www.carleton.ca/~kbstoreywww.carleton.ca/~kbstorey

PETERPETERL.L.

LUTZLUTZ

Red-eared sliderTrachemys scripta elegans

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

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

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

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

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

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

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%)

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

PP

PP PP

PP

PiPiProtein

Phosphatase

ATPATP ADPADPProtein Kinase

P & deP enzymesseparate on ionexchange columns

ReversibleReversiblephosphorylationphosphorylation

control of enzymescontrol of enzymes

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.

Estivation

METABOLIC RATE METABOLIC RATE DEPRESSIONDEPRESSION

Diapause

Freezing

Anoxia

Hibernation

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%)

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

cDNA ArrayscDNA Arrays- Methods- Methods- MaterialsMaterials- SourcesSources- Publications- Publications

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

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

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

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

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

CONTROL REGION OF A CONTROL REGION OF A TYPICAL EUKARYOTIC GENETYPICAL EUKARYOTIC GENE

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

• 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

• 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)

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

• 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

• 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

• 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

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

• 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

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

THE BRAINS OF THE THE BRAINS OF THE OPERATIONOPERATION

GENE PROTEIN

PHYSIOLOGY BIOCHEMISTRY

INTEGRATION

PETER LUTZ TERRRTLE

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

Hypoxic CascadeHypoxic Cascade

MAMMAL TURTLE CARP

30 min 12 h 12 h TIME IN ANOXIA

Lutz PL & Milton SL. 2004. J Exp Biol 207: 3141-3147

ANOXIA SURVIVALANOXIA SURVIVAL IN TURTLE BRAININ TURTLE BRAIN

Lutz PL & Milton SL. 2004. J Exp Biol 207: 3141-3147

ANOXIA SURVIVALANOXIA SURVIVAL IN TURTLE BRAININ TURTLE BRAIN

Lutz PL & Milton SL. 2004. J Exp Biol 207: 3141-3147

ANOXIA SURVIVALANOXIA SURVIVAL IN TURTLE BRAININ TURTLE BRAIN

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

Lutz PL & Milton SL. 2004. J Exp Biol 207: 3141-3147

ANOXIA SURVIVALANOXIA SURVIVAL IN TURTLE BRAININ TURTLE BRAIN

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

BRAIN GENES BRAIN GENES

• GABA transporter• GABA receptor• Adenosine receptor• 5’Nucleotidase• Serotonin receptor

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

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

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

cDNA ARRAY SCREENINGcDNA ARRAY SCREENING

AEROBIC ANOXIC

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)

Overgaard, J. et al. J Exp Biol 2007;210:1687-1699

Major effects of anoxia on cellular energetic turnover

TRANSPORTERS / RECEPTORSTRANSPORTERS / RECEPTORS