www.carleton.ca/~kbstorey. peterl.lutz red-eared slider trachemys scripta elegans
TRANSCRIPT
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www.carleton.ca/~kbstoreywww.carleton.ca/~kbstorey
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PETERPETERL.L.
LUTZLUTZ
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Red-eared sliderTrachemys scripta elegans
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Lutz PL, Storey KB. 1997. Handbook of Physiology(Dantzler WH, ed) Oxford Univ. Press, Vol. 2, pp. 1479-1522.
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TURTLE HYPOXIATURTLE HYPOXIA
• Metabolism reduced Metabolism reduced to 10 % of normoxia to 10 % of normoxia
• Anoxic survival for Anoxic survival for months at 7°C months at 7°C
• Glycogen catabolized; Glycogen catabolized; up to 200 mM lactate up to 200 mM lactate accumulatedaccumulated
• Shell dissolves to Shell dissolves to buffer acid load andbuffer acid load and lactate stored in shell
Herbert & Jackson (1985) Physiol Zool 58:655
Winter submergence at 3°C
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GENESGENES
Transcription
RNAsRNAs
Control bytranscriptional regulation
Control bytranslational regulation
Translation
PROTEINSPROTEINS(ENZYMES)(ENZYMES)
Control byproteases INACTIVEINACTIVE
ENZYMEENZYME
NoModification
FUNCTIONALFUNCTIONALENZYMESENZYMES
CovalentModification
Degradation
Control by post-translationalmodification
ACTIVEACTIVEENZYMESENZYMES
Inhibitionand
Activation
Control at level ofenzyme function
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METABOLISM IN METABOLISM IN ANOXIA ANOXIA
• mRNA synthesis mRNA synthesis • Protein synthesis Protein synthesis • Ion Pumping Ion Pumping • Fuel use Fuel use • OO22 consumed consumed
ATP turnover to <5% of normalATP turnover to <5% of normal
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PRINCIPLES OF PRINCIPLES OF ANOXIA SURVIVALANOXIA SURVIVAL1. Metabolic rate reduction 1. Metabolic rate reduction
2. Control by protein kinases2. Control by protein kinases(SAPKs, 2(SAPKs, 2nd nd messenger PKs)messenger PKs)
3. Selective gene activation3. Selective gene activation
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Nucleus
GENESON/OFF
mRNAs
[ i + e Factors]
PROTEINS
Ca+2
KINASES (2nd)
PATHWAYS
SMW
CHO
AA
ATP
?SAPK
ATP
ADP
MITOGENES
FAT
[Trans.F] Na
K
ETC
P PROT
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AONXIA INDUCED AONXIA INDUCED CHANGESCHANGES
• Protein Synthesis slows to 1% Protein Synthesis slows to 1% • Pumps & Channels closed Pumps & Channels closed • Energy Production slows to 5% Energy Production slows to 5% • Energy Utilization slows to 2%Energy Utilization slows to 2%• Few ‘SAP’ kinases activatedFew ‘SAP’ kinases activated
• Gene ‘inactivation’ ( mRNA )Gene ‘inactivation’ ( mRNA )• Few Genes activated (1-2%)Few Genes activated (1-2%)
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PROTEIN KINASESPROTEIN KINASES
PROTEIN
nATP nADP
PROTEIN-(P)n
• Covalent modification by phosphorylationCovalent modification by phosphorylation
• Families of protein kinases: PKA (cAMP),Families of protein kinases: PKA (cAMP), PKG (cGMP), CaM (Ca PKG (cGMP), CaM (Ca2+2+), PKC (Ca), PKC (Ca2+2+, PL,DG), PL,DG)
• SAPKs : daisy chain phosphorylations SAPKs : daisy chain phosphorylations
• Regulation is via interconversion of activeRegulation is via interconversion of active vs subactive forms of protein substrates vs subactive forms of protein substrates
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PP
PP PP
PP
PiPiProtein
Phosphatase
ATPATP ADPADPProtein Kinase
P & deP enzymesseparate on ionexchange columns
ReversibleReversiblephosphorylationphosphorylation
control of enzymescontrol of enzymes
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PROTEIN PHOSPHORYLATIONPROTEIN PHOSPHORYLATION& GLYCOLYSIS& GLYCOLYSIS
• Protein kinase A, PKGProtein kinase A, PKG
• Protein kinase C (Brain)Protein kinase C (Brain)
• Protein phosphatase 1, 2A, 2CProtein phosphatase 1, 2A, 2C
Storey, K.B. 1996. Metabolic adaptations supporting anoxia tolerance in reptiles: recent advances. Comp. Biochem. Physiol. B 113, 23-35.
