms, the nitrone adduct (oxidized adduct) and the hydroxylamine (reduced adduct). In the bile of rats treated with DMSO and POBN, the ESR-active form of POBN/(.-)CH(3) was not detected. However, with the addition of the LC/MS technique, we found approximately 0.75 microM POBN/(.-)CH(3) hydroxylamine, which represents a great improvement in radical detection sensitivity and reliability. This novel protocol provides a comprehensive way to characterize and quantify in vitro and in vivo free radical formation and will have many applications in biological research.     

Glutathionylation at Cys-111 induces dissociation of wild type and FALS mutant SOD1 dimers

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 SOD1(WT) dimers, increasing the equilibrium dissociation constant K(d) to ~10-20 μM, comparable to that of the aggressive A4V mutant. SOD1(A4V) is further destabilized by glutathionylation, experiencing an ~30-fold increase in K(d). Dissociation kinetics of glutathionylated SOD1(WT) and SOD1(A4V) are unchanged, as measured by surface plasmon resonance, indicating that glutathionylation destabilizes these variants by decreasing association rate. In contrast, SOD1(I112T) has a modestly increased dissociation rate but no change in K(d) 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.     

Decreased metallation and activity in subsets of mutant superoxide dismutases associated with familial amyotrophic lateral sclerosis

Over 90 different mutations in the gene encoding copper/zinc superoxide dismutase (SOD1) cause approximately 2% of amyotrophic lateral sclerosis (ALS) cases by an unknown mechanism. We engineered 14 different human ALS-related SOD1 mutants and obtained high yields of biologically metallated proteins from an Sf21 insect cell expression system. Both the wild type and mutant "as isolated" SOD1 variants were deficient in copper and were heterogeneous by native gel electrophoresis. By contrast, although three mutant SOD1s with substitutions near the metal binding sites (H46R, G85R, and D124V) were severely deficient in both copper and zinc ions, zinc deficiency was not a consistent feature shared by the as isolated mutants. Eight mutants (A4V, L38V, G41S, G72S, D76Y, D90A, G93A, and E133 Delta) exhibited normal SOD activity over pH 5.5-10.5, per equivalent of copper, consistent with the presumption that bound copper was in the proper metal-binding site and was fully active. The H48Q variant contained a high copper content yet was 100-fold less active than the wild type enzyme and exhibited a blue shift in the visible absorbance peak of bound Cu(II), indicating rearrangement of the Cu(II) coordination geometry. Further characterization of these as-isolated SOD1 proteins may provide new insights regarding mutant SOD1 enzyme toxicity in ALS.     

Release of copper ions from the familial amyotrophic lateral sclerosis-associated Cu,Zn-superoxide dismutase mutants

Point mutations of Cu,Zn-superoxide dismutase (SOD) have been linked to familial amyotrophic lateral sclerosis (FALS). We reported that the Swedish FALS Cu,Zn-SOD mutant, D90A, exhibited an enhanced hydroxyl radical-generating activity, while its dismutation activity was identical to that of the wild-type enzyme (Kim et al. 1998a; 1998b). Transgenic mice that express a mutant Cu,Zn-SOD, Gly93 --> Ala (G93A), have been shown to develop amyotrophic lateral sclerosis (ALS) symptoms. We cloned the cDNA for the FALS G93A mutant, overexpressed the protein in E. coli cells, purified the protein, and studied its enzymic activities. Our results showed that the G93A, the D90A, and the wild-type enzymes have identical dismutation activity. However, the hydroxyl radical-generating activity of the G93A mutant was enhanced relative to those of the D90A and the wild-type enzyme (wild-type < D90A < G93A). These higher free radical-generating activities of mutants facilitated the release of copper ions from their own molecules (wild-type < D90A < G93A). The released copper ions can enhance the Fenton-like reaction to produce hydroxyl radicals and play a major role in the oxidative damage of macromolecules. Thus, the FALS symptoms may be associated with the enhancements in both the free radical-generating activity and the releasing of copper ions from the mutant enzyme.     

Isolation and characterization of the initial radical adduct formed from linoleic acid and alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone in the presence of soybean lipoxygenase.

The spin trapping agent alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) was used to trap the initial radical formed from [U-14C]linoleic acid in the reaction with soybean lipoxygenase. By using low levels of enzyme and relatively short incubation times it was possible to avoid the formation of secondary oxidation products and polymers. The adduct was extracted after methyl esterification, and isolated by a combination of open column chromatography on silicic acid and high pressure liquid chromatography on Spherisorb S5 CN with non-aqueous solvents. The 1:1 POBN-linoleate adduct was characterized by UV, IR and ESR spectra of the appropriate HPLC column fraction, by the ratio of the UV absorption to 14C content, and by mass spectrometry of the reduced (hydroxylamine) form. The results indicated that POBN trapped a linoleic acid carbon-centered radical such that POBN was attached to the fatty acid chain at C-13 or C-9 (two isomers), the linoleate double bonds having become conjugated in the process. The exact locations of the bridges in the two isomers were only tentatively determined. There was no evidence for the presence of oxygen-bridged adducts. The trapped linoleoyl radical adduct provides evidence for the production of a free radical as part of the enzymatic mechanism of soybean lipoxygenase.     

