Copper(2+) binding to the surface residue cysteine 111 of His46Arg human copper-zinc superoxide dismutase, a familial amyotrophic lateral sclerosis mutant

Mutations in copper-zinc superoxide dismutase (CuZnSOD) cause 25% of familial amyotrophic lateral sclerosis (FALS) cases. This paper examines one such mutant, H46R, which has no superoxide dismutase activity yet presumably retains the gain-of-function activity that leads to disease. We demonstrate that Cu(2+) does not bind to the copper-specific catalytic site of H46R CuZnSOD and that Cu(2+) competes with other metals for the zinc binding site. Most importantly, Cu(2+) was found to bind strongly to a surface residue near the dimer interface of H46R CuZnSOD. Cysteine was identified as the new binding site on the basis of multiple criteria including UV-vis spectroscopy, RR spectroscopy, and chemical derivatization. Cysteine 111 was pinpointed as the position of the reactive ligand by tryptic digestion of the modified protein and by mutational analysis. This solvent-exposed residue may play a role in the toxicity of this and other FALS CuZnSOD mutations. Furthermore, we propose that the two cysteine 111 residues, found on opposing subunits of the same dimeric enzyme, may provide a docking location for initial metal insertion during biosynthesis of wild-type CuZnSOD in vivo.     

The structure of holo and metal-deficient wild-type human Cu,Zn superoxide dismutase and its relevance to familial amyotrophic lateral sclerosis

Cu, Zn superoxide dismutase (SOD1) forms a crucial component of the cellular defence against oxidative stress. Zn-deficient wild-type and mutant human SOD1 have been implicated in the disease familial amyotrophic lateral sclerosis (FALS). We present here the crystal structures of holo and metal-deficient (apo) wild-type protein at 1.8A resolution. The P21 wild-type holo enzyme structure has nine independently refined dimers and these combine to form a "trimer of dimers" packing motif in each asymmetric unit. There is no significant asymmetry between the monomers in these dimers, in contrast to the subunit structures of the FALS G37R mutant of human SOD1 and in bovine Cu,Zn SOD. Metal-deficient apo SOD1 crystallizes with two dimers in the asymmetric unit and shows changes in the metal-binding sites and disorder in the Zn binding and electrostatic loops of one dimer, which is devoid of metals. The second dimer lacks Cu but has approximately 20% occupancy of the Zn site and remains structurally similar to wild-type SOD1. The apo protein forms a continuous, extended arrangement of beta-barrels stacked up along the short crystallographic b-axis, while perpendicular to this axis, the constituent beta-strands form a zig-zag array of filaments, the overall arrangement of which has a similarity to the common structure associated with amyloid-like fibrils.     

Calcineurin activity is regulated both by redox compounds and by mutant familial amyotrophic lateral sclerosis-superoxide dismutase

Calcineurin (CN) is a protein phosphatase involved in a wide range of cellular responses to calcium-mobilizing signals, and a role for this enzyme in neuropathology has been postulated. We have investigated the possibility that redox modulation of CN activity is relevant to neuropathological conditions where an imbalance in reactive oxygen species has been described. We have monitored CN activity in cultured human neuroblastoma SH-SY5Y cells and obtained evidence that CN activity is promoted by treatment with ascorbate or dithiothreitol and impaired by oxidative stress. Evidence for the existence of a redox regulation of this enzyme has been also obtained by overexpression of wild-type antioxidant Cu,Zn superoxide dismutase (SOD1) that promotes CN activity and protects it from oxidative inactivation. On the contrary, overexpression of mutant SOD1s associated with familial amyotrophic lateral sclerosis (FALS) impairs CN activity both in transfected human neuroblastoma cell lines and in the motor cortex of brain from FALS-transgenic mice. These data suggest that CN might be a target in the pathogenesis of SOD1-linked FALS.     

