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Teresa Pereira CMB, Karolinska Institutet 2010-06-15

Reactive oxygen species and the Hypoxia-inducible Factor signaling pathway. Teresa Pereira CMB, Karolinska Institutet 2010-06-15. Normoxia - O 2 available is in balance with the demand (21% O 2 in the lab) Hypoxia - unbalance between oxygen supply

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Teresa Pereira CMB, Karolinska Institutet 2010-06-15

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  1. Reactive oxygen species and the Hypoxia-inducible Factor signaling pathway Teresa Pereira CMB, KarolinskaInstitutet 2010-06-15

  2. Normoxia - O2 available is in balance with the demand (21% O2 in the lab) Hypoxia - unbalance between oxygen supply and demand (1% O2 in the lab)

  3. Heart and Lungs Brain Cornea Cartilage Physiological oxygen levels Avascular Tissues 21%O2 5%O2 14%O2

  4. Adaptive responses to hypoxia Glucose and energy metabolism GLUT1,3 Hypoxia Red blood cell production Cell migration E-cadherin Erythropoietin Formation and dynamic regulation of bloodvessels Autophagy BNIP3 VEGF-A pH regulation CA9

  5. Hypoxia and pathology Defective vascularisation leading to low pO2 is a characteristic of a number of diseases - local hypoxia Anemia-systemic hypoxia

  6. Hypoxia and tumor development Carmeliet, P. 2005, Oncology, 69

  7. HIF-1a Hypoxia-inducible factor-1a HIF-1a protein stability is regulated by oxygen levels HIF-1a A B N C bHLH A B bHLH HIF-1b/Arnt

  8. Von Hippel-Lindau Tumor Suppressor Gene Hershko, Cell Death Differ., 2005 Mutated in VHL disease- hereditary cancer syndrome: retinal and CNS hemangioblastomas renal cell carcinomas and pheochromocytomas. Mutated in sporadic renal cell carcinomas and hemangioblastomas. Hypervascularized tumors. Constitutive expression of VEGF in VHL inactivated cells.

  9. Degradation of HIF-1a by pVHL is associated with the tumor supressor function of pVHL Tanimoto et al., EMBO J, 2000,

  10. B Degradation A PHDs Degradation of HIF-1a is regulated by two specific proline residues Normoxia VHL OH OH P P N C bHLH 772 822 331 531 584 1 91 402 563 Superfamily of iron II and 2-oxoglutarate dependent oxygenases. NCB 2007, 3, 144-153

  11. B Degradation A PHDs OH N Hydroxylation of an asparagine residue in HIF-1a inhibits interaction with CBP at normoxia Normoxia FIH VHL OH OH P P N C bHLH 772 822 331 531 584 1 91 402 563 IronII and 2-oxoglutarate dependent oxygenase

  12. O2 PHDs Fe2+ 2-oxoglutarate OH OH P P HIF-a HIF-a OH OH P P VHL VHL Proteasome Oxygen-dependent Regulation of HIF-a Expression HIF-a

  13. O2 PHDs Fe2+ 2-oxoglutarate VHL VHL Oxygen-dependent Regulation of HIF-a Expression HIF-a HIF-a ARNT Coactivators HRE

  14. O2 O2 PHDs 2-oxoglutarate Fe2+ FIH-1 Fe2+ 2-oxoglutarate OH OH OH OH OH P P P HIF-a HIF-a HIF-a OH OH OH N N P P P VHL VHL HIF-a ARNT Proteasome HIF-a ARNT Oxygen-dependent Regulation of HIF-a Activity HIF-a HIF-a Coactivators HRE

  15. Prolylhydroxylase activity decreases progressively with reduction of O2 levels HIF stabilization begins at 5% O2 and increases exponentially up to 0.5% O2 Km- 100 mM JBC, 2006, 281, 28712-20

  16. Increase in ROS production at hypoxia is paradoxical: concentration of O2 decrease at hypoxia O2 is a substrate for ROS production Biochem. J. 2007, 405, 1-9

  17. Generation of ROS in response to hypoxia DCFH- 2’-7’-dichlorofluorescein oxidized by H2O2 but not O2.– Ebselen- glutathione peroxidase mimetic PDTC- thiol reductive agent pyrrolidinedithiocarbamate r0 -cells lacking mitochondrial DNA-derived proteins PNAS, 1998, 95, 11715-720

