teresa pereira cmb, karolinska institutet 2010-06-15 reactive oxygen species and the...
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Teresa PereiraCMB, Karolinska Institutet
2010-06-15
Reactive oxygen species and theHypoxia-inducible Factor
signaling pathway
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)
Heart and Lungs Brain
Cornea
Cartilage
Physiological oxygen levels
Avascular Tissues
5%O2 14%O2
21%O2
Hypoxia
Adaptive responses to hypoxia
Red blood cell production
Formation and dynamicregulation of blood vessels
Glucose andenergy metabolism
Autophagy
Cell migration
pH regulation
Erythropoietin
VEGF-A
GLUT1,3
CA9
BNIP3
E-cadherin
Hypoxia and pathology
Defective vascularisation leading to lowpO2 is a characteristic of a number of diseases - local hypoxia
Anemia-systemic hypoxia
Hypoxia and tumor development
Carmeliet, P. 2005, Oncology, 69
Hypoxia-inducible factor-1aHIF-1a protein stability is regulated by oxygen levels
HIF-1a
HIF-1aN CA BbHLH
A BbHLH
HIF-1b/Arnt
Von Hippel-Lindau Tumor Suppressor Gene
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.
Hershko, Cell Death Differ., 2005
Degradation of HIF-1a by pVHL is associated with the tumor supressor function of pVHL
Tanimoto et al., EMBO J, 2000,
Degradation of HIF-1a is regulated by two specific proline residues
Normoxia
Superfamily of iron II and 2-oxoglutaratedependent oxygenases.
1 822772584531
N C
PP
VHL
A BbHLH
Degradation
33191
OH OH
402 563
PHDs
NCB 2007, 3, 144-153
Normoxia
Iron II and 2-oxoglutaratedependent oxygenase
1 822772584531
N C
PP
VHL
A BbHLH
Degradation
33191
OH OH
402 563
PHDs
N
OH
FIH
Hydroxylation of an asparagine residue in HIF-1a inhibits interaction with CBP at normoxia
HIF-a
O2 PHDs
Fe2+2-oxoglutarate
HIF-a P
P
OH
OH
VHL
VHL
HIF-a P
P
OH
OH
Proteasome
Oxygen-dependent Regulation of HIF- a Expression
HIF-a
O2 PHDs
Fe2+2-oxoglutarate
VHL
VHL
HRE
HIF-a
ARNT
Oxygen-dependent Regulation of HIF- a Expression
Coactivators
HIF-a
O2 PHDs
Fe2+2-oxoglutarate
VHL
VHL
HRE
HIF-a
Oxygen-dependent Regulation of HIF- a Activity
HIF-a P
P
OH
OH
HIF-a P
P
OH
OH
HIF-a
ARNT
OH
N
HIF-a P
P
OH
OH
Proteasome
FIH-1
O2
2-oxoglutarateFe2+
Coactivators
HIF-a
ARNT
OH
N
Km- 100 mM
Prolyl hydroxylase activity decreases progressively withreduction of O2 levels HIF stabilization begins at 5% O2 and increases exponentially up to 0.5% O2
JBC, 2006, 281, 28712-20
Biochem. J. 2007, 405, 1-9
Increase in ROS production at hypoxia is paradoxical:concentration of O2 decrease at hypoxia O2 is a substrate for ROS production
PNAS, 1998, 95, 11715-720
Ebselen- glutathione peroxidase mimeticPDTC- thiol reductive agent pyrrolidine dithiocarbamater0 -cells lacking mitochondrial DNA-derived proteins
DCFH- 2’-7’-dichlorofluoresceinoxidized by H2O2 but not O2
.–
Generation of ROS in response to hypoxia
wikipedia
CFP-69 aa cysteine-containing from the redox-regulated HSP-33- YFP
Oxidation of cysteine thiols causes separation of the CFP and YFP – increase in CFP intensity and decrease in YFP intensityratiometric
Allows measuring cytosolic thiol redox In live cells
Assessment of cytosolic ROS using a FRET sensor
Assessment of cytosolic ROS using a FRET sensor
Cell Metab, 2005, 1, 401-408
Circ Res, 2010, 106, 526-535
Assessment of the effect of hypoxia on redox signaling using a redox-sensitive ratiometric fluorescent
protein sensor RoGFP
GFP with two engineered cysteine thiolsexcitation maxima- 400 nm oxidized - 484 nm reduced
Cyto-RoGFP
Circ Res, 2010, 106, 526-535
mitochondrialintermembrane space
mitochondrialmatrix
Measuring ROS in hypoxia using RoGFP
JBC, 2000, 275, 25130-38
DFO-deferoxamineiron quelator
Stabilization of HIF-1a in response to hypoxia isdependent on ROS
Cell Death Differ, 2008, 15, 660-666
Electron transport chain
Cell Metab, 2005, 1, 393-399Cell Metab, 2005, 1, 401-408
Rotenone-complex I inhibitorMyxothiazol- complex III inhibitorStigmatelin- complex III inhibitor
Role of complex III on HIF-a stabilization
Exp Physiol 2006, 91, 807-819
Generation of ROS by complex III
Partial pressure of oxygen is reduced – mitochondrial electron transferfrom ubiquinol to cyt c1 by the Reiske iron-sulfur protein is delayedallowing electrons to bind to molecular oxygen forming O2
-
Qo
Qi
Cell Metab, 2005, 1, 393-399
HIF-a stabilization at hypoxia is dependent on Cyt C
HIF-a stabilization in hypoxia is dependent on Rieske iron-sulfur protein of complex III
Cell Metab, 2005, 1, 401-408
Measuring FeII and FeIII by EPR spectroscopy
Cell, 2004, 118, 781-794
How is prolyl hydroxylase activity affected by ROS?
-ROS may trigger signal transduction cascate-change PHDs disulfite bond-oxydize enzyme-bound iron
g=6 hemoproteinsg=4.3 free iron
g= 2.24, 2.01 and 1.93dioxygenases
Why do cancer cells use glycolysis instead of oxidative phophorylation to produce ATP?
Warburg effect
Clin Cancer Res 2007, 13, 789-794
2 ATP versus 38 ATPs
cytochrome oxidase 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
Hypoxia and tumor development
Pyruvate dehydrogenase kinase 1 is a HIF-1 target gene
Pyruvate acetyl-CoAPDH
PDK1
Cell Metab 2006, 3, 177-185
HIF-1-induced PDK1 activity inhibits PDH resulting in decreased flux through the TCA cycle
Cell Metab 2006, 3, 177-185
72h hypoxia
Cell Metab 2006, 3, 177-185
Effect of PDK1 on hypoxia-induced ROS production
DCF fluorescence
FEBS letters, 581, 3582-3591, 2007Curr. Opinion Cell Biol. 19, 223-9, 2007
Cellular adaptation to hypoxia
Biochem. J. 2007, 405, 1-9
Cytochrome C oxidase subunit composition is regulated by O2 in yeast and human cells
COX5a high levels of O2COX5b low levels of O2
COX5b increases rate of electron transfer
COX4-1 expression increases production ofROS at hypoxia
siRNA COX4-2 leads to increases levels of ROS at hypoxia
To maintain the efficiency of respiration under conditions of low O2
Regulation of ROS production by the Hypoxia-induciblepathway
-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)
Cardiov Res 2008, 77, 463-470
Role of HIF-1 a in the acute phase of ischaemic preconditioning: production of ROS
Thank you for your attention
naked mole rat
Model to cancer research
subterranean mole rat