Superoxide dismutase 1 modulates expression of transferrin receptor

Copper–zinc superoxide dismutase (SOD1) plays a protective role against the toxicity of superoxide, and studies in Saccharomyces cerevisiae and in Drosophila have suggested an additional role for SOD1 in iron metabolism. We have studied the effect of the modulation of SOD1 levels on iron metabolism in a cultured human glial cell line and in a mouse motoneuronal cell line. We observed that levels of the transferrin receptor and the iron regulatory protein 1 were modulated in response to altered intracellular levels of superoxide dismutase activity, carried either by wild-type SOD1 or by an SOD-active amyotrophic lateral sclerosis (ALS) mutant enzyme, G93A-SOD1, but not by a superoxide dismutase inactive ALS mutant, H46R-SOD1. Ferritin expression was also increased by wild-type SOD1 overexpression, but not by mutant SOD1s. We propose that changes in superoxide levels due to alteration of SOD1 activity affect iron metabolism in glial and neuronal cells from higher eukaryotes and that this may be relevant to diseases of the nervous system.     

Cloning,production and characterisation of a recombinant Cu/Zn superoxide dismutase from Taenia solium

A full-length complementary DNA clone encoding a cytosolic Cu/Zn superoxide dismutase with a M(r) of 15,588 Da was isolated from a Taenia solium larvae complementary DNA library. Comparison analysis of its deduced amino acid sequence revealed a 71% identity with Schistosoma mansoni, 57.2-59.8% with mammalian and less than 54% with other helminth cytosolic Cu/Zn superoxide dismutase. The characteristic motifs and the amino acid residues involved in coordinating copper and zinc enzymatic function are conserved. The T. solium Cu/Zn superoxide dismutase was expressed in the pRSET vector. Enzymatic and filtration chromatographic analysis showed a recombinant enzyme with an activity of 2,941 U/mg protein and a native M(r) of 37 kDa. Inhibition assays using KCN, H(2)O(2), NaN(3) and SDS indicated that Cu/Zn is the metallic cofactor in the enzyme. Thiabendazole (500 microM) and albendazole (300 microM) completely inhibited the activity of T. solium Cu/Zn superoxide dismutase. Thiabendazole had no effect on bovine Cu/Zn superoxide dismutase; in contrast, albendazole had a moderate effect on it at same concentrations. Antibodies against T. solium Cu/Zn superoxide dismutase did not affect the enzymatic function; nevertheless, it cross reacts with several Taenia species, but not with trematodes, nematodes, pig, human and bovine Cu/Zn superoxide dismutase enzymes. Western blot analysis indicated the enzyme was expressed in all stages. These results indicate that T. solium possesses a Cu/Zn superoxide dismutase enzyme that can protect him from oxidant-damage caused by the superoxide anion.     

Oxidative inactivation of calcineurin by Cu,Zn superoxide dismutase G93A, a mutant typical of familial amyotrophic lateral sclerosis

Calcineurin is a serine/threonine phosphatase involved in a wide range of cellular responses to calcium mobilizing signals. Previous evidence supports the notion of the existence of a redox regulation of this enzyme, which might be relevant for neurodegenerative processes, where an imbalance between generation and removal of reactive oxygen species could occur. In a recent work, we have observed that calcineurin activity is depressed in two models for familial amyotrophic lateral sclerosis (FALS) associated with mutations of the antioxidant enzyme Cu,Zn superoxide dismutase (SOD1), namely in neuroblastoma cells expressing either SOD1 mutant G93A or mutant H46R and in brain areas from G93A transgenic mice. In this work we report that while wild-type SOD1 has a protective effect, calcineurin is oxidatively inactivated by mutant SOD1s in vitro; this inactivation is mediated by reactive oxygen species and can be reverted by addition of reducing agents. Furthermore, we show that calcineurin is sensitive to oxidation only when it is in an 'open', calcium-activated conformation, and that G93A-SOD1 must have its redox-active copper site available to substrates in order to exert its pro-oxidant properties on calcineurin. These findings demonstrate that both wild-type and mutant SOD1s can interfere directly with calcineurin activity and further support the possibility of a relevant role for calcineurin-regulated biochemical pathways in the pathogenesis of FALS.     