Elevated "Hydroxyl Radical" Generation In Vivo in an Animal Model of Amyotrophic Lateral Sclerosis

Abstract: Mutations in the enzyme copper/zinc superoxide dismutase-1 (SOD1) are associated with familial amyotrophic lateral sclerosis (FALS). The means by which the mutations cause FALS appears to be due to an adverse property of the mutant SOD1 protein that may involve increased generation of free radicals. We used in vivo microdialysis to measure the conversion of 4-hydroxybenzoic acid to 3,4-dihydroxybenzoic acid (3,4-DHBA) as a measure of "hydroxyl radical-like" production in transgenic amyotrophic lateral sclerosis (ALS) mice with the G93A mutation as well as littermate controls. The conversion of 4-hydroxybenzoic acid to 3,4-DHBA was significantly increased in the striatum of transgenic ALS mice at baseline but not in mice overexpressing wild-type human SOD1. Following administration of 3-nitropropionic acid 3,4-DHBA generation was significantly increased as compared with baseline, and the increase in the transgenic ALS mice was significantly greater than those in controls, whereas the increase in mice overexpressing wild-type human SOD1 was significantly attenuated. The present results provide in vivo evidence that expression of mutations in SOD1 can lead to increased generation of "hydroxyl radical-like" activity, which further implicates oxidative damage in the pathogenesis of ALS.     

Acute methanol intoxication generates free radicals in rats: an ESR spin trapping investigation

Electron spin resonance (ESR) spectroscopy has been used to investigate free radical generation in rats with acute methanol poisoning. The spin trapping technique was used where a spin trapping agent, alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone (POBN), reacted with the corresponding alcohol-derived or alcohol-dependent radical to form radical adducts. One radical adduct was detected in both bile and urine samples 2 h after acute methanol poisoning in male Sprague Dawley rats. The hyperfine coupling constants for the radical adduct from [(13)C]-labeled methanol detected in the bile were a(N) = 15.58, a(beta)(H) = 2.81 G, and a(beta)(13C) = 4.53 G, which unambiguously identified this species as POBN/*CH@OH. The same radical adduct was detected in urine. The identification of a methanol-derived radical adduct in samples from bile and urine provided strong direct evidence for the generation of the alcohol-derived radicals during acute intoxication by methanol. Simultaneous administration of the alcohol dehydrogenase inhibitor 4-methylpyrazole and methanol resulted in an increase in the generation of the free radical metabolite detected in the bile. This is the first ESR evidence of methanol-derived free radical generation in an animal model of acute methanol intoxication.     

Mutated human SOD1 causes dysfunction of oxidative phosphorylation in mitochondria of transgenic mice

A growing body of evidence suggests that impaired mitochondrial energy production and increased oxidative radical damage to the mitochondria could be causally involved in motor neuron death in amyotrophic lateral sclerosis (ALS) and in familial ALS associated with mutations of Cu,Zn superoxide dismutase (SOD1). For example, morphologically abnormal mitochondria and impaired mitochondrial histoenzymatic respiratory chain activities have been described in motor neurons of patients with sporadic ALS. To investigate further the role of mitochondrial alterations in the pathogenesis of ALS, we studied mitochondria from transgenic mice expressing wild type and G93A mutated hSOD1. We found that a significant proportion of enzymatically active SOD1 was localized in the intermembrane space of mitochondria. Mitochondrial respiration, electron transfer chain, and ATP synthesis were severely defective in G93A mice at the time of onset of the disease. We also found evidence of oxidative damage to mitochondrial proteins and lipids. On the other hand, presymptomatic G93A transgenic mice and mice expressing the wild type form of hSOD1 did not show significant mitochondrial abnormalities. Our findings suggest that G93A-mutated hSOD1 in mitochondria may cause mitochondrial defects, which contribute to precipitating the neurodegenerative process in motor neurons.     

Do oxidatively modified proteins cause ALS?

