Two Structurally Similar Maize Cytosolic Superoxide Dismutase Genes, Sod4 and Sod4A, Respond Differentially to Abscisic Acid and High Osmoticum1

The maize (Zea mays) superoxide dismutase genes Sod4 and Sod4A are highly similar in structure but each responds differentially to environmental signals. We examined the effects of the hormone abscisic acid (ABA) on the developmental response of Sod4 and Sod4A. Although both Sod4 and Sod4A transcripts accumulate during late embryogenesis, only Sod4 is up-regulated by ABA and osmotic stress. Accumulation of Sod4 transcript in response to osmotic stress is a consequence of increased endogenous ABA levels in developing embryos. Sod4 mRNA is up-regulated by ABA in viviparous-1 mutant embryos. Sod4 transcript increases within 4 h with ABA not only in developing embryos but also in mature embryos and in young leaves. Sod4A transcript is up-regulated by ABA only in young leaves, but neither Sod4 nor Sod4A transcripts changed in response to osmotic stress. Our data suggest that in leaves Sod4 and Sod4A may respond to ABA and osmotic stress via alternate pathways. Since the Sod genes have a known function, we hypothesize that the increase in Sod mRNA in response to ABA is due in part to ABA-mediated metabolic changes leading to changes in oxygen free radical levels, which in turn lead to the induction of the antioxidant defense system.

A Cyanobacterium Lacking Iron Superoxide Dismutase Is Sensitized to Oxidative Stress Induced with Methyl Viologen but Is Not Sensitized to Oxidative Stress Induced with Norflurazon1

A strain of Synechococcus sp. strain PCC 7942 with no functional Fe superoxide dismutase (SOD), designated sodB−, was characterized by its growth rate, photosynthetic pigments, and cyclic photosynthetic electron transport activity when treated with methyl viologen or norflurazon (NF). In their unstressed conditions, both the sodB− and wild-type strains had similar chlorophyll and carotenoid contents and catalase activity, but the wild type had a faster growth rate and higher cyclic electron transport activity. The sodB− was very sensitive to methyl viologen, indicating a specific role for the FeSOD in protection against superoxide generated in the cytosol. In contrast, the sodB− mutant was less sensitive than the wild type to oxidative stress imposed with NF. This suggests that the FeSOD does not protect the cell from excited singlet-state oxygen generated within the thylakoid membrane. Another up-regulated antioxidant, possibly the MnSOD, may confer protection against NF in the sodB− strain. These results support the hypothesis that different SODs have specific protective functions within the cell.

Mutations in copper-zinc superoxide dismutase that cause amyotrophic lateral sclerosis alter the zinc binding site and the redox behavior of the protein.

A series of mutant human and yeast copper-zinc superoxide dismutases has been prepared, with mutations corresponding to those found in familial amyotrophic lateral sclerosis (ALS; also known as Lou Gehrig's disease). These proteins have been characterized with respect to their metal-binding characteristics and their redox reactivities. Replacement of Zn2+ ion in the zinc sites of several of these proteins with either Cu2+ or Co2+ gave metal-substituted derivatives with spectroscopic properties different from those of the analogous derivative of the wild-type proteins, indicating that the geometries of binding of these metal ions to the zinc site were affected by the mutations. Several of the ALS-associated mutant copper-zinc superoxide dismutases were also found to be reduced by ascorbate at significantly greater rate than the wild-type proteins. We conclude that similar alterations in the properties of the zinc binding site can be caused by mutations scattered throughout the protein structure. This finding may help to explain what is perhaps the most perplexing question in copper-zinc superoxide dismutase-associated familial ALS-i.e., how such a diverse set of mutations can result in the same gain of function that causes the disease.

Nitration and inactivation of manganese superoxide dismutase in chronic rejection of human renal allografts.

Inflammatory processes in chronic rejection remain a serious clinical problem in organ transplantation. Activated cellular infiltrate produces high levels of both superoxide and nitric oxide. These reactive oxygen species interact to form peroxynitrite, a potent oxidant that can modify proteins to form 3-nitrotyrosine. We identified enhanced immunostaining for nitrotyrosine localized to tubular epithelium of chronically rejected human renal allografts. Western blot analysis of rejected tissue demonstrated that tyrosine nitration was restricted to a few specific polypeptides. Immunoprecipitation and amino acid sequencing techniques identified manganese superoxide dismutase, the major antioxidant enzyme in mitochondria, as one of the targets of tyrosine nitration. Total manganese superoxide dismutase protein was increased in rejected kidney, particularly in the tubular epithelium; however, enzymatic activity was significantly decreased. Exposure of recombinant human manganese superoxide dismutase to peroxynitrite resulted in a dose-dependent (IC50 = 10 microM) decrease in enzymatic activity and concomitant increase in tyrosine nitration. Collectively, these observations suggest a role for peroxynitrite during development and progression of chronic rejection in human renal allografts. In addition, inactivation of manganese superoxide dismutase by peroxynitrite may represent a general mechanism that progressively increases the production of peroxynitrite, leading to irreversible oxidative injury to mitochondria.