Expression of Human A4V Mutant Cu,Zn Superoxide Dismutase in Schizosaccharomyces pombe: Investigations of its Toxic Properties

Cu,Zn superoxide dismutase (SOD1) is an antioxidant enzyme that catalyzes the removal of superoxide radicals generated in various biological oxidations. Amyotrophic lateral sclerosis (ALS) is one of the most common neurodegenerative disorders, occurring in families (FALS) and sporadically (SALS). FALS and SALS are distinguishable genetically but not clinically. More than 100 point mutations in the human SOD 1 gene have been identified that cause FALS. In order to determine the effects of mutant SOD protein, we first cloned wild-type and A4V mutant human SOD1 into Schizosaccharomyces pombe. This study shows viabilities and some antioxidant properties including SOD, catalase, proteasomal activity, and protein carbonyl levels of transformants in SOD1 deleted strain (MN415); and its parental strain (JY741) at different stress conditions. There was no more oxidative damage in the human mutant SOD carrying the transformant strain compared with other strains. These results may help to explain whether ALS progresses as a consequence of cellular oxidative damage.     

Role of the electrostatic loop of Cu,Zn superoxide dismutase in the copper uptake process.

Cu,Zn superoxide dismutases are characterized by the presence of four highly conserved charged residues (Lys120, Glu/Asp130, Glu131 and Lys134), which are placed at the edge of the active site channel and have been shown to be individually involved in the electrostatic attraction of the substrate toward the catalytically active copper ion. By genetic engineering we mutated these four residues into neutrally charged ones (Leu120, Gln130, Gln131, Thr134). The effects of these mutations on the rate of superoxide dismutation were not dramatic. In fact, at two different pH and ionic strength values, the mutant enzyme had a catalytic constant even higher with respect to the wild-type protein, showing that electrostatic interaction at these surface sites is not essential for high catalytic efficiency of the enzyme. The mutant and the wild-type enzyme showed the same degree of inhibition by CN(-), and both were not affected by I(-), showing that mutations did not alter the sensitivity of the enzyme to anions. On the other hand, reconstitution of active enzyme from either the wild-type or mutant copper-free enzymes with a copper(I)-glutathione [Cu(I)-GSH] complex showed that metal uptake by the mutant was much slower than by the wild-type enzyme. The demonstration that the 'electrostatic loop' is apparently conserved to assure optimal copper uptake by the enzyme, rather than fast dismutation, may provide further support to the idea that Cu,Zn superoxide dismutase is a bifunctional protein, acting in cellular defense against oxidative stress both as a copper buffer and as a superoxide radical scavenger.     

The role of arginine 143 in the electrostatics and mechanism of Cu,Zn superoxide dismutase: Computational and experimental evaluation by mutational analysis

Cu, Zn superoxide dismutase protects cells from oxidative damage by removing superoxide radicals in one of the fastest enzyme reactions known. The redox reaction at the active-site Cu ion is rate-limited by diffusion and enhanced by electrostatic guidance. To quantitatively define the electrostatic and mechanistic contributions of sequence-invariant Arg-143 in human Cu, Zn superoxide dismutase, single-site mutants at this position were investigated experimentally and computationally. Rate constants for several Arg-143 mutants were determined at different pH and ionic strength conditions using pulse radiolytic methods and compared to results from Brownian dynamics simulations. At physiological pH, substitution of Arg-143 by Lys caused a 2-fold drop in rate, neutral substitutions (Ile, Ala) reduced the rate about 10-fold, while charge-reversing substitutions (Asp, Glu) caused a 100-fold decrease. Position 143 mutants showed pH dependencies not seen in other mutants. At low pH, the acidic residue mutations exhibited pro-tonation/deprotonation effects. At high pH, all enzymes showed typical decreases in rate except the Lys mutant in which the rate dropped off at an unusually low pH. Increasing ionic strength at acidic pH decreased the rates of the wild-type enzyme and Lys mutant, while the rate of the Glu mutant was unaffected. Increasing ionic strength at higher pH (>10) increased the rates of the Lys and Glu mutants while the rate of the wild-type enzyme was unaffected. Reaction simulations with Brownian dynamics incorporating electrostatic effects tested computational predictability of ionic strength dependencies of the wild-type enzyme and the Lys, Ile, and Glu mutants. The calculated and experimental ionic strength profiles gave similar slopes in all but the Glu mutant, indicating that the electrostatic attraction of the substrate is accurately modeled. Differences between the calculated and experimental rates for the Glu and Lys mutants reflect the mechanistic contribution of Arg-143. Results from this joint analysis establish that, aside from the Cu ligands, Arg-143 is the single most important residue in Cu, Zn superoxide dismutase both electrostatically and mechanistically, and provide an explanation for the evolutionary selection of arginine at position 143. © 1994 Wiley-Liss, Inc.     