Over 90 individual mutations in SOD1 are known to cause familial amyotrophic lateral sclerosis (FALS). It is widely accepted that these mutations exert their toxic effects by a gain of function mechanism, but the nature of these toxic effects is as yet unknown. It has been proposed by several laboratories that reactions of FALS-mutant CuZnSOD are the source of elevated oxidative stress in CuZnSOD-linked FALS. It has also been proposed that aggregates of CuZnSOD are somehow involved in the disease. The hypothesis that aggregates of CuZnSOD cause ALS is particularly attractive because protein aggregates are frequently associated with other neurodegenerative diseases. Recent evidence increasingly suggests that protein aggregates containing CuZnSOD protein play a role in CuZnSOD-linked ALS, but it is not yet know why the aggregates form nor if the CuZnSOD proteins in the aggregates are cleaved, oxidized, demetallated, or otherwise covalently modified.     

Accelerated s-nitrosothiol breakdown by amyotrophic lateral sclerosis mutant copper,zinc-superoxide dismutase

Mutations in copper,zinc-superoxide dismutase (SOD) have been implicated in familial amyotrophic lateral sclerosis (FALS). We have investigated the breakdown of S-nitrosothiols by wild-type (WT) SOD and two common FALS mutants, alanine-4 valine (A4V) SOD and glycine-37 arginine (G37R) SOD. In the presence of glutathione, A4V SOD and G37R SOD catalyzed S-nitrosoglutathione breakdown three times more efficiently than WT SOD. Indeed, A4V SOD catabolized GSNO more efficiently than WT SOD throughout the physiological range of GSH concentrations. Moreover, a variety of additional S-nitrosothiols were catabolized more readily by A4V SOD than by WT SOD. Initial rate data for fully reduced WT SOD and A4V SOD, and data using ascorbic acid as the reductant, suggest that FALS mutations in SOD may influence the efficiency of reduction of the copper center by glutathione. We have identified a potentially toxic gain of function of two common FALS mutations that may contribute to neurodegeneration in FALS.     

Aberrantly increased hydrophobicity shared by mutants of Cu,Zn-superoxide dismutase in familial amyotrophic lateral sclerosis

More than 100 different mutations in the gene encoding Cu,Zn-superoxide dismutase (SOD1) cause preferential motor neuron degeneration in familial amyotrophic lateral sclerosis (ALS). Although the cellular target(s) of mutant SOD1 toxicity have not been precisely specified, evidence to date supports the hypothesis that ALS-related mutations may increase the burden of partially unfolded SOD1 species. Influences that may destabilize SOD1 in vivo include impaired metal ion binding, reduction of the intrasubunit disulfide bond, or oxidative modification. In this study, we observed that metal-deficient as-isolated SOD1 mutants (H46R, G85R, D124V, D125H, and S134N) with disordered electrostatic and zinc-binding loops exhibited aberrant binding to hydrophobic beads in the absence of other destabilizing agents. Other purified ALS-related mutants that can biologically incorporate nearly normal amounts of stabilizing zinc ions (A4V, L38V, G41S, D90A, and G93A) exhibited maximal hydrophobic behavior after exposure to both a disulfide reducing agent and a metal chelator, while normal SOD1 was more resistant to these agents. Moreover, we detected hydrophobic SOD1 species in lysates from affected tissues in G85R and G93A mutant but not wildtype SOD1 transgenic mice. These findings suggest that a susceptibility to the cellular disulfide reducing environment and zinc loss may convert otherwise stable SOD1 mutants into metal-deficient forms with locally destabilized electrostatic and zinc-binding loops. These abnormally hydrophobic SOD1 species may promote aberrant interactions of the enzyme with itself or with other cellular constituents to produce toxicity in familial ALS.     

Integrative role of cPLA with COX-2 and the effect of non-steriodal anti-inflammatory drugs in a transgenic mouse model of amyotrophic lateral sclerosis

Cyclooxygenase-2 (COX-2) is a key molecule in the inflammatory pathway in amyotrophic lateral sclerosis (ALS). Cytosolic phospholipase A (cPLA2) is an important enzyme providing substrate for cyclooxygenases. We therefore examined cPLA2 expression in human ALS and mutant Cu/Zn superoxide dismutase (SOD1) transgenic mice and its relation to COX-2. Immunohistochemistry and real-time RT-PCR revealed elevated cPLA2 protein and its mRNA levels in the lumbar spinal cord of mutant SOD1 mice. COX-2 immunoreactivity was increased in lumbar spinal cord sections from both familial ALS (FALS) and sporadic ALS (SALS) as compared to controls, and cPLA2 immunoreactivity was increased in a patient with FALS. Oral administration of the non-selective cyclooxygenase (COX) inhibitor, sulindac, extended the survival (by 10%) of G93A SOD1 mice as compared to littermate controls. Sulindac, as well as the selective COX-2 inhibitors, rofecoxib and celecoxib reduced cPLA2 immunoreactivity in the lumbar spinal cord of G93A transgenic mice. Sulindac treatment preserved motor neurons, and reduced microglial activation and astrocytosis, in the spinal cord of G93A SOD1 transgenic mice. These results suggest that cPLA2 plays an important role in supplying arachidonic acid to the COX-2 driven inflammatory pathway in ALS associated with SOD1 mutations.     

