Overexpression of human copper,zinc-superoxide dismutase (SOD1) prevents postischemic injury

Superoxide and superoxide-derived oxidants have been hypothesized to be important mediators of postischemic injury. Whereas copper,zinc-superoxide dismutase, SOD1, efficiently dismutates superoxide, there has been controversy regarding whether increasing intracellular SOD1 expression would protect against or potentiate cellular injury. To determine whether increased SOD1 protects the heart from ischemia and reperfusion, studies were performed in a newly developed transgenic mouse model in which direct measurement of superoxide, contractile function, bioenergetics, and cell death could be performed. Transgenic mice with overexpression of human SOD1 were studied along with matched nontransgenic controls. Immunoblotting and immunohistology demonstrated that total SOD1 expression was increased 10-fold in hearts from transgenic mice compared with nontransgenic controls, with increased expression in both myocytes and endothelial cells. In nontransgenic hearts following 30 min of global ischemia a reperfusion-associated burst of superoxide generation was demonstrated by electron paramagnetic resonance spin trapping. However, in the transgenic hearts with overexpression of SOD1 the burst of superoxide generation was almost totally quenched, and this was accompanied by a 2-fold increase in the recovery of contractile function, a 2.2-fold decrease in infarct size, and a greatly improved recovery of high energy phosphates compared with that in nontransgenic controls. These results demonstrate that superoxide is an important mediator of postischemic injury and that increasing intracellular SOD1 dramatically protects the heart from this injury. Thus, increasing intracellular SOD1 expression may be a highly effective approach to decrease the cellular injury that occurs following reperfusion of ischemic tissues.

Crystal structures of the copper and nickel complexes of RNase A: Metal-induced interprotein interactions and identification of a novel copper binding motif

We report the crystal structures of the copper and nickel complexes of RNase A. The overall topology of these two complexes is similar to that of other RNase A structures. However, there are significant differences in the mode of binding of copper and nickel. There are two copper ions per molecule of the protein, but there is only one nickel ion per molecule of the protein. Significant changes occur in the interprotein interactions as a result of differences in the coordinating groups at the common binding site around His-105. Consequently, the copper- and nickel-ion-bound dimers of RNase A act as nucleation sites for generating different crystal lattices for the two complexes. A second copper ion is present at an active site residue His-119 for which all the ligands are from one molecule of the protein. At this second site, His-119 adopts an inactive conformation (B) induced by the copper. We have identified a novel copper binding motif involving the α-amino group and the N-terminal residues.

Identification of the prooxidant site of human ceruloplasmin: A model for oxidative damage by copper bound to protein surfaces

Free transition metal ions oxidize lipids and lipoproteins in vitro; however, recent evidence suggests that free metal ion-independent mechanisms are more likely in vivo. We have shown previously that human ceruloplasmin (Cp), a serum protein containing seven Cu atoms, induces low density lipoprotein oxidation in vitro and that the activity depends on the presence of a single, chelatable Cu atom. We here use biochemical and molecular approaches to determine the site responsible for Cp prooxidant activity. Experiments with the His-specific reagent diethylpyrocarbonate (DEPC) showed that one or more His residues was specifically required. Quantitative [14C]DEPC binding studies indicated the importance of a single His residue because only one was exposed upon removal of the prooxidant Cu. Plasmin digestion of [14C]DEPC-treated Cp (and N-terminal sequence analysis of the fragments) showed that the critical His was in a 17-kDa region containing four His residues in the second major sequence homology domain of Cp. A full length human Cp cDNA was modified by site-directed mutagenesis to give His-to-Ala substitutions at each of the four positions and was transfected into COS-7 cells, and low density lipoprotein oxidation was measured. The prooxidant site was localized to a region containing His426 because CpH426A almost completely lacked prooxidant activity whereas the other mutants expressed normal activity. These observations support the hypothesis that Cu bound at specific sites on protein surfaces can cause oxidative damage to macromolecules in their environment. Cp may serve as a model protein for understanding mechanisms of oxidant damage by copper-containing (or -binding) proteins such as Cu, Zn superoxide dismutase, and amyloid precursor protein.

