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Free Radical Biology & Medicine Apr 2022Cu/Zn-superoxide dismutase (SOD1) is a homodimer with two identical subunits, each of which binds a copper and zinc ion in the native state. In contrast to such a text...
Cu/Zn-superoxide dismutase (SOD1) is a homodimer with two identical subunits, each of which binds a copper and zinc ion in the native state. In contrast to such a text book case, SOD1 proteins purified in vitro or even in vivo have been often reported to bind a non-stoichiometric amount of the metal ions. Nonetheless, it is difficult to probe how those metal ions are distributed in the two identical subunits. By utilizing native mass spectrometry, we showed here that addition of a sub-stoichiometric copper/zinc ion to SOD1 led to the formation of a homodimer with a stochastic combination of the subunits binding 0, 1, and even 2 metal ions. We also found that the homodimer was able to bind four copper or four zinc ions, implying the binding of a copper and zinc ion at the canonical zinc and copper site, respectively. Such ambiguity in the metal quota and selectivity could be avoided when an intra-subunit disulfide bond in SOD1 was reduced before addition of the metal ions. Apo-SOD1 in the disulfide-reduced state was monomeric and was found to bind only one zinc ion per monomer. By binding a zinc ion, the disulfide-reduced SOD1 became conformationally compact and acquired the ability to dimerize. Based upon the results in vitro, we describe the pathway in vivo enabling SOD1 to bind copper and zinc ions with high accuracy in their quota and selectivity. A failure of correct metallation in SOD1 will also be discussed in relation to amyotrophic lateral sclerosis.
Topics: Amyotrophic Lateral Sclerosis; Copper; Humans; Mass Spectrometry; Mutation; Superoxide Dismutase; Superoxide Dismutase-1; Zinc
PubMed: 35314356
DOI: 10.1016/j.freeradbiomed.2022.03.014 -
Genetics Oct 2023Oxidative stress can damage DNA and thereby contribute to genome instability. To avoid an imbalance or overaccumulation of reactive oxygen species (ROS), cells are...
Oxidative stress can damage DNA and thereby contribute to genome instability. To avoid an imbalance or overaccumulation of reactive oxygen species (ROS), cells are equipped with antioxidant enzymes that scavenge excess ROS. Cells lacking the RecQ-family DNA helicase Sgs1, which contributes to homology-dependent DNA break repair and chromosome stability, are known to accumulate ROS, but the origin and consequences of this oxidative stress phenotype are not fully understood. Here, we show that the sgs1 mutant exhibits elevated mitochondrial superoxide, increased mitochondrial mass, and accumulation of recombinogenic DNA lesions that can be suppressed by antioxidants. Increased mitochondrial mass in the sgs1Δ mutant is accompanied by increased mitochondrial branching, which was also inducible in wildtype cells by replication stress. Superoxide dismutase Sod2 genetically interacts with Sgs1 in the suppression of nuclear chromosomal rearrangements under paraquat (PQ)-induced oxidative stress. PQ-induced chromosome rearrangements in the absence of Sod2 are promoted by Rad51 recombinase and the polymerase subunit Pol32. Finally, the dependence of chromosomal rearrangements on the Rev1/Pol ζ mutasome suggests that under oxidative stress successful DNA synthesis during DNA break repair depends on translesion DNA synthesis.
Topics: Antioxidants; DNA; Genomic Instability; Oxidative Stress; Reactive Oxygen Species; RecQ Helicases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Superoxide Dismutase
PubMed: 37638880
DOI: 10.1093/genetics/iyad147 -
ACS Infectious Diseases Jun 2018Superoxide anion radical is generated as a natural byproduct of aerobic metabolism but is also produced as part of the oxidative burst of the innate immune response... (Review)
Review
Superoxide anion radical is generated as a natural byproduct of aerobic metabolism but is also produced as part of the oxidative burst of the innate immune response design to kill pathogens. In living systems, superoxide is largely managed through superoxide dismutases (SODs), families of metalloenzymes that use Fe, Mn, Ni, or Cu cofactors to catalyze the disproportionation of superoxide to oxygen and hydrogen peroxide. Given the bursts of superoxide faced by microbial pathogens, it comes as no surprise that SOD enzymes play important roles in microbial survival and virulence. Interestingly, microbial SOD enzymes not only detoxify host superoxide but also may participate in signaling pathways that involve reactive oxygen species derived from the microbe itself, particularly in the case of eukaryotic pathogens. In this Review, we will discuss the chemistry of superoxide radicals and the role of diverse SOD metalloenzymes in bacterial, fungal, and protozoan pathogens. We will highlight the unique features of microbial SOD enzymes that have evolved to accommodate the harsh lifestyle at the host-pathogen interface. Lastly, we will discuss key non-SOD superoxide scavengers that specific pathogens employ for defense against host superoxide.
