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MOLECULAR MECHANISMS OF ANOXIA TOLERANCE. www.carleton.ca/~kbstorey. PETER L. LUTZ. Red-eared slider Trachemys scripta elegans. Lutz PL, Storey KB. 1997. Handbook of Physiology (Dantzler WH, ed) Oxford Univ. Press, Vol. 2, pp. 1479-1522. TURTLE HYPOXIA. Winter submergence at 3°C.

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  1. MOLECULAR MECHANISMS OF ANOXIA TOLERANCE www.carleton.ca/~kbstorey

  2. PETER L. LUTZ

  3. Red-eared sliderTrachemys scripta elegans

  4. Lutz PL, Storey KB. 1997. Handbook of Physiology (Dantzler WH, ed) Oxford Univ. Press, Vol. 2, pp. 1479-1522.

  5. TURTLE HYPOXIA Winter submergence at 3°C • Metabolism reduced to 10 % of normoxia • Anoxic survival for months at 7°C • Glycogen catabolized; up to 200 mM lactate accumulated • Shell dissolves to buffer acid load and lactate stored in shell Herbert & Jackson (1985) Physiol Zool 58:655

  6. GENES Control by transcriptional regulation Transcription RNAs Control by translational regulation Translation No Modification Control by proteases PROTEINS (ENZYMES) INACTIVE ENZYME Degradation Control by post- translational modification Covalent Modification FUNCTIONAL ENZYMES Inhibition and Activation Control at level of enzyme function ACTIVE ENZYMES

  7. METABOLISM IN ANOXIA • mRNA synthesis • Protein synthesis • Ion Pumping • Fuel use • O2 consumed ATP turnover to <5% of normal

  8. PRINCIPLES OF ANOXIA SURVIVAL 1. Metabolic rate reduction 2. Control by protein kinases(SAPKs, 2nd messenger PKs) 3. Selective gene activation

  9. Ca+2 GENES [ i + e Factors] Nucleus mRNAs GENES ON/OFF CHO PROTEINS [Trans.F] Na ATP K PATHWAYS AA SAPK P PROT ? SMW FAT ADP ATP KINASES (2nd) MITO ETC

  10. AONXIA INDUCED CHANGES • Protein Synthesis slows to 1% • Pumps & Channels closed • Energy Production slows to 5% • Energy Utilization slows to 2% • Few ‘SAP’ kinases activated • Gene ‘inactivation’ ( mRNA ) • Few Genes activated (1-2%)

  11. PROTEIN PROTEIN-(P)n nATP nADP PROTEIN KINASES • Covalent modification by phosphorylation • Families of protein kinases: PKA (cAMP), PKG (cGMP), CaM (Ca2+), PKC (Ca2+, PL,DG) • SAPKs : daisy chain phosphorylations • Regulation is via interconversion of active vs subactive forms of protein substrates

  12. Protein Kinase ATP ADP P P P P Protein Phosphatase Pi Reversiblephosphorylation control of enzymes P & deP enzymesseparate on ionexchange columns

  13. Storey, K.B. 1996. Metabolic adaptations supporting anoxia tolerance in reptiles: recent advances. Comp. Biochem. Physiol. B 113, 23-35. PROTEIN PHOSPHORYLATION& GLYCOLYSIS • Protein kinase A, PKG • Protein kinase C (Brain) • Protein phosphatase 1, 2A, 2C

  14. Anoxia Hibernation Estivation Freezing Diapause METABOLIC RATE DEPRESSION

  15. ANOXIA INDUCED CHANGES • Protein Synthesis slows to 1% • Pumps & channels closed • Energy Production slows to 5% • Energy Utilization slows to 2% • Few ‘SAP’ kinases activated • Gene ‘inactivation’ ( mRNA ) • Few Genes activated (1-2%)

  16. ROLE OF TRANSCRIPTION Global rate of mRNA synthesis depressed. Method: nuclear run-on Are selected genes up-regulated ? TO ASSESS GENE UPREGULATION: What new mRNAs are created? - cDNA library - Gene Chip

