Seminar on SOD's

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Seminar on SOD's

  1. 1. SeminarSuperoxide dismutase’s and their role in plants and in some human diseases By AZFAR ALI BAJWA
  2. 2. Contents• Introduction• Terms• Superoxide Dismutase -history -function SOD as antioxidant• Types Structure of SOD’s• Mechanism (reaction)• Location of SOD’s in plant cell• Human SOD’s• Role in Plants - ROS production - Values of ROS production -ROS-scavenging System -SOD and stress tolerance 2
  3. 3. • Role in Humans -cardiovascular diseases - downs syndrome• SOD as a pharmaceutical product• SOD from food• Role in cosmetics• Future prospects -SOD as future gene therapy 3
  4. 4. Introduction• Formation of reactive oxygen species(ROS) is inevitable consequence of life - An inevitable consequence of life - unavoidable by-products of aerobic metabolism. - ROS produced from metabolic events, such as respiration or fatty acid oxidation or due to environmental processes.• ROS that leads to oxidative stress. - Oxidative stress occurs when the rate of generation of reactive compounds exceeds the detoxification capacity of the cell (provided by SOD’s). - If not disposed off efficiently, ROS can cause cellular and genetic damage leading to carcinogenesis, senescence, and neurodegenerative disorders . - Because cells under oxidative stress are at risk for lethal or mutagenic damage, all aerobic organisms have evolved defense mechanisms to cope with ROS.(e.g SOD’s) 4
  5. 5. Introduction• Superoxide dismutase (SOD) as a defence mechanism against the ROS - Part of the response against ROS involves the reprogramming of gene expression to increase levels of antioxidant enzymes such as Superoxide dismutase which limit the levels of superoxide (O2˙-) Catalase which limit the levels of hydrogen peroxide (H2O2) 5
  6. 6. Terms• FREE RADICALS: Free radicals are chemical species that have a single unpaired electron in an outer orbit Energy created by this unstable configuration is released through reactions with adjacent molecules (proteins, lipids, carbohydrates and nucleic acids), causing damage to these molecules• ANTI OXIDANT : any molecule capable of stabilizing or deactivating free radicals before they attack cells. 6
  7. 7. TermsReactive oxygen species (ROS)• Reactive oxygen species (ROS) : all highly reactive, oxygen-containing molecules, including free radicals.• The mediators of oxidative stress (O2˙-, H2O2, and OH-) are known as reactive oxygen species (ROS).• Types of ROS include 1. hydroxyl radical 2. superoxide anion radical 3. hydrogen peroxide 4. singlet oxygen 5. nitric oxide radical 6. hypochlorite radical 7. various lipid peroxides. 7
  8. 8. More about ROS• React with membrane lipids, nucleic acids, proteins and enzymes, and other small molecules, resulting in cellular damage .• ROS are generated by exposure to radiation, heavy metals, and redox active compounds, and also occur under normal metabolism .• ROS affect normal cell functions by activating a number of enzymatic cascades and pathological processes in many diseases by inducing oxidative stress• free radicals are involved in the initiation of cellular injury observed in neurodegenerative diseases such as: Alzheimers’s disease (AD) Huntington’s disease (HD) Parkinson’s disease (PD)• ROS can act in all stages of carcinogenesis leading to cancer development 8
  9. 9. Super oxide Dismutase• An enzyme (EC 1.15.1.1)• Superoxide Dismutase (SOD) enzymes form important defence line in living organisms.• Acts as an antioxidant• SOD is an enzyme that repairs cells and reduces the damage done to them by superoxide (the most common free radical in the body)• Acts as an anti inflammatory• catalyze the dismutation of superoxide into oxygen and hydrogen peroxide 9