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Estivation
METABOLIC RATE METABOLIC RATE DEPRESSIONDEPRESSION
Diapause
Freezing
Anoxia
Hibernation
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ANOXIA INDUCED ANOXIA INDUCED CHANGESCHANGES
• Protein Synthesis slows to 1% Protein Synthesis slows to 1% • Pumps & channels closed Pumps & channels closed • Energy Production slows to 5% Energy Production slows to 5% • Energy Utilization slows to 2%Energy Utilization slows to 2%• Few ‘SAP’ kinases activatedFew ‘SAP’ kinases activated
• Gene ‘inactivation’ ( mRNA )Gene ‘inactivation’ ( mRNA )• Few Genes activated (1-2%)Few Genes activated (1-2%)
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ROLE OF ROLE OF TRANSCRIPTIONTRANSCRIPTION
• Global rate of mRNA synthesis depressed. Global rate of mRNA synthesis depressed. Method: nuclear run-onMethod: nuclear run-on
• Are selected genes up-regulated ?Are selected genes up-regulated ?
• TO ASSESS GENE UPREGULATION:TO ASSESS GENE UPREGULATION:
What new mRNAs are created?What new mRNAs are created? - cDNA library - cDNA library - Gene Chip - Gene Chip
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cDNA ArrayscDNA Arrays- Methods- Methods- MaterialsMaterials- SourcesSources- Publications- Publications
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GENE CHANGES IN GENE CHANGES IN TURTLE ANOXIATURTLE ANOXIA
• ccDNA LibraryDNA Library & Chip & Chip (~2% putative up-regulated) (~2% putative up-regulated)
-Transcription Factors-Transcription Factors
- Mitochondrial Genes- Mitochondrial Genes - - Protease inhibitors Protease inhibitors - Shock proteins (Hsps) - Shock proteins (Hsps) - Antioxidant enzymes - Antioxidant enzymes - Ferritin H & L - Ferritin H & L
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ANTIOXIDANT DEFENSEANTIOXIDANT DEFENSE
• Iron storage:Iron storage: - Ferritin (H & L chains) - Ferritin (H & L chains) - Transferrin receptor 2 - Transferrin receptor 2
• Antioxidant enzymesAntioxidant enzymes - SOD (1) - SOD (1) - GST (M5, A2) - GST (M5, A2) - GPX (1, 4) - GPX (1, 4) - Peroxiredoxin 1 - Peroxiredoxin 1
C. picta C. picta hatchlingshatchlingsliver & heartliver & heart
Storey KB. 2005. Gene hunting in hypoxia and exercise. In: R.C. Roach et al., eds. Hypoxia and Exercise, Springer, NY
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The Good And The Bad Of OxygenThe Good And The Bad Of Oxygen
1) Fuels normal aerobic metabolism1) Fuels normal aerobic metabolism2) More than 200 enzymes use O2) More than 200 enzymes use O22
3) Eliminates toxins (xenobiotics) 3) Eliminates toxins (xenobiotics) via cytochrome P450via cytochrome P450
4) Produce O4) Produce O22 via photosynthesis via photosynthesis
The GoodThe Good
1) Reactive oxygen species (ROS) ) Reactive oxygen species (ROS) damage macromolecules, damage macromolecules, depletedeplete GSH, vitamins GSH, vitamins
2) ROS produced by normal aerobic 2) ROS produced by normal aerobic metabolism & must be destroyed metabolism & must be destroyed
3) Heavy metals catalyze formation of3) Heavy metals catalyze formation of particularly dangerous ROS particularly dangerous ROS
4) Associated with disease & ageing4) Associated with disease & ageing
The BadThe Bad
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Reactive Oxygen Species: Reactive Oxygen Species: The Bad GuysThe Bad Guys
SuperoxideSuperoxide - forms when O - forms when O22 acquires a single electron acquires a single electron
- relatively short-lived- relatively short-lived
Hydrogen PeroxideHydrogen Peroxide - formed from superoxide - formed from superoxide - not a radical, is long-lived- not a radical, is long-lived - passes readily through membranes - passes readily through membranes