Topics: Animals; Bacteria; Biological Warfare; Chemical Warfare; Fungi; Host-Pathogen Interactions; Humans; Hydrogen Peroxide; Metals; NADPH Oxidases; Oxidation-Reduction; Parasites; Reactive Oxygen Species; Superoxide Dismutase; Superoxides
PubMed: 29517910
DOI: 10.1021/acsinfecdis.8b00026 -
International Journal of Biological... Aug 2015The ability of marine organism to cope with oxidative stress is one of the main factors that influence its survival in the marine environment, when senescence conditions... (Review)
Review
The ability of marine organism to cope with oxidative stress is one of the main factors that influence its survival in the marine environment, when senescence conditions prevail. The antioxidative defense system includes enzymatic and non-enzymatic components. Among the enzymatic system, superoxide dismutases are the first and most important of the antioxidant metalloenzymes. Four different types of metal centers have been detected in SODs, dividing this family into Cu/Zn, Ni, Mn and Fe-SODs. Its use was limited to non-drug applications in humans (include: cosmetic, food, agriculture, and chemical industries) and drug applications in animals. This paper is a review of the recent literatures on sources of marine SODs, the need for SOD and different applications in industry, covering the last decades. The most recent paper, patents and reviews on characterization and application are reviewed.
Topics: Animals; Aquatic Organisms; Bacterial Proteins; Cyanobacteria; Fungal Proteins; Humans; Oxidation-Reduction; Photobacterium; Superoxide Dismutase
PubMed: 26047895
DOI: 10.1016/j.ijbiomac.2015.05.053 -
Journal of Basic Microbiology Aug 2017A novel superoxide dismutase gene from Antarctic yeast Rhodotorula mucilaginosa AN5 was cloned, sequenced, and then expressed in Escherichia coli. The R. mucilaginosa...
A novel superoxide dismutase gene from Antarctic yeast Rhodotorula mucilaginosa AN5 was cloned, sequenced, and then expressed in Escherichia coli. The R. mucilaginosa AN5 SOD (RmFeSOD) gene was 639 bp open reading frame in length, which encoded a protein of 212 amino acids with a deduced molecular mass of 23.5 kDa and a pI of 7.89. RmFeSOD was identified as iron SOD type with a natural status of homodimer. The recombinant RmFeSOD showed good pH stability in the pH 1.0-9.0 after 1 h incubation. Meanwhile, it was found to behave relatively high thermostability, and maintained more than 80% activity at 50 °C for 1 h. By addition of 1 mM metal ions, the enzyme activity increased by Zn , Cu , Mn , and Fe , and inhibited only by Mg . RmFeSOD showed relatively low tolerance to some compounds, such as PMSF, SDS, Tween-80, Triton X-100, DMSO, β-ME, and urea. However, DTT showed no inhibition to enzyme activity. Using copper stress experiment, the RmFeSOD recombinant E. coli exhibited better growth than non-recombinant bacteria, which revealed that RmFeSOD might play an important role in the adaptability of heavy metals.
Topics: Antarctic Regions; Cloning, Molecular; Enzyme Stability; Escherichia coli; Gene Expression; Hydrogen-Ion Concentration; Metals, Heavy; Molecular Weight; Open Reading Frames; Recombinant Proteins; Rhodotorula; Superoxide Dismutase; Temperature; Urea
PubMed: 28639705
DOI: 10.1002/jobm.201700165 -
Fish & Shellfish Immunology Aug 2019Manganese superoxide dismutase (MnSOD) is a sort of important metalloenzyme that can catalyze ROS in the organisms. In this study, MnSOD cDNA of C. plicata, designated...