  10. 10. • The history of discovery of superoxide dismutase• Superoxide dismutases discovered by American biochemist Irwin Fridovich and his graduate student Joe McCord in 1969 .• Interestingly, the original paper was cited 39 times in just the year 2009 - forty years after original publication.• PubMed lists over 49,000 papers published on superoxide dismutase since its discovery in 1965.• Function of superoxide dismutase - Previously thought to be several metalloproteins with unknown function (for example, CuZnSOD was known as erythrocuprein). - But now it is proven that SOD catalyzes the conversion of superoxide anions to dioxygen and hydrogen peroxide; the latter being broken in turn to water by catalase or peroxidase. - SOD neutralizes superoxide ions by going through successive oxidative and reductive cycles of transition metal ions at its active site 10
  11. 11. • Now in what conditions the SOD’s act ... - Under stress conditions - specifically oxidative stress - pathogen atttack - due to the production of ROS by multiple factors (environmental) 11
  12. 12. SOD as an anti oxidant• Nutritional antioxidants act through different mechanisms and in different compartments, but are mainly free radical scavengers:• 1) they directly neutralize free radicals, 2) they reduce the peroxide concentrations and repair oxidized membranes, 3) they quench iron to decrease reactive oxygen species production, 4) via lipid metabolism, short-chain free fatty acids and cholesteryl esters neutralize reactive oxygen species . 12
  13. 13. Types of SOD’s• Four types of SOD enzymes Based on the type of metal cofactor• Copper, zinc SOD (Cu,Zn SOD)• Manganese SOD (Mn SOD)• Iron SOD (Fe SOD)• Nickel SOD (Ni SOD) 13
  14. 14. Copper, zinc SOD (Cu,Zn SOD)• Found in all eukaryotic species• Distributed in prokaryotes as well• In humans present as cytosolic and extracellular SOD• The coordination environment of the active site in the CuZnSOD enzymes is different• The cofactor involved directly in the catalytic cycle is the Cu(II) ion [this is the oxidised, while the Cu(I) ion is the reduced state].• Square pyramidal coordination environment around the Cu(II) ion - four histidine molecules in the (nearly) equatorial plane coordinating to the copper ion through one of their imidazole nitrogens. 14
  15. 15. • The fifth position is filled by a weakly bound water molecule.• The Zn(II) has its role in keeping the structure of the active site and restoring it at the end of the catalytic cycle• One of the histidine molecules is bound to the Zn(II) ion through its other imidazole nitrogen .• zinc(II) ion is also bonded to an aspartic acid and two histidine molecules 15
  16. 16. Iron SOD (Fe SOD)• FeSOD is found in bacteria and several higher plants• Present in human mitochondria• Reduced FeSOD enzymes contain a nonheme iron as a five-coordinated active site ligated by three histidine units, an aspartate group, and a H2O/OH- ligand (pKa = 8.5) supported by a conserved H-bonding network 16
  17. 17. Mn SOD• present in many bacteria, mitochondria, and chloroplasts• Present in human mitochondria• as well as in the cytosol of eukaryotic cells• FeSODs and MnSODs are homologous, the coordination environment at the metallic centres are the same in both enzymes 17
  18. 18. Nickel SOD (Ni SOD)• NiSOD enzymes are identified in several bacteria of the Streptomyces genera and several Actinomycetes• homohexamer consisting of four-helix-bundle subunits.• The catalytic centre resides in the terminal active-site loop, where the Ni(III) ion is coordinated by the amino group of histidine-1, the amide group of cysteine-2, two thiolate groups of cysteine-2 and cysteine-6, and the imidazolate nitrogen of histidine-1 as an axial ligand, which is lost in the chemically reduced state . 18
  19. 19. Mechanistic Features• ping-pong mechanism• involves O2.- oxidation at the oxidised form of the catalytically active metal ion followed by proton- induced O2.- reduction at the reduced form• SOD redox potential about half-way between the potentials at which superoxide is oxidized and reduced• the redox active metal centre of SOD is able to both oxidise and reduce the superoxide radical anion depending on 1. protonation states of the nearby residues 2. oxidation state of the metal 19