The perplexing role of copper-zinc superoxide dismutase in amyotrophic lateral sclerosis (Lou Gehrig's disease)

The existence of a link between some cases of familial amyotrophic lateral sclerosis (FALS) and copper-zinc superoxide dismutase (CuZnSOD) has been understood for almost a decade. However, beyond the fact that mutations in CuZnSOD cause FALS by a toxic gain of function, the mechanism whereby specific mutations in the protein structure result in development of the disease has remained almost a complete mystery to date. We have undertaken a critical survey of in vitro characteristics of over 30 of the 90 different CuZnSOD mutant proteins that are known to cause FALS in order to determine the differences that exist between mutant and wild-type properties. As-isolated metal content analysis, SOD activity assays, and thermal stability determinations of a significant fraction of the mutants show that the FALS mutant SOD proteins can be classified distinctly into one of two groups. Members of the first group, termed wild-type-like, have physical properties and enzymatic activities that are strikingly similar to those of wild-type CuZnSOD. The second group, however, show aberrant metal content in the as-isolated forms, compromised SOD activities, and unusual DSC thermoscans. All mutations in the members of this second group occur in or near the metal binding sites of the protein and thus they are termed metal binding region mutants. We have also compared the relative rates of self-inactivation caused by reaction of the wild-type protein and several FALS-linked CuZnSOD mutants with hydrogen peroxide, as a measure of relative peroxidative activities. Results and implications of the role of CuZnSOD in FALS are discussed.     

Expression of a familial amyotrophic lateral sclerosis-associated mutant human superoxide dismutase in yeast leads to decreased mitochondrial electron transport

Strains of Saccharomyces cerevisiae that express either the wild type or the amyotrophic lateral sclerosis-associated mutant human copper-zinc superoxide dismutase (SOD1) proteins A4V and G93A, respectively, in a yeast SOD1-deficient parent strain were used to investigate the hypothesis that expression of a mutant SOD1 protein causes deficient mitochondrial electron transport as a possible mechanism for disease induction. Mitochondria isolated from the wild type SOD1-expressing yeast were identical to mitochondria from the parent strain in heme content and activities of complexes II, III, and IV. Mitochondria isolated from the A4V-expressing yeast had decreased rates of electron transport in complexes II+III, III, and IV and corresponding decreases in hemes b, c-c1, and a-a3 content compared to mitochondria from wild type human SOD1-expressing yeast. Mitochondria isolated from G93A-expressing yeast had decreased rates of electron transport in complex IV and probably in complex II with a corresponding decrease in heme a-a3 content. These results suggest that mutant SOD1-expression causes defective electron transport complex assembly and that the yeast system will provide an excellent model for the study of the mechanism of mutant SOD1-induced mitochondrial electron transport defects.     

ALS-causing SOD1 mutations promote production of copper-deficient misfolded species

Point mutations scattered throughout the sequence of Cu,Zn superoxide dismutase (SOD1) cause a subset of amyotrophic lateral sclerosis (ALS) cases. SOD1 is a homodimer in which each subunit binds one copper atom and one zinc atom. Inclusions containing misfolded SOD1 are seen in motor neurons of SOD1-associated ALS cases. The mechanism by which these diverse mutations cause misfolding and converge on the same disease is still not well understood. Previously, we developed several time-resolved techniques to monitor structural changes in SOD1 as it unfolds in guanidine hydrochloride. By measuring the rates of Cu and Zn release using an absorbance-based assay, dimer dissociation through chemical cross-linking, and β-barrel conformation changes by tryptophan fluorescence, we established that wild-type SOD1 unfolds by a branched pathway involving a Zn-deficient monomer as the dominant intermediate of the major pathway, and with various metal-loaded and Cu-deficient dimers populated along the minor pathway. We have now compared the unfolding pathway of wild-type SOD1 with those of A4V, G37R, G85R, G93A, and I113T ALS-associated mutant SOD1. The kinetics of unfolding of the mutants were generally much faster than those of wild type. However, all of the mutants utilize the minority pathway to a greater extent than the wild-type protein, leading to greater populations of Cu-deficient intermediates and decreases in Zn-deficient intermediates relative to the wild-type protein. The greater propensity of the mutants to populate Cu-deficient states potentially implicates these species as a pathogenic form of SOD1 in SOD1-associated ALS and provides a novel target for therapeutic intervention.