  17. cDNA Arrays- Methods • Materials • Sources- Publications

  18. GENE CHANGES IN TURTLE ANOXIA • cDNA Library & Chip (~2% putative up-regulated) • -Transcription Factors • - Mitochondrial Genes- Protease inhibitors - Shock proteins (Hsps) - Antioxidant enzymes - Ferritin H & L

  19. ANTIOXIDANT DEFENSE • Iron storage: - Ferritin (H & L chains) - Transferrin receptor 2 • Antioxidant enzymes - SOD (1) - GST (M5, A2) - GPX (1, 4) - Peroxiredoxin 1 C. picta hatchlingsliver & heart Storey KB. 2005. Gene hunting in hypoxia and exercise. In: R.C. Roach et al., eds. Hypoxia and Exercise, Springer, NY

  20. The Bad The Good 1) Reactive oxygen species (ROS) damage macromolecules, deplete GSH, vitamins 2) ROS produced by normal aerobic metabolism & must be destroyed 3) Heavy metals catalyze formation of particularly dangerous ROS 4) Associated with disease & ageing 1) Fuels normal aerobic metabolism 2) More than 200 enzymes use O2 3) Eliminates toxins (xenobiotics) via cytochrome P450 4) Produce O2 via photosynthesis The Good And The Bad Of Oxygen

  21. e- e-, 2H+ e-, metals O2 Oxygen O2 Superoxide H2O2 Hydrogen Peroxide OH Hydroxyl Radical Reactive Oxygen Species: The Bad Guys Superoxide - forms when O2 acquires a single electron - relatively short-lived Hydrogen Peroxide - formed from superoxide - not a radical, is long-lived - passes readily through membranes Hydroxyl Radical - formed from H2O2 ( with Fe2+ or Cu+ ) - HIGHLY REACTIVE - very short-lived

  22. GENE CHANGES IN TURTLE ANOXIA • cDNA Library & Chip (~2% putative up-regulated) • -Transcription Factors • - Mitochondrial Genes- Protease inhibitors - Shock proteins (Hsps) - Antioxidant enzymes - Ferritin H & L

  23. CONTROL REGION OF A TYPICAL EUKARYOTIC GENE

  24. Changes in Gene Expression and Regulation We Study: • Transcriptional regulation • Changes in mRNA levels • Translational regulation • Changes in protein levels • Post-translational regulation • Changes in post-translational modifications • Changes in subcellular distribution

  25. Transcription Factors • Master regulators of gene expression • Respond to intra- or extracellular signals • Bind to promoter regions of specific genes • Mediate DNA transcription to mRNA

  26. Nuclear Factor kappa B (NF-kB) • Dimeric transcription factor, composed of subunits including p65, p50, p52, c-Rel and Rel B • Activated by: Stress ,Cytokines Free radicals, UV • Functions : • - Immune response, Development • - Cell growth, Apoptosis, Stress response

  27. Target genes NF-kB CONTROL: NF-kB dimer is sub-active in cytoplasm, bound to IkB STRESS:IkB phosphorylated & degraded • Free NF-kB dimer:- moves to nucleus - binds to DNA - transcription of downstream genes

  28. IkBPhosphorylation P-IkB protein levels in turtle tissues Relative protein levels Time course for IkB phosphorylation in liver Relative protein levels • Elevated (P) of IkB frees NF-kB dimer to move into the nucleus IkB is (P) in liver & brain at 5h of anoxia

  29. Turtles: NF-kB Protein Levels NF-kB dimer protein levels Relative protein levels NF-kB p65 and p50 are upregulated during 5 h of anoxia p65 p50

  30. P65: Subcellular distribution NF-kB Relative protein levels P65: DNA-binding activity NF-kB pathway is activated in turtle liver and brain in anoxia ! Relative DNA-binding activity P65 moves into nucleus in anoxia DNA-binding activity of p65 elevated after 5 & 20 h of anoxia

  31. Relative mRNA levels ofNF-kB target genes Relative mRNA levels Ferritin H HO-1 NF-kB: Target Gene Levels Ferritin heavy chain and Heme oxygenase-1 (HO-1) are upregulated after 5 h of anoxia