  20. 20. • The SOD catalytic cycle does not require an external source of electrons• The redox potentials of the SOD metal ion couples are pH-dependent, therefore, the catalytic cycle may be driven through changing the available proton concentration• The active sites of SOD enzymes are buried within the protein, which makes it easier to control proton delivery 20
  21. 21. Models Cu,ZnSOD• the superoxide anion must bind to the Cu(II) centre, displacing the axial water for electron transfer.• This binding is assisted by a positive arginine residue weak binding of the axial water ligand• Electron transfer results in -loss of oxygen -creation of a Cu(I) centre that is three-coordinated -the protonated imidazolate that binds only to the zinc ion 21
  22. 22. Models Cu,ZnSOD• A second superoxide anion would then bind to the open coordination position on Cu(I),• followed by inner-sphere electron transfer to generate peroxide which is protonated by re-forming the imidazolate bridge• Additional protonation would lead to rapid loss of H2O2 and further turnover 22
  23. 23. ModelsCu,ZnSOD 23
  24. 24. Models Cu,ZnSOD• problems with this mechanism are - breaking and forming the imidazolate bridge; - the turnover rate is too high under saturating superoxide conditions - if the zinc ion is removed, there is still dismutation with only a limited reduction in the turnover rate. 24
  25. 25. Models MnSOD and FeSOD• The mechanism for the manganese and iron containing enzymes is similar to that of Cu,ZnSOD• The major difference is that 1. water binds as hydroxide anion to the oxidized site and thus would not be displaced by superoxide anion; instead, the superoxide anion binds to increase the coordination number of the metal.2. Reduction then leads to protonation of the bound hydroxide anion to form water3. hydroxide anion upon oxidation by a second superoxide anion transfers its proton to the resulting peroxide anion. 25
  26. 26. 26
  27. 27. Models Ni SOD• Mechanistic concept concerning how the NiSOD enzyme works does not exist. 27
  28. 28. Location of SOD’s in plant cell 28
  29. 29. Human SOD’s• In humans (as in all other mammals and most chordates), three forms of superoxide dismutase are present.• SOD1: - It is a dimer - located in the cytoplasm, - containing Cu, Zn - molecular weight of 32 kDa - each of the subunit contains the active site, - active site is a dinulcear metal cluster constituted by copper and zinc ions 29
  30. 30. Human SOD’s Crystallographic structure of the human SOD1 enzymeFigure:SOD1 enzyme(N-terminus = blue, C-terminus = red)complexed withcopper (blue-green sphere)and zinc (grey spheres) 30
  31. 31. Human SOD’s• SOD2: - mitochondrial SOD - Mn (manganese) in its reactive centre - homotetramer - molecular weight of 96 kDa - one manganese atom per subunit - it cycles from Mn(III) to Mn(II), and back to Mn(III) Structure of the active site of human superoxide dismutase 2 31
  32. 32. Human SOD’s • SOD3: - Extracellular superoxide dismutase - a tetramer - contains Cu, Zn (copper and zinc)Figure:Crystallographic structure ofhuman SOD3 enzymecomplexed with copper andzinc cations (orange and greyspheres respectively) 32
  33. 33. Role in plants• Act as anti oxidant• Reduces the oxidative stress• Before getting into the detail we will discuss the ROS (reactive oxygen species) - their production - ROS formation - Sources of ROS - ROS-scavenging System-Defense Mechanism of ROS and pathways• Plant Aging Increases with Oxidative Stress 33
  34. 34. ROS productionROS are formed by several different mechanisms.• Biological interaction of ionising radiation can occur with molecules of all aerobic system.• They can be toxic by-products of cellular respiration, electrons which leak from ETC.• The dismutation of two superoxide anions produces H2O2, which cause β-oxidation of fatty acids and peroxisomal photorespiration reactions . 34
