Heat-stable chloroplastic Cu/Zn superoxide dismutase in Chenopodium murale

Chenopodium murale is a weed species having wide adaptation to different climatic regimes and experiences a temperature range of 5-45 degrees C during its life span. Higher temperatures may result in heat stress, which induces higher ROS production leading to oxidative stress in the plant. Superoxide dismutase enzyme (SOD, EC.1.15.1.1) is ubiquitous, being widely distributed among O(2)(-) consuming organisms and is the first line of defense against oxidative stress. In this study, we have characterized the thermostability of the SOD isozymes from C. murale in vitro. The leaf protein extracts, thylakoidal and stromal fractions were subjected to elevated temperatures ranging from 50 degrees C to boiling and analyzed for activity and isoform pattern of SOD. Out of six SOD isoforms, SOD V showed stability even after boiling the extract for 10min. Under high temperature treatment (>60 degrees C) there was an appearance of a new SOD band with higher electrophoretic mobility. The inhibitor studies and subcellular analysis revealed that the SOD V isoform was a chloroplastic Cu/Zn SOD. The stromal Cu/Zn SOD (SOD V) was more stable than the co-migrating thylakoidal isozyme at 80 degrees C and boiling for 10min. Hence, we report an unusual, constitutive thermostable chloroplastic Cu/Zn SOD from C. murale, which may contribute towards its heat tolerance.     

Increased heart rate variability in mice overexpressing the Cu/Zn superoxide dismutase

Cu/Zn superoxide dismutase (SOD1) is implicated in various pathological conditions including Down's syndrome, neurodegenerative diseases, and afflictions of the autonomic nervous system (ANS). To assess the SOD1 contribution to ANS dysfunction, especially its influence on cardiac regulation, we studied the heart rate variability (HRV) and cardiac arrhythmias in conscious 12-month-old male and female transgenic mice for the human SOD1 gene (TghSOD1). TghSOD1 mice presented heart rate reduction as compared with control FVB/N individuals. All HRV parameters reflecting parasympathetic activity were increased in TghSOD1. Pharmacological studies confirmed that the parasympathetic tone was exacerbated and the sympathetic pathway was functional in TghSOD1 mice. A high frequency of atrioventricular block and premature ventricular contractions was observed in TghSOD1. By biochemical assays we found that SOD1 activities were multiplied by 9 and 4 respectively in the heart and brainstem of transgenic mice. A twofold decrease in cholinesterase activity was observed in the heart but not in the brainstem. We demonstrate that SOD1 overexpression induces an ANS dysfunction by an exacerbated vagal tone that may be related to impaired cardiac activity of the cholinesterases and may explain the high occurrence of arrhythmias.     

Drosophila Cu,Zn superoxide dismutase gene confers resistance to paraquat in Escherichia coli

Superoxide dismutase (SOD) is known to protect organisms from reactive oxygen metabolites. We tested the hypothesis that the Drosophila Cu,Zn SOD is capable of protecting Escherichia coli from oxidative damage caused by the herbicide paraquat. The Cu,Zn Sod gene of Drosophila sechellia was subcloned into pET-20b(+) expression vector. Transformation of E. coli with the constructed vector resulted in an overexpression of this eukaryotic superoxide dismutase, as evidenced by dramatically increased levels of the Cu,Zn SOD polypeptide in bacterial cytosolic extracts. As well, the E. coli transformants showed resistance to paraquat-mediated inhibition of growth and survival. Paraquat is known to promote formation of the superoxide radical anion inside cells and thus the data have been interpreted as indicating that the cloned superoxide dismutase provides protection in E. coli against damage attributable to free radicals.     

Cloning and DNA sequencing of cytosolic Cu/Zn superoxide dismutase gene from chinese cabbage

A cDNA clone for the cytosolic Cu/Zn superoxide dismutase (Cu/Zn SOD) from Chinese cabbage (Brassica campestris ssp.pekinensis) was isolated and its DNA sequence was determined. The cDNA clone contains a complete coding sequence which encodes a protein of 152 amino acids and a 3-untranslated region including a poly A signal. The deduced amino acid sequence shows that it is highly homologous to the Cu/Zn SODs from other plants (60–90%). The lack of a putative chloroplast targeting transit peptide indicates that the clone represents a cytosolic form of Cu/Zn SOD. Genomic Southern hybridization suggests that cytosolic Cu/Zn SOD genes are present in 1 or 2 copies per genome.     