In vivo formation of Cu,Zn superoxide dismutase disulfide bond in Escherichia coli

We have found that the in vivo folding of periplasmic Escherichia coli Cu,Zn superoxide dismutase is assisted by DsbA, which catalyzes the efficient formation of its single disulfide bond, whose integrity is essential to ensure full catalytic activity to the enzyme. In line with these findings, we also report that the production of recombinant Xenopus laevis Cu,Zn superoxide dismutase is enhanced when the enzyme is exported in the periplasmic space or is expressed in thioredoxin reductase mutant strains. Our data show that inefficient disulfide bond oxidation in the bacterial cytoplasm inhibits Cu,Zn superoxide dismutase folding in this cellular compartment.     

Proteasome activation and nNOS down-regulation in neuroblastoma cells expressing a Cu,Zn superoxide dismutase mutant involved in familial ALS

Reactive oxygen and nitrogen species have emerged as predominant effectors of neurodegeneration. We demonstrated that expression of the fully active G93A Cu,Zn superoxide dismutase mutant in neuroblastoma cells is associated with an increased level of oxidatively modified proteins, in terms of carbonylated residues. A parallel increase in proteasome activity was detected and this was mandatory in order to assure cell viability. In fact, proteasome inhibition by lactacystin or MG132 resulted in programmed cell death. Nitrosative stress was not involved in the oxidative unbalance, as a decrease in neuronal nitric oxide production and down-regulation of neuronal nitric oxide synthase (nNOS) level were detected. The nNOS down-regulation was correlated to increased proteolytic degradation by proteasome, because comparable levels of nNOS were detected in G93A and parental cells upon treatment with lactacystin. The altered rate of proteolysis observed in G93A cells was specific for nNOS as Cu,Zn superoxide dismutase (Cu,Zn SOD) degradation by proteasome was influenced neither by its mutation nor by increased proteasome activity. Treatment with the antioxidant 5,5'-dimethyl-1-pyrroline N-oxide resulted in inhibition of protein oxidation and decrease in proteasome activity to the basal levels. Overall these results confirm the pro-oxidant activity of G93A Cu,Zn SOD mutant and, at the same time, suggest a cross-talk between reactive oxygen and nitrogen species via the proteasome pathway.     

Differential role of superoxide and glutathione in S‐nitrosoglutathione-mediated apoptosis: a rationale for mild forms of familial amyotrophic lateral sclerosis associated with less active Cu,Zn superoxide dismutase mutants

SH-SY5Y cells transfected with the enzymatically inactive Cu,Zn superoxide dismutase mutant H46R were more resistant to S-nitrosoglutathione (GSNO)-induced apoptosis. Cytochrome c release from mitochondria, caspase 3 activation, p53 up-regulation, p21 cleavage and Bcl-2 modulation, all involved in the apoptotic process, were significantly less altered with respect to untransfected cells. The H46R resistance to NO was associated with a higher content of reduced glutathione (GSH) and was abolished by blockage of glutathione synthesis. On the other hand, H46R cells were as sensitive as SH-SY5Y cells to puromycin-induced apoptosis; furthermore, they were more susceptible to apoptosis elicited by the superoxide-generating drug paraquat and to cell necrosis provoked by t-butyl hydroperoxide. These results confirm that the level of superoxide dismutase activity is fundamental for protecting cells against oxygen free radical challenge. Its impairment is not detrimental to cells exposed to NO, as long as the overall reducing power represented by GSH is assured. These results are relevant to explain a milder progression of the familial amyotrophic lateral sclerosis disease when associated with the H46R mutation.     