  35. 35. ROS production• The oxygen taken by living organisms is converted to O2 - , H2O2, and hydroxyl radical (OH-) and other molecules by various enzymatic metabolism systems.• OH- and O2 - have the shortest life spans.• OH- has the highest reactivity and it reacts with metal ions and nitric oxide (NO) to quench the physiological activities such as vascular relaxation.• Meanwhile, O2 - generates peroxinitrite (ONOO) that causes oxidative damage, and it is transformed into OH- to react indirectly with lipids, proteins and nucleotides. 35
  36. 36. ROS production• Generation of ROS by enzymes in phagocytic cells, like neutrophils and macrophages, which kill some types of bacteria. For example : - xanthine oxdiase - aldehyde oxidase - flavin dehydrogenases ROS arise normally during metabolism and all living organisms can handle them under some circumstances. However, if the ROS production becomes excessive, damage can occur. 36
  37. 37. ROS production• Various environmental factors cause increse in ROS : -intense light, -drought -temperature stress• Plants produce ROS via oxidase enzymes when attacked by pathogens (the oxidative burst).• Plasma membrane localised NADP oxidase (NOX) produces ROS .• Other sources of ROS belong to pathways enhanced during abiotic stresses , such as glycolate oxidases amino oxidases cell-cell-bound peroxidases, 37
  38. 38. Values of production of ROS• The production of ROS in chloroplasts of cells• Under normal growth conditions, a. 240 μΜ O2- b. 0.5 μΜ H2O2• under environmental stress conditions, the cellular homeostasis of cells can be disrupted in response to an elevated production of ROS a. 240-720 μΜ O2- b. 5-15 μΜ H2O2• ROS can be considered as cellular indicators of stresses and as secondary messengers involved in the stress-response signal transduction pathways 38
  39. 39. ROS-scavenging System (Defense Mechanism of ROS)• Both the mitochondrion and the chloroplast contain ROS- scavenging mechanisms.• The protective system of enzymes such as SOD, APX and CAT is a major ROS-scavenging mechanism of plants• SOD acts as first line of defense to catalyse the dismutation of superoxide radicals into molecular oxygen and hydrogen peroxide. Then, APX and CAT remove H2O2 very efficiently. 39
  40. 40. • SODs and CATs are most important, efficient antioxidant enzymes in cells. Their combined action converts the potentially dangerous O2- and H2O2 to water and molecular oxygen, thus averting cellular damage.• The oxidative stresses cause the proliferation of peroxisomes, which might be highly efficient in scavenging of ROS, especially H2O2• Antioxidants such as ascorbic acid and glutathione are crucial for plant defense against oxidative stresses 40
  41. 41. SOD and stress tolerance• Plants experience stresses 1. shade or high light levels 2. sub-zero, low or high temperatures, 3. drought, 4. flooding, 5. high salinity, 6. inorganic nutrient imbalance, 7. infection, 8. predation, 9. natural or man-made toxic compounds 41
  42. 42. SOD and stress tolerance• Stress responses are elicited through several pathways and that these pathways are cross-wired• At least four signal transduction chains exist in plants for responding to drought, salinity and low temperature• An abscisic acid (ABA)-dependent pathway responds to drought and salinity signals. This pathway is itself complex, because some ABA-inducible stress responses depend on protein synthesis, but others utilize existing components of the signaling transduction chain 42
  43. 43. SOD and stress tolerancePhotoinhibition (light-induced damage to PSII)• in which the absorbed light energy exceeds the capacity of the photosystems to direct it through photosynthetic electron transport.• The high light intensities lead to a reduction of photosynthetic capability of plants.• SODs reduce the effect of photoinhibition 43
  44. 44. SOD and stress tolerance• Waterlogging and Drought• Waterlogging imposes an oxygen shortage on submerged plant organs.• Increasing ABA(abscisis acid) stimulates stomatal guard cells to close, reducing water loss and creating ROS 44
