FREEZE TOLERANCE: ITS ALL IN THE GENES

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FREEZE TOLERANCE ITS ALL IN THE GENES
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ADAPTATIONS TO COLD
Below 0C
Above 0C
Migration
Stay warm
Freeze Avoidance
Freeze Tolerance
Hibernation
Supercool
Mammals
Some reptiles amphibians
Others
Invertebrates
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VERTEBRATE FREEZE TOLERANCE
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WOOD FROGRana sylvatica
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WOOD FROGRana sylvatica
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Painted turtle hatchlings Chrysemys
picta marginata
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SURVIVING FREEZING

• Extracellular freezing only
• Up to 70 ofbody water frozen
• High polyols
• Acclimation required
• Glucose
• Glycerol
• Sorbitol

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TO SURVIVE FREEZING

• Alter metabolism to synthesize
  cryoprotectants (polyols, sugars)
• Defend against intracellular desiccation
• Suppress metabolic rate
• ACCOMPLISHED BY
• Activate signaling enzymes in every cell
• - SAP kinases
• - Role reversible controls on cell
  processes
• Up-regulate selected genes

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i e Factors
mRNAs
CHO
PROTEINS
Na
ATP
K
PATHWAYS
AA
PROT
?
SMW
FAT
ADP
ATP
KINASES (2nd)
MITO
ETC
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FREEZE INDUCED CHANGES

• Gene inactivation
• Protein Synthesis slows to 1
• Pumps channels closed
• Energy Production slows to 5
• Energy Utilization slows to 2
• Few SAP kinases activated
• Few Genes activated

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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

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Frozen
Control
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• cDNA Arrays- Methods
• Materials
• Sources- Publications

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FREEZE-INDUCED GENES WOOD FROGS
cDNA Library / Gene Chip

• Mito ETC Transporters
• AOE Shock proteins
• Transcription Factors
• The Unknowns Fr10, Li16, FR47

Storey KB 2004. Strategies for exploration of
freeze responsive gene expression advances in
vertebrate freeze tolerance. Cryobiology 48,
134-145
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TRANSCRIPTION FACTORS

• ATF (Glucose Regulated Proteins)
• HIF (O2), HSF (Hsp)
• NFkB (IkB-P), Nrf-2 (GST), NRF-1
• PPAR, PGC, RXR, chREBP, CREB-P
• STAT, SMAD, p53-P, HNF, AP (1,2)
• Methods EMSA, CHiP

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CONTROL REGION OF A TYPICAL EUKARYOTIC GENE
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ATF Cell Stress

• Thermal stress (cold, freezing)
• Hypoxia / anoxia
• Ischemia / reperfusion
• Oxidative stress

BUILD-UP OF MISFOLDED PROTEINS IN ER
Homeostasis perturbed
UPR Unfolded Protein Response
ER stress
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GRP78 structure and mechanism of action

• ER-related protein
• Member of HSP70 family

• Functions
• Protein folding / chaperone
• Protein stabilization
• Anti-apoptotic function

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GLUCOSE-REGULATED PROTEIN 78 WOOD FROG FREEZING
Relative to Control
PROTEIN ANOXIA, 24 h DEHYDRATE, 40
Heart Others
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MODEL OF UNFOLDED PROTEIN RESPONSE
ATP depletion - ER Calcium depletion - Amino acid
deprivation - Hypoxia - Ischemia - Oxidative
stress Freezing Estivation - Hibernation
ER STRESS
PERK (kinase)
eIF2a (P)
Protein synthesis inhibition
Translationally regulated
Transcription activation
PP1
CHOP
GRP78
GADD34
VEGF
Target genes
Protein folding
Pro-apoptosis
Pro-survival
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ATF6 Pathway

• During stress
• Increase protein folding capacity via GRP78/94
• Reduce folding load via EDEM up-regulation
• Induce apoptosis via GADD153

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ATF6 pathway in Wood Frogs
Muscle
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ATF6 pathway

• Muscle
• Active ATF6 increased
• EDEM increased
• Active XBP1 decreased
• GADD153 decreased

Conclusions

• Tissue specificity of response, similar to GRP
  response
• Decreased GADD153 absence of apoptosis
• Increased EDEM reduction of folding load via
  degradation
• Increased protein folding capacity via GRPs by
  ATF6

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ATF6 pathway Frozen, Dehydrated, Anoxic

• ATF6 Summary Anoxic and dehydrated
• key pathway marker
• Muscle - ATF6 response in dehydration and anoxia
  the same - differs from freezing
• Liver - ATF6 the same in freezing and
  dehydration - differs from anoxia

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The TURTLE
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Hatchling painted turtles Chrysemys picta
marginata

• Overwinter in natal nests, less
• than 10 cm underground

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TURTLE FREEZE TOLERANCE GENE RESPONSE

• Ferritin light heavy chain (HIF-1a)
• Hemoglobin (a, ß) (HIF-1a)
• K/Cl- solute carrier
• Antioxidant enzymes Peroxiredoxin
  Glutathione peroxidase 1
• Serpins (anti-proteases) C1, D1, G1

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OXYRADICAL DAMAGE TO PROTEINS
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ANTIOXIDANT ENZYMES
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SERPINS

• Serine Proteinase Inhibitors

• irreversible inhibitors
• intra- extracellular forms
• 40-50 kD (large family)
• 2 of plasma proteins

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SERPIN ACTION

• Trap protease in complex Serpin Protease
  S P
• Large conformational change crush protease
• Loss of structure, loss of protease activity
• Involved in blood coagulation, fibrinolysis,
  inflammation, etc.