Nitration of γ-tocopherol and oxidation of α-tocopherol by copper-zinc superoxide dismutase/H2O2/NO2−: Role of nitrogen dioxide free radical

Copper-zinc superoxide dismutase (Cu,ZnSOD) is the antioxidant enzyme that catalyzes the dismutation of superoxide (O2•−) to O2 and H2O2. In addition, Cu,ZnSOD also exhibits peroxidase activity in the presence of H2O2, leading to self-inactivation and formation of a potent enzyme-bound oxidant. We report in this study that lipid peroxidation of l-α-lecithin liposomes was enhanced greatly during the SOD/H2O2 reaction in the presence of nitrite anion (NO2−) with or without the metal ion chelator, diethylenetriaminepentacetic acid. The presence of NO2− also greatly enhanced α-tocopherol (α-TH) oxidation by SOD/H2O2 in saturated 1,2-dilauroyl-sn-glycero-3-phosphatidylcholine liposomes. The major product identified by HPLC and UV-studies was α-tocopheryl quinone. When 1,2-diauroyl-sn-glycero-3-phosphatidylcholine liposomes containing γ-tocopherol (γ-TH) were incubated with SOD/H2O2/NO2−, the major product identified was 5-NO2-γ-TH. Nitrone spin traps significantly inhibited the formation of α-tocopheryl quinone and 5-NO2-γ-TH. NO2− inhibited H2O2-dependent inactivation of SOD. A proposed mechanism of this protection involves the oxidation of NO2− by an SOD-bound oxidant to the nitrogen dioxide radical (•NO2). In this study, we have shown a new mechanism of nitration catalyzed by the peroxidase activity of SOD. We conclude that NO2− is a suitable probe for investigating the peroxidase activity of familial Amyotrophic Lateral Sclerosis-linked SOD mutants.

hCTR1: A human gene for copper uptake identified by complementation in yeast

The molecular mechanisms responsible for the cellular uptake of copper in mammalian cells are unknown. We describe isolation of a human gene involved in this process by complementation of the yeast high-affinity copper uptake mutant, ctr1. Besides complementing ctr1 growth defect on nonfermentable media, the human gene also rescues iron transport and SOD1 defects in ctr1 yeast. Overexpression of the gene in yeast leads to vulnerability to the toxicity of copper overload. In addition, its expression in ctr1 yeast significantly increases the level of cellular copper, as demonstrated by atomic absorption. We propose this gene as a candidate for high-affinity copper uptake in humans and by analogy have named it hCTR1. The hCTR1 and yeast CTR1 predicted transmembrane proteins are 29% identical, but the human protein is substantially smaller in both the extracellular metal-binding and intracellular domains. An additional human gene similar to hCTR1, here named hCTR2, was identified in a database search. Both hCTR1 and hCTR2 are expressed in all human tissues examined, and both genes are located in 9q31/32. These studies, together with the previously recognized functional and sequence similarity between the Menkes/Wilson copper export proteins and CCC2 in yeast, demonstrate that similar copper homeostatic mechanisms are used in these evolutionarily divergent organisms.

Interaction of the copper chaperone HAH1 with the Wilson disease protein is essential for copper homeostasis

The delivery of copper to specific sites within the cell is mediated by distinct intracellular carrier proteins termed copper chaperones. Previous studies in Saccharomyces cerevisiae suggested that the human copper chaperone HAH1 may play a role in copper trafficking to the secretory pathway of the cell. In this current study, HAH1 was detected in lysates from multiple human cell lines and tissues as a single-chain protein distributed throughout the cytoplasm and nucleus. Studies with a glutathione S-transferase-HAH1 fusion protein demonstrated direct protein–protein interaction between HAH1 and the Wilson disease protein, which required the cysteine copper ligands in the amino terminus of HAH1. Consistent with these in vitro observations, coimmunoprecipitation experiments revealed that HAH1 interacts with both the Wilson and Menkes proteins in vivo and that this interaction depends on available copper. When these studies were repeated utilizing three disease-associated mutations in the amino terminus of the Wilson protein, a marked diminution in HAH1 interaction was observed, suggesting that impaired copper delivery by HAH1 constitutes the molecular basis of Wilson disease in patients harboring these mutations. Taken together, these data provide a mechanism for the function of HAH1 as a copper chaperone in mammalian cells and demonstrate that this protein is essential for copper homeostasis.

The heme-copper oxidases of Thermus thermophilus catalyze the reduction of nitric oxide: Evolutionary implications

We show that the heme-copper terminal oxidases of Thermus thermophilus (called ba3 and caa3) are able to catalyze the reduction of nitric oxide (NO) to nitrous oxide (N2O) under reducing anaerobic conditions. The rate of NO consumption and N2O production were found to be linearly dependent on enzyme concentration, and activity was abolished by enzyme denaturation. Thus, contrary to the eukaryotic enzyme, both T. thermophilus oxidases display a NO reductase activity (3.0 ± 0.7 mol NO/mol ba3 × min and 32 ± 8 mol NO/mol caa3 × min at [NO] ≈ 50 μM and 20°C) that, though considerably lower than that of bona fide NO reductases (300–4,500 mol NO/mol enzyme × min), is definitely significant. We also show that for ba3 oxidase, NO reduction is associated to oxidation of cytochrome b at a rate compatible with turnover, suggesting a mechanism consistent with the stoichiometry of the overall reaction. We propose that the NO reductase activity of T. thermophilus oxidases may depend on a peculiar CuB+ coordination, which may be revealed by the forthcoming three-dimensional structure. These findings support the hypothesis of a common phylogeny of aerobic respiration and bacterial denitrification, which was proposed on the basis of structural similarities between the Pseudomonas stutzeri NO reductase and the cbb3 terminal oxidases. Our findings represent functional evidence in support of this hypothesis.