Identification of vascular endothelial genes differentially responsive to fluid mechanical stimuli: cyclooxygenase-2, manganese superoxide dismutase, and endothelial cell nitric oxide synthase are selectively up-regulated by steady laminar shear stress.

Early atherosclerotic lesions develop in a topographical pattern that strongly suggests involvement of hemodynamic forces in their pathogenesis. We hypothesized that certain endothelial genes, which exhibit differential responsiveness to distinct fluid mechanical stimuli, may participate in the atherogenic process by modulating, on a local level within the arterial wall, the effects of systemic risk factors. A differential display strategy using cultured human endothelial cells has identified two genes, manganese superoxide dismutase and cyclooxygenase-2, that exhibit selective and sustained up-regulation by steady laminar shear stress (LSS). Turbulent shear stress, a nonlaminar fluid mechanical stimulus, does not induce these genes. The endothelial form of nitric oxide synthase also demonstrates a similar LSS-selective pattern of induction. Thus, three genes with potential atheroprotective (antioxidant, antithrombotic, and antiadhesive) activities manifest a differential response to distinct fluid mechanical stimuli, providing a possible mechanistic link between endothelial gene expression and early events in atherogenesis. The activities of these and other LSS-responsive genes may have important implications for the pathogenesis and prevention of atherosclerosis.

Neurodegeneration, myocardial injury, and perinatal death in mitochondrial superoxide dismutase-deficient mice.

Manganese superoxide dismutase (SOD2) converts superoxide to oxygen plus hydrogen peroxide and serves as the primary defense against mitochondrial superoxide. Impaired SOD2 activity in humans has been associated with several chronic diseases, including ovarian cancer and type I diabetes, and SOD2 overexpression appears to suppress malignancy in cultured cells. We have produced a line of SOD2 knockout mice (SOD2m1BCM/SOD2m1BCM) that survive up to 3 weeks of age and exhibit several novel pathologic phenotypes including severe anemia, degeneration of neurons in the basal ganglia and brainstem, and progressive motor disturbances characterized by weakness, rapid fatigue, and circling behavior. In addition, SOD2m1BCM/SOD2m1BCM mice older than 7 days exhibit extensive mitochondrial injury within degenerating neurons and cardiac myocytes. Approximately 10% of SOD2m1BCM/SOD2m1BCM mice exhibit markedly enlarged and dilated hearts. These observations indicate that SOD2 deficiency causes increased susceptibility to oxidative mitochondrial injury in central nervous system neurons, cardiac myocytes, and other metabolically active tissues after postnatal exposure to ambient oxygen concentrations. Our SOD2-deficient mice differ from a recently described model in which homozygotes die within the first 5 days of life with severe cardiomyopathy and do not exhibit motor disturbances, central nervous system injury, or ultrastructural evidence of mitochondrial injury.

Constitutive overexpression of Cu/Zn superoxide dismutase exacerbates kainic acid-induced apoptosis of transgenic-Cu/Zn superoxide dismutase neurons.

Cu/Zn superoxide dismutase (Cu/Zn SOD) is a key enzyme in the metabolism of oxygen free radicals. The gene resides on chromosome 21 and is overexpressed in patients with Down syndrome. Cultured neurons of transgenic Cu/Zn SOD (Tg-Cu/Zn SOD) mice with elevated activity of Cu/Zn SOD were used to determine whether constitutive overexpression of Cu/Zn SOD creates an indigenous oxidative stress that predisposes the Tg-Cu/Zn SOD neurons to added insults. Neurons from three independently derived Tg-Cu/Zn SOD strains showed higher susceptibility than nontransgenic neurons to kainic acid (KA)-mediated excitotoxicity, reflected by an earlier onset and enhanced apoptotic cell death. This higher susceptibility of transgenic neurons to KA-mediated apoptosis was associated with a chronic prooxidant state that was manifested by reduced levels of cellular glutathione and altered [Ca2+]i homeostasis. The data are compatible with the thesis that overexpression of Cu/Zn SOD creates chronic oxidative stress in the transgenic neurons, which exacerbates their susceptibility to additional insults such as KA-mediated excitotoxicity.