  45. 45. SOD and stress tolerance• Herbicides• Paraquat(viologen) and other herbicides that directly affect chloroplast activity can stimulate processes that induce ROS and block photosynthetic electron transport• Paraquat is a redox-active compound that is photoreduced by PSI and subsequently reoxidised by transfer of its electrons to oxygen to form ROS• Studies have shown that SOD’s amount increase during the action of herbicides. - In Nicotiana plumbaginifolia, chloroplastic, cytosolic, and mitochondrial SOD expression was anyalysed at mRNA level, all three were strongly induced by paraquat 45
  46. 46. SOD and stress tolerance• Temperature• low temperature affect uptake and conductance of water• lead to changes in stomatal opening• changes in the concentration of leaf-internal carbon dioxide, consecutively affecting the carbon reduction cycle, light reactions, energy charge, and proton pumping• consumption of reducing power become favoured, and development and growth may become altered• as a result ROS are produced which lead to increase in SOD’s .• SOD’s play role avoiding temprature stress 46
  47. 47. Senecence (aging)• Senescence or biological aging is the change in the biology of an organism as it ages after its maturity.• Oxidative stresses can cause plant aging before normal senescence occurred 47
  48. 48. Role In humans• Cancer due to excession in amount of ROS• Cardiovascular diseases• Amyotrophic lateral sclerosis (ALS, a form of motor neuron disease).• Overexpression of SOD1 has been linked to the neural disorders seen in Downs syndrome.• In recent years it has become more apparent that in mice the extracellular superoxide dismutase (SOD3, ecSOD) is critical in the development of hypertension.• In other studies, diminished SOD3 activity was linked to lung diseases such as Acute Respiratory Distress Syndrome (ARDS) or Chronic obstructive pulmonary disease (COPD). 48
  49. 49. superoxide dismutase and cardiovascular disease• Oxidative stress due to ROS• Major source of reactive oxygen species in vascular tissues is a membrane-bound NADPH oxidase• Reactive oxygen species cause the oxidation of lipids in particular low-density lipoprotein (LDL) , a process that is central to atherosclerotic lesion formation 49
  50. 50. superoxide dismutase and cardiovascular disease• Nitric oxide (NO) dilates blood vessels and inhibits platelet aggregation• Nitric oxide is released by NO synthase (NOS)• Nitric oxide reacts with the O2- and produce nitrite nitrate and peroxynitrite anion(ONOO-)• The reaction rate is three times faster then the O2- and the SOD’s and 10000 times faster then the reaction of O2- with normal antioxidant enzymes (vit A, E, C) 50
  51. 51. superoxide dismutase and cardiovascular disease• At physiological pH, peroxynitrite is protonated to form peroxynitrous acid, which can yield nitrogen dioxide and a hydroxyl-like radical• NO2 and (ONOO-) lead to lipid peroxidation and membrane damage• SOD prevents the formation of peroxynitrite 51
  52. 52. SOD’s and Downs syndrome• Additional copy of chromosome 21• Overexpression of genes coded for on this chromosome• Studies have shown increased levels of SOD’s in the Down syndrome patient• Overexpresion of SOD causes more H2O2 production• Superoxide dismutase converts the superoxide radical O2- to hydrogen peroxide, which may then form the particularly toxic hydroxyl radical OH- in the presence of metal ions via the Fenton reaction• Free radicals can cause lipoperoxidation of cell membranes (damage to fats), (damage to proteins & DNA) 52
  53. 53. SOD as a pharmaceutical product• by scavenging of free oxygen radicals SOD might interrupt inflammatory cascades and thereby limit further disease progression• SOD’s were used in the treatment of - systemic inflammatory diseases - skin ulcer lesions - arthritic inflammation - hair growth - radiation-induced fibrosis in cancer - hepatitis C related fibrosis - myocardial ischemia - Peyronies Disease - Diabetic retinopathy - to treat reperfusion injury 53