OO22
OxygenOxygenOO22
SuperoxideSuperoxide
ee-- ee--, 2H, 2H++
HH22OO22
HydrogenHydrogenPeroxidePeroxide
OHOHHydroxylHydroxylRadical Radical
ee--, metals, metals
Hydroxyl RadicalHydroxyl Radical - formed from H - formed from H22OO22 ( with Fe ( with Fe2+ 2+ or Cuor Cu++ ) )
- - HIGHLY REACTIVEHIGHLY REACTIVE - very short- - very short-livedlived
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GENE CHANGES IN GENE CHANGES IN TURTLE ANOXIATURTLE ANOXIA
• ccDNA LibraryDNA Library & Chip & Chip (~2% putative up-regulated) (~2% putative up-regulated)
-Transcription Factors-Transcription Factors
- Mitochondrial Genes- Mitochondrial Genes - - Protease inhibitors Protease inhibitors - Shock proteins (Hsps) - Shock proteins (Hsps) - Antioxidant enzymes - Antioxidant enzymes - Ferritin H & L - Ferritin H & L
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CONTROL REGION OF A CONTROL REGION OF A TYPICAL EUKARYOTIC GENETYPICAL EUKARYOTIC GENE
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Changes in Gene Expression and RegulationChanges 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
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• Master regulators Master regulators of gene of gene expressionexpression
• Respond to intra- Respond to intra- or extracellular or extracellular signalssignals
• Bind to promoter Bind to promoter regions of specific regions of specific genesgenes
• Mediate DNA Mediate DNA transcription to transcription to mRNAmRNA
Transcription FactorsTranscription Factors
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• Dimeric transcription factor, composed of Dimeric transcription factor, composed of subunits including p65, p50, p52, c-Rel and subunits including p65, p50, p52, c-Rel and Rel B Rel B
• Activated by: Stress ,CytokinesActivated by: Stress ,Cytokines Free radicals, UV Free radicals, UV
• Functions :Functions :
- Immune response, Development- Immune response, Development
- Cell growth, Apoptosis, Stress response- Cell growth, Apoptosis, Stress response
Nuclear Factor kappa B (NF-kB)Nuclear Factor kappa B (NF-kB)
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CONTROL:CONTROL:
NF-kB dimer is sub-NF-kB dimer is sub-active in cytoplasm, active in cytoplasm, bound to IkBbound to IkB
STRESS:STRESS: IkB phosphorylated IkB phosphorylated & degraded& degraded
• Free NF-kB dimer:Free NF-kB dimer:- moves to nucleus - moves to nucleus - binds to DNA - binds to DNA - transcription of - transcription of downstream genesdownstream genes
Target genes
NF-kBNF-kB
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• IkB is (P) in liver & IkB is (P) in liver & brain at 5h of brain at 5h of anoxiaanoxia
• Elevated (P) of Elevated (P) of IkB frees NF-kB IkB frees NF-kB dimer to move dimer to move into the nucleusinto the nucleus
IkB IkB PhosphorylationPhosphorylation
P-IkB protein levels in turtle tissues
Time course for IkB phosphorylation in liver
Rel
ativ
e p
rote
in le
vels
Rel
ativ
e p
rote
in le
vels
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• NF-kB p65 and NF-kB p65 and p50 are p50 are upregulated upregulated during 5 h of during 5 h of anoxiaanoxia
NF-kB dimer protein levelsNF-kB dimer protein levels
p65
p50
Rel
ativ
e p
rote
in le
vels
Turtles: NF-kB Protein LevelsTurtles: NF-kB Protein Levels
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• P65 moves into P65 moves into nucleus in nucleus in anoxiaanoxia
• DNA-binding DNA-binding activity of p65 activity of p65 elevated after 5 & elevated after 5 & 20 h of anoxia20 h of anoxia
NF-kB pathway NF-kB pathway is activated in is activated in turtle liver and turtle liver and brain in anoxia !brain in anoxia !