  18. Assessment of cytosolic ROS using a FRET sensor CFP-69 aacysteine-containing from the redox-regulated HSP-33- YFP Oxidation of cysteinethiols causes separation of the CFP and YFP – increase in CFP intensity and decrease in YFP intensity ratiometric Allows measuring cytosolicthiolredox In live cells wikipedia

  19. Assessment of cytosolic ROS using a FRET sensor Cell Metab, 2005, 1, 401-408

  20. Assessment of the effect of hypoxia on redox signaling using a redox-sensitive ratiometric fluorescent protein sensor RoGFP Cyto-RoGFP GFP with two engineered cysteinethiols excitation maxima- 400 nm oxidized - 484 nm reduced Circ Res, 2010, 106, 526-535

  21. Measuring ROS in hypoxia using RoGFP mitochondrial intermembrane space mitochondrial matrix Circ Res, 2010, 106, 526-535

  22. Stabilization of HIF-1a in response to hypoxia is dependent on ROS DFO-deferoxamine iron quelator JBC, 2000, 275, 25130-38

  23. Electron transport chain Cell Death Differ, 2008, 15, 660-666

  24. Role of complex III on HIF-a stabilization Rotenone-complex I inhibitor Myxothiazol- complex III inhibitor Stigmatelin- complex III inhibitor Cell Metab, 2005, 1, 393-399 Cell Metab, 2005, 1, 401-408

  25. Generation of ROS by complex III Qo Qi Partial pressure of oxygen is reduced – mitochondrial electron transfer from ubiquinol to cyt c1 by the Reiske iron-sulfur protein is delayed allowing electrons to bind to molecular oxygen forming O2- Exp Physiol 2006, 91, 807-819

  26. HIF-a stabilization at hypoxia is dependent on Cyt C Cell Metab, 2005, 1, 393-399

  27. HIF-a stabilization in hypoxia is dependent on Rieske iron-sulfur protein of complex III Cell Metab, 2005, 1, 401-408

  28. How is prolylhydroxylase activity affected by ROS? -ROS may trigger signal transduction cascate -change PHDsdisulfite bond -oxydize enzyme-bound iron g=6 hemoproteins g=4.3 free iron g= 2.24, 2.01 and 1.93 dioxygenases Measuring FeII and FeIII by EPR spectroscopy Cell, 2004, 118, 781-794

  29. Why do cancer cells use glycolysis instead of oxidative phophorylation to produce ATP? Warburg effect 2 ATP versus 38 ATPs cytochromeoxidase activity is only limited by O2 availability when O2 is lower than 1 mM (0.1% O2) support cell growth- pyruvate used in lipid synthesis for membrane assembly Clin Cancer Res 2007, 13, 789-794

  30. Hypoxia and tumor development

  31. Pyruvatedehydrogenasekinase 1 is a HIF-1 target gene Pyruvate acetyl-CoA PDH PDK1 Cell Metab 2006, 3, 177-185

  32. HIF-1-induced PDK1 activity inhibits PDH resulting in decreased flux through the TCA cycle Cell Metab 2006, 3, 177-185

  33. Effect of PDK1 on hypoxia-induced ROS production 72h hypoxia DCF fluorescence Cell Metab 2006, 3, 177-185

  34. Cellular adaptation to hypoxia FEBS letters, 581, 3582-3591, 2007 Curr. Opinion Cell Biol. 19, 223-9, 2007

  35. Cytochrome C oxidase subunit composition is regulated by O2 in yeast and human cells To maintain the efficiency of respiration under conditions of low O2 COX5a high levels of O2 COX5b low levels of O2 COX5b increases rate of electron transfer COX4-1 expression increases production of ROS at hypoxia siRNA COX4-2 leads to increases levels of ROS at hypoxia Biochem. J. 2007, 405, 1-9

  36. Regulation of ROS production by the Hypoxia-inducible pathway -induction of glycolytic enzymes and LDH -induction of PDK1 –reducing flux through the TCA cycle -induction of COX4-2 and inhibition of COX4-1 –efficient respiration -inhibition of genes involved in mitochondria biogenesis (PGC1a)

  37. Role of HIF-1a in the acute phase of ischaemic preconditioning: production of ROS Cardiov Res 2008, 77, 463-470

  38. Thank you for your attention naked mole rat subterranean mole rat Model to cancer research

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