Manganese superoxide dismutase (MnSOD) is a sort of important metalloenzyme that can catalyze ROS in the organisms. In this study, MnSOD cDNA of C. plicata, designated as CpMnSOD (accession no. MK465057), was cloned from hemocytes. The full-length cDNA of MnSOD was 1096 bp with a 672 bp open reading frame encoding 223 amino acids. The deduced amino acid sequence contained a mitochondrial-targeting sequence (MTS) of 18 amino acids in the N-terminus, and four conserved amino acids for manganese binding (H, H, D, H). CpMnSOD showed a high level (65-73%) of sequence similarity to MnSODs from other species. The results of Real-time quantitative PCR revealed that CpMnSOD mRNA constitutively expressed in tissues. The highest expression level was in hepatopancreas, followed by muscle, mantle and gill, and the lowest expression level was in hemocytes. After microcystin challenge, the expression levels of CpMnSOD mRNA were up-regulated in hemocytes and hepatopancreas. The cDNA of CpMnSOD was cloned into the plasmid pColdI-ZZ, and the recombinant protein was expressed in Escherichia coli BL21 (DE3). The enzyme stability assay showed that the purified CpMnSOD protein maintained more than 80% enzyme activity at temperature up to 70 °C, at pH 2.0-10.0, and resistant to 8 mol/L urea or 8% SDS.
Topics: Amino Acid Sequence; Animals; Base Sequence; Escherichia coli; Gene Expression Profiling; Gene Expression Regulation; Immunity, Innate; Phylogeny; Recombinant Proteins; Sequence Alignment; Superoxide Dismutase; Unionidae
PubMed: 31078645
DOI: 10.1016/j.fsi.2019.04.307 -
The Journal of Biological Chemistry Mar 2018The copper-containing superoxide dismutases (SODs) represent a large family of enzymes that participate in the metabolism of reactive oxygen species by... (Review)
Review
The copper-containing superoxide dismutases (SODs) represent a large family of enzymes that participate in the metabolism of reactive oxygen species by disproportionating superoxide anion radical to oxygen and hydrogen peroxide. Catalysis is driven by the redox-active copper ion, and in most cases, SODs also harbor a zinc at the active site that enhances copper catalysis and stabilizes the protein. Such bimetallic Cu,Zn-SODs are widespread, from the periplasm of bacteria to virtually every organelle in the human cell. However, a new class of copper-containing SODs has recently emerged that function without zinc. These copper-only enzymes serve as extracellular SODs in specific bacteria ( Mycobacteria), throughout the fungal kingdom, and in the fungus-like oomycetes. The eukaryotic copper-only SODs are particularly unique in that they lack an electrostatic loop for substrate guidance and have an unusual open-access copper site, yet they can still react with superoxide at rates limited only by diffusion. Copper-only SOD sequences similar to those seen in fungi and oomycetes are also found in the animal kingdom, but rather than single-domain enzymes, they appear as tandem repeats in large polypeptides we refer to as CSRPs (copper-only SOD-repeat proteins). Here, we compare and contrast the Cu,Zn copper-only SODs and discuss the evolution of copper-only SOD protein domains in animals and fungi.
Topics: Copper; Fungal Proteins; Fungi; Metalloproteins; Mycobacterium; Oomycetes; Periplasmic Proteins; Superoxide Dismutase; Zinc
PubMed: 29259135
DOI: 10.1074/jbc.TM117.000182 -
Plant Physiology and Biochemistry : PPB Feb 2023The serine-threonine protein phosphatases PP2A regulate many cellular processes, however their role in oxidative stress responses and defence is less known. We show the...
The serine-threonine protein phosphatases PP2A regulate many cellular processes, however their role in oxidative stress responses and defence is less known. We show the involvement of its C (catalytic) and B" (a regulatory) subunits. The c3c4 (C subunit) and fass (B") subunit mutants and Col wt of Arabidopsis were used. Controls and treatments with the PP2A inhibitor microcystin-LR (MCY-LR) and reactive oxygen species (ROS) inducer diquat (DQ) were employed. ROS levels of primary roots were largely genotype dependent and both C and B" subunit mutants had increased sensitivity to MCY-LR and DQ indicating the involvement of these subunits in oxidative stress induction. Superoxide dismutases (SOD), mainly the Cu/Zn-SOD isoform, as key enzymes involved in ROS scavenging are also showing altered (mostly increased) activities in both c3c4 and fass mutants and have opposite relations to ROS induction. This indicates that the two types of subunits involved have partially different regulatory roles. In relation to this, control and MCY-LR/DQ treated B" subunit mutants were proven to have altered levels of phosphorylation of histone H2AX. γH2AX, the phosphorylated form indicates double stranded DNA damage during oxidative stress. Overall we point out the probable pivotal role of several PP2A subunits in the regulation of oxidative stress responses in plants and pave the way for future research to reveal the signaling pathways involved.