A gain-of-function of an amyotrophic lateral sclerosis-associated Cu,Zn-superoxide dismutase mutant: An enhancement of free radical formation due to a decrease in Km for hydrogen peroxide.

Cu,Zn-superoxide dismutase (SOD) is known to be a locus of mutation in familial amyotrophic lateral sclerosis (FALS). Transgenic mice that express a mutant Cu,Zn-SOD, Gly-93--> Ala (G93A), have been shown to develop amyotrophic lateral sclerosis (ALS) symptoms. We cloned the FALS mutant, G93A, and wild-type cDNA of human Cu,Zn-SOD, overexpressed them in Sf9 insect cells, purified the proteins, and studied their enzymic activities for catalyzing the dismutation of superoxide anions and the generation of free radicals with H2O2 as substrate. Our results showed that both enzymes contain one copper ion per subunit and have identical dismutation activity. However, the free radical-generating function of the G93A mutant, as measured by the spin trapping method, is enhanced relative to that of the wild-type enzyme, particularly at lower H2O2 concentrations. This is due to a small, but reproducible, decrease in the value of Km for H2O2 for the G93A mutant, while the kcat is identical for both enzymes. Thus, the ALS symptoms observed in G93A transgenic mice are not caused by the reduction of Cu,Zn-SOD activity with the mutant enzyme; rather, it is induced by a gain-of-function, an enhancement of the free radical-generating function. This is consistent with the x-ray crystallographic studies showing the active channel of the FALS mutant is slightly larger than that of the wild-type enzyme; thus, it is more accessible to H2O2. This gain-of-function, in part, may provide an explanation for the association between ALS and Cu,Zn-SOD mutants.

The Golgi apparatus of spinal cord motor neurons in transgenic mice expressing mutant Cu,Zn superoxide dismutase becomes fragmented in early, preclinical stages of the disease.

Dominant mutations of the SOD1 gene encoding Cu,Zn superoxide dismutase have been found in members of certain families with familial amyotrophic lateral sclerosis (ALS). To better understand the contribution of SOD1 mutations in the pathogenesis of familial ALS, we developed transgenic mice expressing one of the mutations found in familial ALS. These animals display clinical and pathological features closely resembling human ALS. Early changes observed in these animals were intra-axonal and dendritic vacuoles due to dilatation of the endoplasmic reticulum and vacuolar degeneration of mitochondria. We have reported that the Golgi apparatus of spinal cord motor neurons in patients with sporadic ALS is fragmented and atrophic. In this study we show that spinal cord motor neurons of transgenic mice for an SOD1 mutation display a lesion of the Golgi apparatus identical to that found in humans with sporadic ALS. In these mice, the stacks of the cisternae of the fragmented Golgi apparatus are shorter than in the normal organelle, and there is a reduction in Golgi-associated vesicles and adjacent cisternae of the rough endoplasmic reticulum. Furthermore, the fragmentation of the Golgi apparatus occurs in an early, presymptomatic stage and usually precedes the development of the vacuolar changes. Transgenic mice overexpressing the wild-type human superoxide dismutase are normal. In familial ALS, an early lesion of the Golgi apparatus of motor neurons may have adverse functional effects, because newly synthesized proteins destined for fast axoplasmic transport pass through the Golgi apparatus.

The contrasting roles of ICE family proteases and interleukin-1beta in apoptosis induced by trophic factor withdrawal and by copper/zinc superoxide dismutase down-regulation.

We compare here the mechanisms of apoptotic death of PC12 cells induced by down-regulation of Cu2+,Zn2+ superoxide dismutase (SOD1) and withdrawal of trophic support (serum/nerve growth factor). Our previous results indicated that the initiating causes of death are different in each paradigm. However, bcl-2 rescues cells in either paradigm, suggesting common downstream elements to the cell death pathway. To determine whether the ICE [interleukin 1beta converting enzyme] family of proteases, which is required for apoptosis on trophic factor withdrawal, is also required for apoptosis induced by oxidative stress, we have developed a novel peptide inhibitor that mimics the common catalytic site of these enzymes and thereby blocks their access to substrates. This differs from the more usual pseudosubstrate approach to enzyme inhibition. Blockade of ICE family proteases by either this inhibitor or by a permeant competitive ICE family antagonist rescues PC12 cells from apoptotic death following apoptosis induced by down-regulation of SOD1, as well as from trophic factor/nerve growth factor deprivation. SOD1 down-regulation results in an increase in interleukin 1beta (IL- 1beta) production by the cells, and cell death under these conditions can be prevented by either blocking antibodies against IL-1beta or the IL-1 receptor antagonist (IL-1Ralpha). In contrast, trophic factor withdrawal does not increase IL-1beta secretion, and the blocking antibody failed to protect PC12 cells from trophic factor withdrawal, whereas the receptor antagonist was only partially protective at very high concentrations. There were substantial differences in the concentrations of pseudosubstrate inhibitors which rescued cells from SOD1 down-regulation and trophic factor deprivation. These results suggest the involvement of different members of the ICE family, different substrates, or both in the two different initiating causes of cell death.

