Prion-like activity of Cu/Zn superoxide dismutase

Neurodegenerative diseases belong to a larger group of protein misfolding disorders, known as proteinopathies. There is increasing experimental evidence implicating prion-like mechanisms in many common neurodegenerative disorders, including Alzheimer disease, Parkinson disease, the tauopathies, and amyotrophic lateral sclerosis (ALS), all of which feature the aberrant misfolding and aggregation of specific proteins. The prion paradigm provides a mechanism by which a mutant or wild-type protein can dominate pathogenesis through the initiation of self-propagating protein misfolding. ALS, a lethal disease characterized by progressive degeneration of motor neurons is understood as a classical proteinopathy; the disease is typified by the formation of inclusions consisting of aggregated protein within and around motor neurons that can contribute to neurotoxicity. It is well established that misfolded/oxidized SOD1 protein is highly toxic to motor neurons and plays a prominent role in the pathology of ALS. Recent work has identified propagated protein misfolding properties in both mutant and wild-type SOD1, which may provide the molecular basis for the clinically observed contiguous spread of the disease through the neuroaxis. In this review we examine the current state of knowledge regarding the prion-like properties of SOD1 and comment on its proposed mechanisms of intercellular transmission.     

Cu/Zn superoxide dismutase can form pore-like structures

Mutations in Cu/Zn superoxide dismutase (SOD) are associated with familial amyotrophic lateral sclerosis (FALS), a neurodegenerative disease that is characterized by the selective death of motor neurons. Despite the genetic association made between the protein and the disease, the mechanism by which the mutant SOD proteins become toxic is still a mystery. Using wild-type SOD and three pathogenic mutants (A4V, G37R, and G85R), we show that the copper-induced oxidation of metal-depleted SOD causes its in vitro aggregation into pore-like structures, as determined by atomic force microscopy. Because toxic pores have been recently implicated in the pathogenic mechanism of other neurodegenerative diseases, these results raise the possibility that the aberrant self-assembly of oxidatively damaged SOD mutants into toxic oligomers or pores may have a pathological role in FALS.     

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.     

Role of the JNK/c-Jun/AP-1 signaling pathway in galectin-1-induced T-cell death

Galectin-1 (gal-1), an endogenous β-galactoside-binding protein, triggers T-cell death through several mechanisms including the death receptor and the mitochondrial apoptotic pathway. In this study we first show that gal-1 initiates the activation of c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase kinase 4 (MKK4), and MKK7 as upstream JNK activators in Jurkat T cells. Inhibition of JNK activation with sphingomyelinase inhibitors (20 μM desipramine, 20 μM imipramine), with the protein kinase C-δ (PKCδ) inhibitor rottlerin (10 μM), and with the specific PKCθ pseudosubstrate inhibitor (30 μM) indicates that ceramide and phosphorylation by PKCδ and PKCθ mediate gal-1-induced JNK activation. Downstream of JNK, we observed increased phosphorylation of c-Jun, enhanced activating protein-1 (AP-1) luciferase reporter, and AP-1/DNA-binding in response to gal-1. The pivotal role of the JNK/c-Jun/AP-1 pathway for gal-1-induced apoptosis was documented by reduction of DNA fragmentation after inhibition JNK by SP600125 (20 μM) or inhibition of AP-1 activation by curcumin (2 μM). Gal-1 failed to induce AP-1 activation and DNA fragmentation in CD3-deficient Jurkat 31-13 cells. In Jurkat E6.1 cells gal-1 induced a proapoptotic signal pattern as indicated by decreased antiapoptotic Bcl-2 expression, induction of proapoptotic Bad, and increased Bcl-2 phosphorylation. The results provide evidence that the JNK/c-Jun/AP-1 pathway plays a key role for T-cell death regulation in response to gal-1 stimulation.     