Topics: Reactive Oxygen Species; Arabidopsis; Protein Phosphatase 2; Oxidative Stress; Superoxide Dismutase
PubMed: 36640685
DOI: 10.1016/j.plaphy.2022.12.031 -
Molecules and Cells Mar 2016A balance between production and degradation of reactive oxygen species (ROS) is critical for maintaining cellular homeostasis. Increased levels of ROS during oxidative...
A balance between production and degradation of reactive oxygen species (ROS) is critical for maintaining cellular homeostasis. Increased levels of ROS during oxidative stress are associated with disease conditions. Antioxidant enzymes, such as extracellular superoxide dismutase (EC-SOD), in the extracellular matrix (ECM) neutralize the toxicity of superoxide. Recent studies have emphasized the importance of EC-SOD in protecting the brain, lungs, and other tissues from oxidative stress. Therefore, EC-SOD would be an excellent therapeutic drug for treatment of diseases caused by oxidative stress. We cloned both the full length (residues 1-240) and truncated (residues 19-240) forms of human EC-SOD (hEC-SOD) into the donor plasmid pFastBacHTb. After transposition, the bacmid was transfected into the Sf9-baculovirus expression system and the expressed hEC-SOD purified using FLAG-tag. Western blot analysis revealed that hEC-SOD is present both as a monomer (33 kDa) and a dimer (66 kDa), as detected by the FLAG antibody. A water-soluble tetrazolium (WST-1) assay showed that both full length and truncated hEC-SOD proteins were enzymatically active. We showed that a potent superoxide dismutase inhibitor, diethyldithiocarbamate (DDC), inhibits hEC-SOD activity.
Topics: Animals; Circular Dichroism; Cloning, Molecular; Ditiocarb; Humans; Oxidative Stress; Protein Structure, Secondary; Sf9 Cells; Superoxide Dismutase
PubMed: 26912083
DOI: 10.14348/molcells.2016.2271 -
Scientific Reports Oct 2023Global warming is a critical challenge limiting crop productivity. Heat stress during cucumber growing stages caused deterioration impacts on the flowering, fruit, and...
Global warming is a critical challenge limiting crop productivity. Heat stress during cucumber growing stages caused deterioration impacts on the flowering, fruit, and yield stages. In this study, "inbred line 1 and hybrid P1 × P2" (heat-tolerant) and "Barracuda" (heat-sensitive) were utilized to determine the heat tolerance in summer season. The heat injury index was used to exhibit the heat tolerance performance. The heat injury index for heat tolerant (HT) genotypes, on leaves (HIIL%) and female flowers (HIIF%), was less than 25 and 15 % in HT, compared to heat sensitive (HS) was more than 75 and 85%, respectively. Moreover, the content of leaf chlorophyll, proline, brassinosteroid (BRs), abscisic acid content (ABA), the activity of catalase (CAT, EC 1.11. 1.6), peroxidase (POD, EC 1.11.1.7) and superoxide dismutase (SOD, EC 1.15.1.1) increased with the heat stress responses in HT plants. Expression pattern analyses of eight genes, related to POD (CSGY4G005180 and CSGY6G015230), SOD (CSGY4G010750 and CSGY1G026400), CAT (CsGy4G025230 and CsGy4G025240), and BR (CsGy6G029150 and CsGy6G004930) showed a significant increase in HT higher than in HS plants. This study furnishes valuable markers for heat tolerance genotypes breeding in cucumber and provides a basis for understanding heat-tolerance mechanisms.
Topics: Cucumis sativus; Thermotolerance; Plant Breeding; Superoxide Dismutase; Heat-Shock Response
PubMed: 37907590
DOI: 10.1038/s41598-023-45163-7