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SERPIN FAMILIES

• 16 Clades, 6 subgroups
• Protection from proteases duringmetabolic rate
  depression
• D1 inhibits thrombin
• A1 a1-antitrypsin, anti-elastase - most
  common plasma serpin
• C1 anti-thrombin III
• F1 anti-plasmin

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TURTLE HYPOXIA GENE RESPONSE

• Ferritin light heavy chain (HIF)
• Antioxidant enzymes Glutathione peroxidase 1 /
  4 Glutathione S-transferase M5 / A2
  Peroxiredoxin
• Serpins

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GENE REGULATION

• Genes
• Transcription Factors (Tf) as Regulators -
  Coordinate regulation of downstream genes
  of Tf - Genes up as a functional unit
  (CASSETTE)
• Attenuation of Tf signals - Activators /
  inhibitors, Phosphorylation - mRNA processing
  / export / degradation - Protein synthesis
  regulation / Proteolysis - Mix Match Tf
  responses
• Up-regulation of Tf DNA binding (EMSA)

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FREEZE TOLERANCE

• J. STOREY
• D. McNALLY
• J. MacDONALD
• T. CHURCHILL
• S. GREENWAY
• C. HOLDEN
• S. WU
• J. NILES

• J. DU
• A. DeCROOS
• L. ZHENHONG
• Q. CAI
• F. SCHUELER
• S. BROOKS
• B. RUBINSKY
• R. BROOKS

Funded by NSERC Canada
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GENES
Control by transcriptional regulation
Transcription
RNAs
Control by translational regulation
Translation
Control by proteases
No Modification
PROTEINS (ENZYMES)
INACTIVE ENZYME
Degradation
Covalent modification
Control by post- translational modification
FUNCTIONAL ENZYMES
Inhibition and Activation
Control at level of enzyme function
ACTIVE ENZYMES
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IRE1/XBP1 pathway
Data trends

• Muscle Tissue
• Active XBP1 levels decreased.
• EDEM levels increased significantly.
• Levels of GADD153 decreased.

• Liver Tissue
• Active XBP1 levels increased.
• EDEM levels decreased.
• GADD153 also decreased.

Conclusions

• Similar to the previous pathway in all aspects.
• Increased folding capacity.
• Decreased folding load
• Absence or lack of apoptosis

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IRE1/XBP1 pathway summary data
IRE1/XBP1 pathway summary
Summary of Anoxic and Dehydration data

• Levels of XBP1 protein (key pathway Marker)
• In the muscle, the trends of XBP1 levels during
  freezing and dehydration were similar to one
  another, while differing from the anoxic stress.
• In liver tissue, XBP1 trends were similar between
  the all three stresses (freezing, anoxia and
  dehydration).

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IRE1/XBP1 pathway Data
Control
Thawed
Freezing
Muscle
Liver
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XBP-1 pathway
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ERAD
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PERK/eIF2a/ATF4 pathway (A)

• Pathway options during stress
• Initially reduces folding load by attenuating
  translation
• Increase protein folding capacity through GRP78
• Reinitiates translation by dephosphorylating
  eIF2a through GADD34 and PP1
• Induction of apoptosis through GADD153

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IRE1/XBP1 Pathway (C)

• Pathway options during stress
• Similar to the ATF6 pathway
• Increases folding capacity through GRP78 and
  GRP94
• Reduces folding load through EDEM (ERAD)
• Induction of apoptosis through GADD153
• Activation of pathway differs from previous
  pathway.
• IRE1 splicing of XBP1

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PERK/eIF2a/ATF4 pathwayData
Thawed
Freezing
Control
Muscle
Liver
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PERK/eIF2a/ATF4 pathway
Data trends

• Muscle Tissue
• Increase of ATF4 protein levels.
• GADD 34 protein levels did not significantly
  change.
• The ATF3 levels increased.
• A decrease in GADD153.

• Liver Tissue
• Increase of ATF4 protein levels.
• GADD 34 protein levels increased.
• The ATF3 levels increased.
• A decrease in GADD153.

Conclusions

• Decrease in GADD153 levels support decrease or
  even lack of apoptosis in both tissues.
• Increase ATF4 supports the increase in folding
  capacity in both tissues.
• The increase of liver GADD34 leads to
  reinitiation of translation.

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PERK/eIF2a/ATF4 pathwaysummary data
PERK/eIF2a/ATF4 pathway summary
Summary of Anoxic and Dehydration data

• Levels of ATF4 protein (key pathway Marker)
• ATF4 trends in muscle were similar between the
  anoxic and freezing stresses. The dehydrated
  trends were opposite form the others.
• Liver trends for ATF4 were similar between
  freezing and dehydration but differed between the
  two previous and the anoxic stress.