Divalent-metal-dependent nucleolytic activity of Cu, Zn superoxide dismutase

The known action of Cu, Zn superoxide dismutase (holo SOD) that converts O₂ ⁻ to O₂ and H₂O₂ plays a crucial role in protecting cells from toxicity of oxidative stress. However, the overproduction of holo SOD does not result in increased protection but rather creates a variety of unfavorable effects, suggesting that too much holo SOD may be injurious to the cells. In the in vitro study, we report a finding that the holo SOD from bovine erythrocytes and its apo form possess a divalent-metal-dependent nucleolytic activity, which was confirmed by UV–vis absorption titration of calf thymus DNA (ctDNA) with the holo SOD, quenching of holo SOD intrinsic fluorescence by ctDNA, and by gel electrophoresis monitoring conversion of DNA from the supercoiled DNA to nicked and linear forms, and fragmentation of a linear λDNA. Moreover, the DNA cleavage activity was examined in detail under certain reaction conditions. The steady-state study indicates that DNA cleavage supported by both forms of SOD obeys Michaelis–Menten kinetics. On the other hand, the assays with some other proteins indicate that this new function is specific to some proteins including the holo SOD. Therefore, this study reveals that the divalent-metal-dependent DNA cleavage activity is an intrinsic property of the holo SOD, which is independent of its natural metal (copper and zinc) sites, and may provide an alternative insight into the link between SOD enzymes and neurodegenerative disorders.     

Solvent effects on the distribution of conformational substates in native and azide reacted Cu, Zn superoxide dismutase

Native and azide reacted Cu, Zn superoxide dismutase in aqueous and mixed water-glycerol solution have been investigated by EPR spectroscopy at low temperature. An accurate computer simulation, based on a well established theoretical model which has been reformulated for rhombic symmetry, has shown that the EPR spectrum of the copper ion in the native protein shows a significant g and A strain in the parallel region. The strain arises from a distribution of the ligand field strengths onto the metal ion and this could be traced back to the existence of a multiplicity of conformational states in the protein molecule. The strain is reduced in the presence of azide which is known to bind directly to the copper atom and to give rise to a more relaxed configuration corresponding to a square pyramidal geometry in which the apical ligand occupies an elongated position. In both samples, addition of glycerol further reduces the strain, indicating that the solvent is directly coupled to the protein matrix, thereby modulating the structural heterogeneity displayed by the protein molecule. Received: 6 June 1996 / Accepted: 9 April 1997     

Selective loss of neurofilament expression in Cu/Zn superoxide dismutase (SOD1) linked amyotrophic lateral sclerosis

Neurofilament pathology is a hallmark of sporadic and familial amyotrophic lateral sclerosis (SALS and FALS). The disease mechanisms underlying this pathology are presently unclear, but recent evidence in SALS patients suggest that reductions in neurofilament light subunit (NFL) mRNA may contribute to the death of motor neurones. Mutations in the gene encoding Cu-Zn superoxide dismutase (SOD1) represent the best-studied cause of FALS, and a number of laboratory models of SOD1-mediated disease exist. Here we have used microdissected lumbar spinal cord motor neurones from human SOD1 FALS patients as well as G93A SOD1 transgenic mice and demonstrated that reduced NFL mRNA levels are seen in both. To probe the molecular mechanisms underpinning these observations, we generated NSC34 motor neurone-like cell lines expressing wild-type and mutant SOD1. NSC34 cells expressing G37R or G93A SOD1 showed selective reductions in NFL and NFM mRNA and protein. These data suggest that NFL mRNA reductions are common to SALS and FALS patients, and that cells and mice expressing mutant SOD1 may enable us to characterize the molecular mechanism(s) responsible for the loss of neurofilament mRNA.     

Heavy metal-mediated activation of the rat Cu/Zn superoxide dismutase gene via a metal-responsive element

The Cu/Zn superoxide dismutase (SOD1) catalyzes the dismutation of superoxide radicals produced in the course of biological oxidations. When placed under the control of the rat SOD1 gene promoter and transfected into human HepG2 hepatoma cells, the activity of a chloramphenicol acetyltransferase reporter gene was found to increase three- to four-fold in the presence of heavy metals (cadmium, zinc and copper). Functional analysis of mutant derivatives of the SOD1 gene promoter and the use of a heterologous promoter system confirmed that the induction of the SOD1 gene by metal ions requires a metal-responsive element (MRE) located between positions −273 and −267 (GCGCGCA). It was also shown by gel mobility shift assays that an MRE binding protein is induced by the exposure of the human liver cell line HepG2 to heavy metals. These results suggest that the MRE participates in the induction of the SOD1 gene by heavy metals. Received: 5 February 1999 / Accepted: 21 May 1999     

1H and 31P NMR studies of the binding of low-affinity anions to Cu,Zn superoxide dismutase