Chloride is an allosteric effector of copper assembly for the yeast multicopper oxidase Fet3p: An unexpected role for intracellular chloride channels

GEF1 is a gene in Saccharomyces cerevisiae, which encodes a putative voltage-regulated chloride channel. gef1 mutants have a defect in the high-affinity iron transport system, which relies on the cell surface multicopper oxidase Fet3p. The defect is due to an inability to transfer Cu+ to apoFet3p within the secretory apparatus. We demonstrate that the insertion of Cu into apoFet3p is dependent on the presence of Cl−. Cu-loading of apoFet3p is favored at acidic pH, but in the absence of Cl− there is very little Cu-loading at any pH. Cl− has a positive allosteric effect on Cu-loading of apoFet3p. Kinetic studies suggest that Cl− may also bind to Fet3p and that Cu+ has an allosteric effect on the binding of Cl− to the enzyme. Thus, Cl− may be required for the metal loading of proteins within the secretory apparatus. These results may have implications in mammalian physiology, as mutations in human intracellular chloride channels result in disease.

Copper binding to the prion protein: Structural implications of four identical cooperative binding sites

Evidence is growing to support a functional role for the prion protein (PrP) in copper metabolism. Copper ions appear to bind to the protein in a highly conserved octapeptide repeat region (sequence PHGGGWGQ) near the N terminus. To delineate the site and mode of binding of Cu(II) to the PrP, the copper-binding properties of peptides of varying lengths corresponding to 2-, 3-, and 4-octarepeat sequences have been probed by using various spectroscopic techniques. A two-octarepeat peptide binds a single Cu(II) ion with Kd ≈ 6 μM whereas a four-octarepeat peptide cooperatively binds four Cu(II) ions. Circular dichroism spectra indicate a distinctive structuring of the octarepeat region on Cu(II) binding. Visible absorption, visible circular dichroism, and electron spin resonance spectra suggest that the coordination sphere of the copper is identical for 2, 3, or 4 octarepeats, consisting of a square-planar geometry with three nitrogen ligands and one oxygen ligand. Consistent with the pH dependence of Cu(II) binding, proton NMR spectroscopy indicates that the histidine residues in each octarepeat are coordinated to the Cu(II) ion. Our working model for the structure of the complex shows the histidine residues in successive octarepeats bridged between two copper ions, with both the Nɛ2 and Nδ1 imidazole nitrogen of each histidine residue coordinated and the remaining coordination sites occupied by a backbone amide nitrogen and a water molecule. This arrangement accounts for the cooperative nature of complex formation and for the apparent evolutionary requirement for four octarepeats in the PrP.

High-resolution microPET imaging of carcinoembryonic antigen-positive xenografts by using a copper-64-labeled engineered antibody fragment

Rapid imaging by antitumor antibodies has been limited by the prolonged targeting kinetics and clearance of labeled whole antibodies. Genetically engineered fragments with rapid access and high retention in tumor tissue combined with rapid blood clearance are suitable for labeling with short-lived radionuclides, including positron-emitting isotopes for positron-emission tomography (PET). An engineered fragment was developed from the high-affinity anticarcinoembryonic antigen (CEA) monoclonal antibody T84.66. This single-chain variable fragment (Fv)-CH3, or minibody, was produced as a bivalent 80 kDa dimer. The macrocyclic chelating agent 1,4,7,10-tetraazacyclododecane-N,N′,N′′, N′′′-tetraacetic acid (DOTA) was conjugated to the anti-CEA minibody for labeling with copper-64, a positron-emitting radionuclide (t1/2 = 12.7 h). In vivo distribution was evaluated in athymic mice bearing paired LS174T human colon carcinoma (CEA positive) and C6 rat glioma (CEA negative) xenografts. Five hours after injection with 64Cu-DOTA-minibody, microPET imaging showed high uptake in CEA-positive tumor (17.9% injected dose per gram ± 3.79) compared with control tumor (6.0% injected dose per gram ± 1.0). In addition, significant uptake was seen in liver, with low uptake in other tissues. Average target/background ratios relative to neighboring tissue were 3–4:1. Engineered antibody fragments labeled with positron-emitting isotopes such as copper-64 provide a new class of agents for PET imaging of tumors.