Cu/Zn superoxide dismutase and catalase activities in Pinus mugo needles growing at elevated stands in the mountains, and their photochemical efficiency of PSII

Pinus mugo needles were sampled at different altitudes (1420, 1590 and 1920 m a.s.l.) to analyse levels of oxidative stress and changes in maximum photochemical efficiency of PSII. Polyacrylamide gel electrophoresis demonstrated that almost all superoxide dismutase activity represented Cu/Zn superoxide dismutase, and only 4-6% represents Mn superoxide dismutase. In extracts from plants sampled at 1590 and 1920 m a.s.l., lower activity of Cu/Zn superoxide dismutase was found. Comparing these data with immunoblots, it can be concluded that the differences in superoxide dismutase activity was related to protein amount. In needles from higher altitudes, a decrease in catalase activity was detected, as opposed to the protein amount, which was higher in needles from the higher stands. Considering the decrease in catalase and Cu/Zn superoxide dismutase activities in needles collected at 1590 and 1920 m a.s.l., we suggest that higher levels of oxidative stress may induce changes in photochemical efficiency of PSII.     

Vibrio cholerae periplasmic superoxide dismutase: isolation of the gene and overexpression of the protein

Superoxide dismutases are ubiquitous enzymes which play an important role in protecting cells against oxidative damage and which have also been shown to contribute to the pathogenicity of many bacterial species. Here we demonstrate that Vibrio cholerae, the causative agent of cholerae, expresses an active periplasmic Cu,Zn superoxide dismutase. Moreover, we have set up an expression system yielding large amounts of V. cholerae recombinant Cu,Zn superoxide dismutase in the periplasm of Escherichia coli and a procedure to obtain the enzyme in a highly purified form. Unlike the bovine enzyme, V. cholerae Cu,Zn superoxide dismutase has been proved to be highly resistant to inactivation by hydrogen peroxide. This property, which appears to be common to other bacterial enzymes of this class, might improve the ability of Cu,Zn superoxide dismutase to protect bacteria against the reactive oxygen species produced by phagocytes.     

Regulation of the synthesis of superoxide dismutases in rat lungs during oxidant and hyperthermic stresses

Heat shock proteins are induced at normal temperatures by oxidants and during reoxygenation following hypoxia. We now report cyanide-resistant O2 consumption increased 30-50% in rat lungs exposed to heat shock or reoxygenation following hypoxia. The synthesis of Cu,Zn superoxide dismutase, but not Mn superoxide dismutase, was increased in rat lung slices by in vivo hyperthermia (39 degrees C), by in vitro heat shock (41 degrees C), and during incubation of lung slices with the Cu chelator diethyldithiocarbamate, which decreased the activity of Cu,Zn superoxide dismutase. The heat shock-induced increase in Cu,Zn superoxide dismutase developed 2 h later than the induction of heat shock proteins and was not blocked by actinomycin D. The rates of synthesis of both superoxide dismutases were decreased 50% by hypoxia and failed to increase during reoxygenation. During hypoxia the activity of Cu,Zn superoxide dismutase decreased about 50%, but the activity of Mn superoxide dismutase remained unchanged. We conclude that hyperthermia increases the synthesis of Cu,Zn superoxide dismutase, the synthesis of Cu,Zn superoxide dismutase and Mn superoxide dismutase are not coordinately regulated by hyperthermia or by the oxidant stress produced by lowering the activity of Cu,Zn superoxide dismutase, and the synthesis of heat shock proteins and Cu,Zn superoxide dismutase are regulated at different levels of gene expression.     

Effect of overexpression of human Cu/Zn-SOD on activation-induced lymphocyte proliferation and apoptosis