Anions that do not coordinate to the catalytically active copper ion of Cu,Zn superoxide dismutase, but still affect the activity of the enzyme by weaker interactions with the protein moiety surrounding the active site (low affinity anions), uniformly perturbed the 1H NMR line of the NH group of the copper ligand His 46. This effect was detected on the enzyme having Co(II) substituted for the native Zn(II), in which the resonances of residues bound to the copper are detected because of the antiferromagnetic coupling between Cu(II) and Co(II). The interaction with the enzyme of phosphate, a good representative of low-affinity anions, was also studied by 31P NMR of the native enzyme and of enzyme samples covalently modified at all lysines or at the Arg 141, which is 5 A away from the copper. The results obtained indicate that Arg 141 is a likely candidate for binding of low-affinity anions in the vicinity of the copper and that the 1H NMR line of His 46 NH is diagnostic for such an interaction.     

Mechanism of the peroxidase activity of Cu,Zn superoxide dismutase

In addition to its very efficient catalysis of the dismutation of superoxide ( O₂^- ) into O₂ plus H₂O₂, Cu, Zn SOD acts less efficiently as a non-specific peroxidase. This peroxidase activity is CO₂ dependent although very slow peroxidation of some substrates occurs in the absence of CO2. The mechanism of that CO₂ dependence is explained by the generation of a strong oxidant at the copper site by two sequential reactions with H₂O₂, followed by the oxidation of CO₂ to the carbonate radical that then diffuses into the bulk solution. This diffusible carbonate radical is then responsible for the diverse oxidations that have been reported. A different mechanism that involves the reduction of peroxymonocarbonate by the reduced superoxide dismutase to yield carbonate radical has been proposed. We will demonstrate that this mechanism is not supported by the available data. It seems likely that generation of the carbonate radical has relevance to the oxidative stress faced by aerobic organisms.     

Zinc binding modulates the entire folding free energy surface of human Cu,Zn superoxide dismutase

Over 100 amino acid replacements in human Cu,Zn superoxide dismutase (SOD) are known to cause amyotrophic lateral sclerosis, a gain-of-function neurodegenerative disease that destroys motor neurons. Supposing that aggregates of partially folded states are primarily responsible for toxicity, we determined the role of the structurally important zinc ion in defining the folding free energy surface of dimeric SOD by comparing the thermodynamic and kinetic folding properties of the zinc-free and zinc-bound forms of the protein. The presence of zinc was found to decrease the free energies of a peptide model of the unfolded monomer, a stable variant of the folded monomeric intermediate, and the folded dimeric species. The unfolded state binds zinc weakly with a micromolar dissociation constant, and the folded monomeric intermediate and the native dimeric form both bind zinc tightly, with subnanomolar dissociation constants. Coupled with the strong driving force for the subunit association reaction, the shift in the populations toward more well-folded states in the presence of zinc decreases the steady-state populations of higher-energy states in SOD under expected in vivo zinc concentrations (approximately nanomolar). The significant decrease in the population of partially folded states is expected to diminish their potential for aggregation and account for the known protective effect of zinc. The ∼ 100-fold increase in the rate of folding of SOD in the presence of micromolar concentrations of zinc demonstrates a significant role for a preorganized zinc-binding loop in the transition-state ensemble for the rate-limiting monomer folding reaction in this β-barrel protein.     

The coupling between disulphide status, metallation and dimer interface strength in Cu/Zn superoxide dismutase

The gain of neurotoxic function in amyotrophic lateral sclerosis (ALS) has been linked to misfolding of the homodimeric enzyme Cu/Zn superoxide dismutase (SOD). Here, we present the crystal structure of fully cysteine-depleted human SOD (SOD(CallA)), representing a reduced, marginally stable intermediate on the folding pathway in vivo that has also been implicated as neurotoxic precursor state. A hallmark of this species is that it fails to dimerize and becomes trapped as a monomer in the absence of the active-site metals. The crystallographic data show that removal of the C57-C146 disulphide bond sets free the interface loop IV in the apo protein, whereas the same loop remains unaffected in the holo protein. Thus, the low dimerisation propensity of disulphide-reduced apoSOD seems to be of entropic origin due to increased loop flexibility in the monomeric state: in the disulphide-reduced holo protein this gain in configurational entropy upon splitting of the dimer interface is reduced by the metal coordination.     