P65: Subcellular distribution
Rel
ativ
e p
rote
in le
vels
P65: DNA-binding activityP65: DNA-binding activityR
elat
ive
DN
A-b
ind
ing
act
ivit
y
NF-kBNF-kB
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Relative mRNA levels ofRelative mRNA levels ofNF-kB target genesNF-kB target genes
Rel
ativ
e m
RN
A le
vels
Ferritin HFerritin H HO-1HO-1
NF-kB: Target Gene LevelsNF-kB: Target Gene Levels
Ferritin heavy chain and Ferritin heavy chain and Heme oxygenase-1 (HO-1) Heme oxygenase-1 (HO-1) are upregulated after 5 h are upregulated after 5 h of anoxiaof anoxia
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• Sequesters ironSequesters iron• Can hold up to 4,500 atoms of ironCan hold up to 4,500 atoms of iron• 24 subunits: light (19 kDa) and heavy (21 kDa) 24 subunits: light (19 kDa) and heavy (21 kDa) • Limits iron-catalyzed ROS production via theLimits iron-catalyzed ROS production via the Fenton reaction Fenton reaction
70’sradicals
Ferritin heavy chainFerritin heavy chain
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Help minimize free iron levels in cellsHelp minimize free iron levels in cells
Ferritin: Binds iron; Heavy & Light chainsFerritin: Binds iron; Heavy & Light chains
Heme oxygenase -1:Heme oxygenase -1: - Degrades heme, a source of redox active iron- Degrades heme, a source of redox active iron - Free iron then stored into ferritin- Free iron then stored into ferritin
Iron can be a source of oxidative stress:• Catalyzes production of Hydroxyl radicals via Fenton reaction:
Ferritin and Heme Oxygenase -1Ferritin and Heme Oxygenase -1
HH22OO22••OH + OHOH + OH--
FeFe22
++Hydroxyl radical is very reactive and Hydroxyl radical is very reactive and responsible for most oxidative stress-responsible for most oxidative stress-mediated damagemediated damage
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THE BRAINS OF THE THE BRAINS OF THE OPERATIONOPERATION
GENE PROTEIN
PHYSIOLOGY BIOCHEMISTRY
INTEGRATION
PETER LUTZ TERRRTLE
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Hypoxia / IschemiaHypoxia / Ischemia• Sensitive AnimalsSensitive Animals (most mammals) (most mammals)
- Energy deficit (high ATP demand)- Energy deficit (high ATP demand)
- Disruption of ions and depolarization- Disruption of ions and depolarization
- Release of excitotoxic GLU, - Release of excitotoxic GLU,
- Excess intracellular Ca- Excess intracellular Ca2+2+
- Oxidative Stress (+ reperfusion )- Oxidative Stress (+ reperfusion )
- Cell Death- Cell Death
• Tolerant AnimalsTolerant Animals (e.g. turtles, carp ) (e.g. turtles, carp )
- Decrease ATP demand (Metabolic Arrest)- Decrease ATP demand (Metabolic Arrest)
- Adenosine as a Retaliatory Molecule- Adenosine as a Retaliatory Molecule
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Hypoxic CascadeHypoxic Cascade
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MAMMAL TURTLE CARP
30 min 12 h 12 h TIME IN ANOXIA