The process of lymphocyte proliferation and apoptosis is known to be linked to oxidative stress. In the present study, we have used a new transgenic mouse model to investigate the effect of human Cu/Zn superoxide dismutase (Cu/Zn-SOD) overexpression on activation-induced lymphocytes proliferation and apoptosis. Cu/Zn-SOD activity was 3.5-fold higher in the spleen of the transgenic mice overexpressing Cu/Zn-SOD (Tg-Cu/Zn-SOD) compared to the wild-type littermates. Proliferative response of lymphocytes to lipopolysaccharide (LPS), Concanavalin A (Con A), and anti-CD3 was measured by [3H]-thymidine incorporation. Activation-induced apoptosis was determined by incubating the T cells with anti-CD3 (primary stimulus) for 72 h, followed by restimulation with Con A (secondary stimulus) for various times. Apoptosis was assessed by measuring DNA fragmentation using a spectrofluorimetric assay and monitoring the expression of the specific apoptotic markers (Fas/CD95 receptor and Fas/CD95 ligand (Fas-L) using flow cytometry. There was no significant difference in proliferative response of lymphocytes to LPS, Con A, or anti-CD3 in transgenic mice overexpressing human Cu/Zn superoxide dismutase (Tg-Cu/Zn-SOD) compared to wild-type littermates. In addition, no significant difference was observed in lymphocyte populations and subsets between Tg-Cu/Zn-SOD mice and wild-type littermates. However, splenic T cells from Tg-Cu/Zn-SOD mice exhibited a significantly (p <.05) higher level of activation-induced DNA fragmentation than T cells from wild-type littermates. The increase in DNA fragmentation was paralleled with an increase in the proportion of T cells expressing Fas and Fas-L molecules. The possible consequences of Cu/Zn-SOD overproduction on activation-induced apoptosis are discussed.     

Cellular response of antioxidant metalloproteins in Cu/Zn SOD transgenic mice exposed to hyperoxia

Ceruloplasmin, metallothionein, and ferritin are metal-binding proteins with potential antioxidant activity. Despite evidence that they are upregulated in pulmonary tissue after oxidative stress, little is known regarding their influence on trace metal homeostasis. In this study, we have used copper- and zinc-containing superoxide dismutase (Cu/Zn SOD) transgenic-overexpressing and gene knockout mice and hyperoxia to investigate the effects of chronic and acute oxidative stress on the expression of these metalloproteins and to identify their influence on copper, zinc, and iron homeostasis. We found that the oxidative stress-mediated induction of ceruloplasmin and metallothionein in the lung had no effect on tissue levels of copper, iron, or zinc. However, Cu/Zn SOD expression had a marked influence on hepatic copper and iron as well as circulating copper homeostasis. These results suggest that ceruloplasmin and metallothionein may function as antioxidants independent of their role in trace metal homeostasis and that Cu/Zn SOD functions in copper homeostasis via mechanisms distinct from its superoxide scavenging properties.     

Studies on the expression of Cu,Zn superoxide dismutase in human tissues during development

The developmental expression of Cu,Zn superoxide dismutase in human lung and erythrocytes has been studied using activity measurements, immunoblotting and immunohistochemistry. Enzyme activity in erythrocytes increased significantly during gestation but no developmental trend was seen in lung. Immunoblotting identified a single enzyme form that was present in a variety of tissues and immunohistochemistry showed the enzyme to have widespread distribution in lung tissue. These data indicate that Cu,Zn superoxide dismutase is consistently expressed during human development and that, unlike in other species, no late-fetal surge in expression occurs.     

Inactivation of cytochrome c oxidase by mutant SOD1s in mouse motoneuronal NSC-34 cells is independent from copper availability but is because of nitric oxide

The copper-enzyme cytochrome c oxidase (Cytox) has been indicated as a primary molecular target of mutant copper, zinc superoxide dismutase (SOD1) in familial amyotrophic lateral sclerosis (fALS); however, the mechanism underlying its inactivation is still unclear. As the toxicity of mutant SOD1s could arise from their selective recruitment to mitochondria, it is conceivable that they might compete with Cytox for the mitochondrial copper pool causing Cytox inactivation. To investigate this issue, we used mouse motoneuronal neuroblastoma × spinal cord cell line-34, stably transfected for the inducible expression of low amounts of wild-type or mutant (G93A, H46R, and H80R) human SOD1s and compared the effects observed on Cytox with those obtained by copper depletion. We demonstrated that all mutants analyzed induced cell death and decreased the Cytox activity, but not the protein content of the Cytox subunit II, at difference with copper depletion that also affected subunit II protein. Copper supplementation did not counteract mutant hSOD1s toxicity. Otherwise, the treatment of neuroblastoma × spinal cord cell line-34 expressing G93A, H46R, or H80R hSOD1 mutants, and showing constitutive expression of iNOS and nNOS, with either a NO scavenger, or NOS inhibitors prevented the inhibition of Cytox activity and rescued cell viability. These results support the involvement of NO in mutant SOD1s-induced Cytox damage, and mitochondrial toxicity.