Sequence and structural determinants of Cu, Zn superoxide dismutase aggregation

Diverse point mutations in the enzyme Cu, Zn superoxide dismutase (SOD1) are linked to its aggregation in the familial form of the disease amyotrophic lateral sclerosis. The disease-associated mutations are known to destabilize the protein, but the structural basis of the aggregation of the destabilized protein and the structure of aggregates are not well understood. Here, we investigate in silico the sequence and structural determinants of SOD1 aggregation: (1) We identify sequence fragments in SOD1 that have a high aggregation propensity, using only the sequence of SOD1, and (2) we perform molecular dynamics simulations of the SOD1 dimer folding and misfolding. In both cases, we identify identical regions of the protein as having high propensity to form intermolecular interactions. These regions correspond to the N- and C-termini, and two crossover loops and two beta-strands in the Greek-key native fold of SOD1. Our results suggest that the high aggregation propensity of mutant SOD1 may result from a synergy of two factors: the presence of highly amyloidogenic sequence fragments ("hot spots"), and the presence of these fragments in regions of the protein that are structurally most likely to form intermolecular contacts under destabilizing conditions. Therefore, we postulate that the balance between the self-association of aggregation-prone sequences and the specific structural context of these sequences in the native state determines the aggregation propensity of proteins.     

Structural and thermodynamic effects of post-translational modifications in mutant and wild type Cu, Zn superoxide dismutase

Aggregation of Cu,Zn superoxide dismutase (SOD1) is implicated in amyotrophic lateral sclerosis. Glutathionylation and phosphorylation of SOD1 is omnipresent in the human body, even in healthy individuals, and has been shown to increase SOD1 dimer dissociation, which is the first step on the pathway toward SOD1 aggregation. We found that post-translational modification of SOD1, especially glutathionylation, promotes dimer dissociation. We discovered an intermediate state in the pathway to dissociation, a conformational change that involves a “loosening” of the β-barrels and a loss or shift of dimer interface interactions. In modified SOD1, this intermediate state is stabilized as compared to unmodified SOD1. The presence of post-translational modifications could explain the environmental factors involved in the speed of disease progression. Because post-translational modifications such as glutathionylation are often induced by oxidative stress, post-translational modification of SOD1 could be a factor in the occurrence of sporadic cases of amyotrophic lateral sclerosis, which represent 90% of all cases of the disease.     

Cu,Zn superoxide dismutase increases intracellular calcium levels via a phospholipase C-protein kinase C pathway in SK-N-BE neuroblastoma cells

The superoxide dismutase isoenzymes (SOD) play a key role in scavenging, O*2- radicals. In contrast with previous studies, recent data have shown that human neuroblastoma cells are able to export the cytosolic Cu,Zn superoxide dismutase (SOD1), thus suggesting a paracrine role exerted by this enzyme in the nervous system. To evaluate whether SOD1 could activate intracellular signalling pathways, the functional interaction between SOD1 and human neuroblastoma SK-N-BE cells was investigated. By analyzing the surface binding of biotinylated SOD1 on SK-N-BE cells and by measuring intracellular calcium concentrations and PKC activity, we demonstrated that SOD1 specifically interacts in a dose-dependent manner with the cell surface membrane of SK-N-BE. This binding was able to activate a PLC-PKC-dependent pathway that increased intracellular calcium concentrations mainly deriving from the intracellular stores. Furthermore, we showed that this effect was independent of SOD1 dismutase activity and was totally inhibited by U73122, the PLC blocker. On the whole, these data indicate that SOD1 carries out a neuromodulatory role affecting calcium-dependent cellular functions.     

Modification of cysteine 111 in Cu/Zn superoxide dismutase results in altered spectroscopic and biophysical properties

Cu/Zn superoxide dismutase (SOD) mutations are involved in about 20% of all cases of familial amyotrophic lateral sclerosis (FALS). Recently, it has been proposed that aberrant copper activity may be occurring within SOD at an alternative binding, and cysteine 111 has been identified as a potential copper ligand. Using a commercial source of human SOD isolated from erythrocytes, an anomalous absorbance at 325 nm was identified. This unusual property, which does not compromise SOD activity, had previously been shown to be consistent with a sulfhydryl modification at a cysteine residue. Here, we utilized limited trypsin proteolysis and mass spectrometry to show that the modification has a mass of 32 daltons and is located at cysteine 111. The reaction of SOD with sodium sulfide, which can react with cysteine to form a persulfide group, and with potassium cyanide, which can selectively remove persulfide bonds, confirmed the addition of a persulfide group at cysteine 111. Gel electrophoresis and glutaraldehyde cross-linking revealed that this modification makes the acid-induced denaturation of SOD fully irreversible. Furthermore, the modified protein exhibits a slower acid-induced unfolding, and is more resistant to oxidation-induced aggregation caused by copper and hydrogen peroxide. Thus, these results suggest that cysteine 111 can have a biochemical and biophysical impact on SOD, and suggest that it can interact with copper, potentially mediating the copper-induced oxidative damage of SOD. It will be of interest to study the role of cysteine 111 in the oxidative damage and aggregation of toxic SOD mutants.