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Lutz PL & Milton SL. 2004. J Exp Biol 207: 3141-3147
ANOXIA SURVIVALANOXIA SURVIVAL IN TURTLE BRAININ TURTLE BRAIN
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Lutz PL & Milton SL. 2004. J Exp Biol 207: 3141-3147
ANOXIA SURVIVALANOXIA SURVIVAL IN TURTLE BRAININ TURTLE BRAIN
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Lutz PL & Milton SL. 2004. J Exp Biol 207: 3141-3147
ANOXIA SURVIVALANOXIA SURVIVAL IN TURTLE BRAININ TURTLE BRAIN
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BRAIN GENES BRAIN GENES Up-regulated in turtle anoxiaUp-regulated in turtle anoxia
(DNA array)(DNA array)
• GABA transporter• GABA receptor
Adult T. s. elegans
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Lutz PL & Milton SL. 2004. J Exp Biol 207: 3141-3147
ANOXIA SURVIVALANOXIA SURVIVAL IN TURTLE BRAININ TURTLE BRAIN
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BRAIN GENES BRAIN GENES Up-regulated in turtle anoxiaUp-regulated in turtle anoxia
(DNA array)(DNA array)
• Adenosine receptor• 5’Nucleotidase
Adult T. s. elegans
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BRAIN GENES BRAIN GENES
• GABA transporter• GABA receptor• Adenosine receptor• 5’Nucleotidase• Serotonin receptor
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GENESGENES
Transcription
RNAsRNAs
Control bytranscriptional regulation
Control bytranslational regulation
Translation
PROTEINSPROTEINS(ENZYMES)(ENZYMES)
Control byproteases INACTIVEINACTIVE
ENZYMEENZYME
NoModification
FUNCTIONALFUNCTIONALENZYMESENZYMES
CovalentModification
Degradation
Control by post-translationalmodification
ACTIVEACTIVEENZYMESENZYMES
Inhibitionand
Activation
Control at level ofenzyme function
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ANOXIAANOXIA• J. STOREYJ. STOREY• S. BROOKSS. BROOKS• Q. CAI Q. CAI • W. WILLMOREW. WILLMORE• H. MEHRANIH. MEHRANI• D. DOUGLASD. DOUGLAS• J. DUNCAN• S. GREENWAYS. GREENWAY• A. KRIVORUCHKOA. KRIVORUCHKO
• M. HERMES-LIMA• T. ENGLISHT. ENGLISH• K. LARADEK. LARADE• E. RUSSELLE. RUSSELL• T. PANNUNZIOT. PANNUNZIO• R. WHITWAM• S. KORYCAN• B. MICHAELIDIS• J. ZHOU
www.carleton.ca/~kbstoreywww.carleton.ca/~kbstorey
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Nucleus
GENESON/OFF
mRNAs
[ i + e Factors]
PROTEINS
Ca+2
KINASES (2nd)
PATHWAYS
SMW
CHO
AA
ATP
?SAPK
ATP
ADP
MITOGENES
FAT
[Trans.F] Na
K
ETC
P PROT
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cDNA ARRAY SCREENINGcDNA ARRAY SCREENING
AEROBIC ANOXIC
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ANTIOXIDANT ENZYMESANTIOXIDANT 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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Overgaard, J. et al. J Exp Biol 2007;210:1687-1699
Major effects of anoxia on cellular energetic turnover
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TRANSPORTERS / RECEPTORSTRANSPORTERS / RECEPTORS