Subunit-destabilizing mutations in Drosophila copper/zinc superoxide dismutase: neuropathology and a model of dimer dysequilibrium.

Mutations in Cu/Zn superoxide dismutase (SOD), a hallmark of familial amyotrophic lateral sclerosis (FALS) in humans, are shown here to confer striking neuropathology in Drosophila. Heterozygotes with one wild-type and one deleted SOD allele retain the expected 50% of normal activity for this dimeric enzyme. However, heterozygotes with one wild-type and one missense SOD allele show lesser SOD activities, ranging from 37% for a heterozygote carrying a missense mutation predicted from structural models to destabilize the dimer interface, to an average of 13% for several heterozygotes carrying missense mutations predicted to destabilize the subunit fold. Genetic and biochemical evidence suggests a model of dimer dysequilibrium whereby SOD activity in missense heterozygotes is reduced through entrapment of wild-type subunits into unstable or enzymatically inactive heterodimers. This dramatic impairment of the activity of wild-type subunits in vivo has implications for our understanding of FALS and for possible therapeutic strategies.

Escherichia coli iron superoxide dismutase targeted to the mitochondria of yeast cells protects the cells against oxidative stress.

A gene encoding a fusion protein consisting of Escherichia coli iron superoxide dismutase (FeSOD) with the mitochondrial targeting presequence of yeast manganese superoxide dismutase (MnSOD) was cloned and expressed in E. coli and in Saccharomyces cerevisiae DL1Mn- yeast cells deficient in MnSOD. In the yeast cells the fusion protein was imported into the mitochondrial matrix. However, the presequence was not cleaved. In a control set of experiments, the E. coli FeSOD gene without the yeast MnSOD leader sequence was also cloned and expressed in S. cerevisiae DL1Mn- cells. In this case the FeSOD was located in the cytosol and was not imported into the mitochondrial matrix. E. coli FeSOD, with and without the yeast MnSOD presequence, proved to be active in yeast, but, whereas the FeSOD targeted to the mitochondria of yeast cells deficient in MnSOD protected the cells from the toxic effects of oxidative stress, FeSOD without the yeast MnSOD presequence did not protect the yeast cells deficient in MnSOD against oxidative stress.

On signal sequence polymorphisms and diseases of distribution.

We report a previously unappreciated property of the signals that target organelle-specific proteins to their subcellular sites of action. Such targeting sequences are shown to be polymorphic. We discovered this polymorphism when we cloned the mitochondrial manganese-containing superoxide dismutase from cell lines of normal individuals and patients with genetic diseases of premature aging and compared their sequences to each other and to those previously reported. The polymorphism consists of a single nucleotide change in the region of the DNA that encodes the signal sequence such that either an alanine or valine is present. Subsequently, eight cell lines were analyzed and all three possible combinations of the two signal sequences were observed. Such signal sequence polymorphisms could result in diseases of distribution, where essential proteins are not properly targeted, thereby leading to absolute or relative deficiencies of critical enzymes within specific cellular compartments. Progeria and related syndromes may be diseases of distribution.

Carboxyfullerenes as neuroprotective agents

Two regioisomers with C3 or D3 symmetry of water-soluble carboxylic acid C60 derivatives, containing three malonic acid groups per molecule, were synthesized and found to be equipotent free radical scavengers in solution as assessed by EPR analysis. Both compounds also inhibited the excitotoxic death of cultured cortical neurons induced by exposure to N-methyl-d-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), or oxygen-glucose deprivation, but the C3 regioisomer was more effective than the D3 regioisomer, possibly reflecting its polar nature and attendant greater ability to enter lipid membranes. At 100 μM, the C3 derivative fully blocked even rapidly triggered, NMDA receptor-mediated toxicity, a form of toxicity with limited sensitivity to all other classes of free radical scavengers we have tested. The C3 derivative also reduced apoptotic neuronal death induced by either serum deprivation or exposure to Aβ1–42 protein. Furthermore, continuous infusion of the C3 derivative in a transgenic mouse carrying the human mutant (G93A) superoxide dismutase gene responsible for a form of familial amyotrophic lateral sclerosis, delayed both death and functional deterioration. These data suggest that polar carboxylic acid C60 derivatives may have attractive therapeutic properties in several acute or chronic neurodegenerative diseases.

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.