Cu/Zn superoxide dismutase plays important role in immune response

Activation of macrophages leads to the secretion of cytokines and enzymes that shape the inflammatory response and increase metabolic processes. This, in turn, results in increased production of reactive oxygen species. The role of Cu/Zn superoxide dismutase (SOD-1), an important enzyme in cellular oxygen metabolism, was examined in activated peritoneal elicited macrophages (PEM) and in several inflammatory processes in vivo. LPS and TNF-alpha induced SOD-1 in PEM. SOD-1 induction by LPS was mainly via extracellular signal-regulated kinase-1 activation. Transgenic mice overexpressing SOD-1 demonstrated a significant increase in the release of TNF-alpha and of the metalloproteinases MMP-2 and MMP-9 from PEM. Disulfiram (DSF), an inhibitor of SOD-1, strongly inhibited the release of TNF-alpha, vascular endothelial growth factor, and MMP-2 and MMP-9 from cultured activated PEM. These effects were prevented by addition of antioxidants, further indicating involvement of reactive oxygen species. In vivo, transgenic mice overexpressing SOD-1 demonstrated a 4-fold increase in serum TNF-alpha levels and 2-fold stronger delayed-type hypersensitivity reaction as compared with control nontransgenic mice. Conversely, oral administration of DSF lowered TNF-alpha serum level by 4-fold, lowered the delayed-type hypersensitivity response in a dose-dependent manner, and significantly inhibited adjuvant arthritis in Lewis rats. The data suggest an important role for SOD-1 in inflammation, establish DSF as a potential inhibitor of inflammation, and raise the possibility that regulation of SOD-1 activity may be important in the treatment of immune-dependent pathologies.     

Post-translational modification of Cu/Zn superoxide dismutase under anaerobic conditions

In eukaryotic organisms, the largely cytosolic copper- and zinc-containing superoxide dismutase (Cu/Zn SOD) enzyme represents a key defense against reactive oxygen toxicity. Although much is known about the biology of this enzyme under aerobic conditions, less is understood regarding the effects of low oxygen levels on Cu/Zn SOD enzymes from diverse organisms. We show here that like bakers' yeast (Saccharomyces cerevisiae), adaptation of the multicellular Caenorhabditis elegans to growth at low oxygen levels involves strong downregulation of its Cu/Zn SOD. Much of this regulation occurs at the post-translational level where CCS-independent activation of Cu/Zn SOD is inhibited. Hypoxia inactivates the endogenous Cu/Zn SOD of C. elegans Cu/Zn SOD as well as a P144 mutant of S. cerevisiae Cu/Zn SOD (herein denoted Sod1p) that is independent of CCS. In our studies of S. cerevisiae Sod1p, we noted a post-translational modification to the inactive enzyme during hypoxia. Analysis of this modification by mass spectrometry revealed phosphorylation at serine 38. Serine 38 represents a putative proline-directed kinase target site located on a solvent-exposed loop that is positioned at one end of the Sod1p β-barrel, a region immediately adjacent to residues previously shown to influence CCS-dependent activation. Although phosphorylation of serine 38 is minimal when the Sod1p is abundantly active (e.g., high oxygen level), up to 50% of Sod1p can be phosphorylated when CCS activation of the enzyme is blocked, e.g., by hypoxia or low-copper conditions. Serine 38 phosphorylation can be a marker for inactive pools of Sod1p.     

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     

Aberrant neuronal and mitochondrial proteins in hippocampus of transgenic mice overexpressing human Cu/Zn superoxide dismutase 1

Mutations of Cu/Zn superoxide dismutase 1 (SOD1), a metalloenzyme catalyzing the conversion of superoxide anion to hydrogen peroxide (H(2)O(2)), are linked to motor neuron degeneration. Transgenic mouse strains overexpressing wild-type human SOD1 (Tg-SOD1) were shown to have mitochondrial swelling, vacuolization, or learning and memory deficits and are widely used for biochemical, genetic, and cognitive studies; this, along with the advent of advanced proteomic methods, made us investigate protein expression in hippocampus. Hippocampal tissues of wild-type, hemizygous, and homozygous Tg-SOD1 mice were isolated and used for two-dimensional gel electrophoresis with subsequent matrix-assisted laser desorption/ionization-time of flight identification. We identified several synaptosomal, neuronal, antioxidant, and mitochondrial proteins in hippocampus, and expression levels of syntaxin-binding protein 1, N-ethylmaleimide-sensitive factor, synaptosomal-associated protein 25, dynamin-1, neurofilament triplet L protein, neurofilament triplet M protein, neuronal tropomodulin, and neuronal protein 25 were significantly decreased in Tg-SOD1. None of the antioxidant proteins were altered except mouse SOD1. Mitochondrial ATP synthase alpha/beta chain and elongation factor Tu were aberrant in Tg-SOD1. We conclude that derangement of neuronal and mitochondrial proteins may indicate synaptosomal and neuronal loss in Tg-SOD1 hippocampus, already reported in morphological terms. This observation is of relevance to understanding brain deficits in Down syndrome, as SOD1 is encoded on chromosome 21.     