  32. Ferritin heavy chain • Sequesters iron • Can hold up to 4,500 atoms of iron • 24 subunits: light (19 kDa) and heavy (21 kDa) • Limits iron-catalyzed ROS production via the Fenton reaction 70’s radicals

  33. Fe2+ Ferritin and Heme Oxygenase -1 • Help minimize free iron levels in cells • Ferritin: Binds iron; Heavy & Light chains • Heme oxygenase -1: • - Degrades heme, a source of redox active iron • - Free iron then stored into ferritin • Iron can be a source of oxidative stress: • Catalyzes production of Hydroxyl radicals via Fenton reaction: H2O2 •OH + OH- Hydroxyl radical is very reactive and responsible for most oxidative stress-mediated damage

  34. GENE PROTEIN INTEGRATION BIOCHEMISTRY PHYSIOLOGY THE BRAINS OF THE OPERATION PETER LUTZ TERRRTLE

  35. Hypoxia / Ischemia • Sensitive Animals (most mammals) - Energy deficit (high ATP demand) - Disruption of ions and depolarization - Release of excitotoxic GLU, - Excess intracellular Ca2+ - Oxidative Stress (+ reperfusion ) - Cell Death • Tolerant Animals (e.g. turtles, carp ) - Decrease ATP demand (Metabolic Arrest) - Adenosine as a Retaliatory Molecule

  36. Hypoxic Cascade

  37. MAMMAL TURTLE CARP 30 min 12 h 12 h TIME IN ANOXIA

  38. ANOXIA SURVIVAL IN TURTLE BRAIN Lutz PL & Milton SL. 2004. J Exp Biol 207: 3141-3147

  39. ANOXIA SURVIVAL IN TURTLE BRAIN Lutz PL & Milton SL. 2004. J Exp Biol 207: 3141-3147

  40. ANOXIA SURVIVAL IN TURTLE BRAIN Lutz PL & Milton SL. 2004. J Exp Biol 207: 3141-3147

  41. BRAIN GENES Up-regulated in turtle anoxia(DNA array) • GABA transporter • GABA receptor Adult T. s. elegans

  42. ANOXIA SURVIVAL IN TURTLE BRAIN Lutz PL & Milton SL. 2004. J Exp Biol 207: 3141-3147

  43. BRAIN GENES Up-regulated in turtle anoxia(DNA array) • Adenosine receptor • 5’Nucleotidase Adult T. s. elegans

  44. BRAIN GENES • GABA transporter • GABA receptor • Adenosine receptor • 5’Nucleotidase • Serotonin receptor

  45. GENES Control by transcriptional regulation Transcription RNAs Control by translational regulation Translation No Modification Control by proteases PROTEINS (ENZYMES) INACTIVE ENZYME Degradation Control by post- translational modification Covalent Modification FUNCTIONAL ENZYMES Inhibition and Activation Control at level of enzyme function ACTIVE ENZYMES

  46. J. STOREY S. BROOKS Q. CAI W. WILLMORE H. MEHRANI D. DOUGLAS J. DUNCAN S. GREENWAY A. KRIVORUCHKO M. HERMES-LIMA T. ENGLISH K. LARADE E. RUSSELL T. PANNUNZIO R. WHITWAM S. KORYCAN B. MICHAELIDIS J. ZHOU ANOXIA www.carleton.ca/~kbstorey

  47. Ca+2 GENES [ i + e Factors] Nucleus mRNAs GENES ON/OFF CHO PROTEINS [Trans.F] Na ATP K PATHWAYS AA SAPK P PROT ? SMW FAT ADP ATP KINASES (2nd) MITO ETC

  48. cDNA ARRAY SCREENING AEROBIC ANOXIC

  49. ANTIOXIDANT ENZYMES Willmore WG & Storey KB - FEBS J 272, 3602-14 (2005) - Am J Physiol 273, R219-25 (1997). - Mol Cell Biochem 170, 177-185 (1997)

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