Chaperone-facilitated copper binding is a property common to several classes of familial amyotrophic lateral sclerosis-linked superoxide dismutase mutants

Mutations in Cu, Zn superoxide dismutase (SOD1) cause the neurodegenerative disease familial amyotrophic lateral sclerosis from an as-yet-unidentified toxic property(ies). Analysis in Saccharomyces cerevisiae of a broad range of human familial amyotrophic lateral sclerosis–linked SOD1 mutants (A4V, G37R, G41D, H46R, H48Q, G85R, G93C, and I113T) reveals one property common to these mutants (including two at residues that coordinate the catalytic copper): Each does indeed bind copper and scavenge oxygen-free radicals in vivo. Neither decreased copper binding nor decreased superoxide scavenging activity is a property shared by all mutants. The demonstration that shows that all mutants tested do bind copper under physiologic conditions supports a mechanism of SOD1 mutant-mediated disease arising from aberrant copper-mediated chemistry catalyzed by less tightly folded (and hence less constrained) mutant enzymes. The mutant enzymes also are shown to acquire the catalytic copper in vivo through the action of CCS, a specific copper chaperone for SOD1, which in turn suggests that a search for inhibitors of this SOD1 copper chaperone may represent a therapeutic avenue.

Functional expression of the Wilson disease protein reveals mislocalization and impaired copper-dependent trafficking of the common H1069Q mutation

Wilson disease is an autosomal recessive disorder of hepatic copper metabolism caused by mutations in a gene encoding a copper-transporting P-type ATPase. To elucidate the function of the Wilson protein, wild-type and mutant Wilson cDNAs were expressed in a Menkes copper transporter-deficient mottled fibroblast cell line defective in copper export. Expression of the wild-type cDNA demonstrated trans-Golgi network localization and copper-dependent trafficking of the Wilson protein identical to previous observations for the endogenously expressed protein in hepatocytes. Furthermore, expression of the Wilson cDNA rescued the mottled phenotype as evidenced by a reduction in copper accumulation and restoration of cell viability. In contrast, expression of an H1069Q mutant Wilson cDNA did not rescue the mottled phenotype, and immunofluorescence studies showed that this mutant Wilson protein was localized in the endoplasmic reticulum. Consistent with these findings, pulse–chase analysis demonstrated a 5-fold decrease in the half-life of the H1069Q mutant as compared with the wild-type protein. Maintenance of these transfected cell lines at 28°C resulted in localization of the H1069Q protein in the trans-Golgi network, suggesting that a temperature-sensitive defect in protein folding followed by degradation constitutes the molecular basis of Wilson disease in patients harboring the H1069Q mutation. Taken together, these studies describe a tractable expression system for elucidating the function and localization of the copper-transporting ATPases in mammalian cells and provide compelling evidence that the Wilson protein can functionally substitute for the Menkes protein, supporting the concept that these proteins use common biochemical mechanisms to effect cellular copper homeostasis.

Caspase-1 is activated in neural cells and tissue with amyotrophic lateral sclerosis-associated mutations in copper-zinc superoxide dismutase

The mechanism by which mutations in the superoxide dismutase (SOD1) gene cause motor neuron degeneration in familial amyotrophic lateral sclerosis (ALS) is unknown. Recent reports that neuronal death in SOD1-familial ALS is apoptotic have not documented activation of cell death genes. We present evidence that the enzyme caspase-1 is activated in neurons expressing mutant SOD1 protein. Proteolytic processing characteristic of caspase-1 activation is seen both in spinal cords of transgenic ALS mice and neurally differentiated neuroblastoma (line N2a) cells with SOD1 mutations. This activation of caspase-1 is enhanced by oxidative challenge (xanthine/xanthine oxidase), which triggers cleavage and secretion of the interleukin 1β converting enzyme substrate, pro-interleukin 1β, and induces apoptosis. This N2a culture system should be an instructive in vitro model for further investigation of the proapoptotic properties of mutant SOD1.

Expression of the Anabaena hetR gene from a copper-regulated promoter leads to heterocyst differentiation under repressing conditions

Heterocyst differentiation in the filamentous cyanobacterium Anabaena PCC 7120 requires a functional hetR gene. Increased expression of the hetR gene is seen in developing and mature heterocysts in response to fixed nitrogen limitation. We mapped four likely transcriptional start sites for hetR and identified a specific transcript that is positively autoregulated. By using the copper-responsive petE promoter from Anabaena PCC 7120 to drive hetR expression, we show that ectopic expression of hetR increases heterocyst frequency and induces heterocyst differentiation under fully repressing conditions. Coexpression of a reporter gene shows that expression from the petE promoter is smoothly induced depending on the amount of copper supplied. In the heterocyst pattern mutant PatA, where terminally positioned heterocysts are formed almost exclusively, expression of the petE∷hetR fusion does not result in the formation of intercalary heterocysts. These results suggest that although the intracellular concentration of HetR has to be elevated for the differentiation decision, PatA plays a role as well. This role may be in the form of posttranslational modification of HetR, because PatA is a member of the response regulator family of proteins.