Food-grade expression of human glutathione S-transferase and Cu/Zn superoxide dismutase in Lactococcus lactis

A food-grade gene expression system in Lactococcus lactis was established by the combination of a vector containing the lacF gene as the selection marker and a strain WZ103 carrying an in-frame deletion of this gene in the chromosome as the host. The human glutathione S-transferase A1-1 (hGSTA1) and Cu/Zn superoxide dismutase (hSOD) genes were respectively cloned into a food-grade vector under the control of the lactococcal inducible promoter P(lacA). The resulting expression plasmids were separately introduced into the lactose-deficient (Lac(-)) host, and the lactose-utilizing (Lac(+)) transformants were directly selected on a chemically defined medium, using lactose as the sole carbon source. The successful food-grade expression of hGSTA1 and hSOD in the L. lactis WZ103 transformed with these plasmids were analyzed by Western blotting and enzymatic activity assay, respectively.     

Cu/Zn superoxide dismutase is differentially regulated in period gene-mutant mice

The circadian clock in the brain coordinates the phase of peripheral oscillators that regulate tissue-specific physiological outputs. Here we report that circadian variations in the expression and activity of Cu/Zn superoxide dismutase (SOD1; EC 1.15.1.1) are present in liver homogenates from mice. The SOD1 mRNA expression from wild-type (WT) mice peaked at Zeitgeber Time 9 (ZT9; 9 h after lights-on time). While there was no rhythmicity in that from period2 (per2) gene knockout (P2K) mice, the level of SOD1 from per1/per2 double knockout (DKO) mice was significantly elevated at ZT5. The enzyme activity of SOD1 was also rhythmic in the mouse liver. Moreover, the total amount of the SOD1 exhibited a rhythmic oscillation with a peak at ZT9 in the liver from WT mice. We also found that tert-butylhydroperoxide (t-BHP)-induced oxidative damage in both WT and P2K mouse embryonic fibroblast (MEF) cells resulted in the up-regulation of SOD1 levels. Our data suggest that the expression of an important antioxidant enzyme, SOD1, is under circadian clock control and that mice are more susceptible to oxidative stress depending on the time of day.     

Cu/Zn superoxide dismutase in yeast mitochondria - a general phenomenon

Fermentative and respiratory yeast strains of genera Saccharomyces, Kluyveromyces, Pichia, Candida and Hansenula have been investigated for mitochondrial localization of Cu/Zn superoxide dismutase (SOD). Pure mitochondrial fractions were obtained and the specific activities of Cu/Zn and Mn SODs were measured in comparison with those in the corresponding cell-free extracts. The Cu/Zn SOD: Mn SOD ratio in mitochondria and crude extracts was calculated and was considered a specific characteristic of all tested strains. Electrophoretical visualization of SOD patterns provided evidence for possible migration of cytosolic Cu/Zn SOD to mitochondria. The characteristic Cu/Zn SOD profile in mitochondria of all tested strains suggested its ubiquity within the fermentative and respiratory yeasts.     

Identification of Cu/Zn superoxide dismutase in cattle and river buffaloes

All air-living organisms produce superoxide dismutase (SOD) and several antioxidant enzymes that limit oxidative stress by detoxifying reactive oxygen species. SOD1 gene has been investigated in Egyptian native cattle and buffalos at the level of genomic DNA and cDNA that were extracted from leucocytes. An unexpected band at approximately 370 bp was obtained in cattle genomic DNA and cDNA as well as in buffalo cDNA. SOD1 amplified sequence of native cattle genomic DNA and cDNA showed a 93% alignment. Native cattle genomic DNA SOD1 amplicon shares sequence homology with mRNAs of Bos taurus “similar to superoxide dismutase” (SOD1) sequence of the GeneBank database. It also shares sequence homology with “similar to superoxide dismutase” on B. taurus chromosome BTA13. The results indicate that the genomic DNA of Egyptian native cattle contains SOD1 processed pseudo gene. SOD1 primers amplified three fragments in buffalo genomic DNA which indicates that buffalo genome has different copies of SOD1 due to alternative splicing. It failed to produce the 370 bp fragments found in cattle DNA. The protein analysis revealed no differences between Egyptian native cattle and B. taurus SOD1 mRNA. However, one amino acid, aspartic acid (Asp), in Egyptian native cattle and B. taurus SOD1, is substituted with asparagine (Asn) (D26N) in buffaloes. This amino acid substitution may be due to non-synonymous single nucleotide polymorphisms (nsSNPs). The nsSNPs detected in buffaloes may affect the function of the encoded protein. This study is the first investigation reporting that the resistance of the buffalo to diseases and parasites that afflict cattle may not be acquired but may have a genetic basis.