  54. 54. SOD as a pharmaceutical product• SOD cream was concluded to be effective in severe burns• “Orgotein” is the pharmaceutical form of SOD and is a potent anti-inflammatory agent• Supplements Of SOD (Super GliSODin) 54
  55. 55. SOD from FOOD• Take SOD from FOOD TIP to stay stess free from ROS ;) Ensure adequate intake of the• Antioxidants in plant materials can antioxidant-type nutrients protect from heart disease and That is take 5-8 servings of fruit and cancer as they can induce O2 – vegetables per day quenching activities, similar to those of SOD. 55
  56. 56. Cosmetic and skin uses• SOD is used in cosmetic products to reduce free radical damage to skin,for example to reduce fibrosis following radiation for breast cancer• Superoxide Dismutase found in our skin generate adequate amounts of skin-building cells called fibroblasts.• if superoxide dismutase is made into a lotion and applied to the skin, -prevent the formation of wrinkles. -heal wounds, -reduce the appearance of scars, -anti aging -lighten skin pigmentation caused by UV rays. 56
  57. 57. SOD and Relevant Research Fields• In plants - diseases - cure• In humans and animals - diseases - cure• In food - nutireints in diet• SOD as a pharmaceutical product• Risk of ROS-related diseases is decreased by reinforcement of the defense mechanism against oxidative stress.• Many researchers are interested in the anti-oxidation qualities of plants and their products, for example, red wine and tea. 57
  58. 58. SOD as future gene therapy• Gene therapy : insertion of genes into an individuals cells and tissues to treat disease, such as cancer where deleterious mutant alleles are replaced with functional ones• germ line gene therapy (sperms or eggs, are modified)• somatic gene therapy ( somatic cells )• Viral approach for gene therapy (use of retrovirus & others)• non-viral approach involves the creation of an artificial lipid sphere with an aqueous core (the liposome). -This liposome, which carries the therapeutic DNA, is capable of passing the DNA through the target cells membrane. 58
  59. 59. SOD as future gene therapy 59
  60. 60. SOD as future gene therapy• Successful reduction and protection against irradiation therapy side effects such as cystitis was reported using manganese superoxide dismutase gene therapy.• Induction of manganese superoxide dismutase by gene therapy using plasmid/liposome complex (MnSOD-PL) contained the complete human manganese SOD (MnSOD) transgene did not prevent disruption of barrier function by irradiation• but led to rapid regeneration of the urothelium and recovery of barrier function 60
  61. 61. Refrences• Microbial superoxide dismutase enzyme as therapeutic agent and future gene therapy Hatem M. El Shafey1*, Saleh A. Bahashwan2, Abdulaziz A. Alghaithy3 and Samah Ghanem3,4• Occurrence and Charactrisation of Superoxide Dismutases in the Female Reproductive Structures of Petunia A thesis by YeYing Wang University of Canterbury 2006• E xtracellular superoxide dismutase and cardiovascular disease Tohru Fukaia ,*, Rodney J. Folzb, Ulf Landmessera, David G. Harrisona aDepartment of Medicine, Division of Cardiology, Emory University, 1639 Pierce Drive, WMB 319, Atlanta, GA 30322, USA bDepartment of Medicine, Division of Pulmonary and Critical Care Medicine, Duke University Medical Center, Durham, NC 27710, USA Received 7 November 2001; accepted 17 January 2002• Plant stress adaptations — making metabolism move Hans J Bohnert* and Elena Sheveleva Department of Biochemistry and Department of Plant Sciences, The University of Arizona• Oxidative Stress and Acclimation Mechanisms in Plants Ruth Grene Department of Plant Pathology, Physiology, and Weed Science, 435 Old Glade Road, Virginia Tech, Blacksburg, VA 24061-0330; email: grene@vt.edu• FUNCTIONAL AND STRUCTURAL MIMICS OF SUPEROXIDE DISMUTASE ENZYMES chapter 10 István Pálinkó* Department of Organic Chemistry, University of Szeged, Dóm tér 8, Szeged, H-6720 Hungary 61

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