FROZEN ALIVE: THE SECRETS IN THE GENES

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FROZEN ALIVE THE SECRETS 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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FREEZE TOLERANT ANIMALS

• TERRESTRIAL INSECTS
• INTERTIDAL MOLLUSCS BARNACLES
• AMPHIBIANS REPTILES - FROGS (6
  species) - HATCHLING PAINTED TURTLES -
  BOX TURTLES - GARTER SNAKES - LIZARDS
  (some)

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Garter snake, Thamnophis sirtalis
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Painted turtle hatchlings Chrysemys picta
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Box turtle, Terrapene carolina
OSCAR
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GRAY TREE FROGHyla versicolor
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CHORUS FROGPseudacris triseriata
SPRING PEEPERPseudacris crucifer
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WOOD FROGRana sylvatica
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WOOD FROGRana sylvatica
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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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SURVIVING FREEZING

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

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WOOD FROG CRYOPROTECTANTS

• Blood glucose rises from 5 mM to 200-400 mM
• Glucose triggered by ice formation
• Made from liver glycogen (180 mg/g)
• Liver is 12 of body mass
• Glucose distribution via Blood Liver gt
  Core organs gt Periphery

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FREEZE INDUCED CHANGES

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

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i e Factors
Nucleus
mRNAs
GENES ON/OFF
CHO
PROTEINS
Trans.F
Na
ATP
K
PATHWAYS
AA
SAPK
P
PROT
?
SMW
FAT
ADP
ATP
KINASES (2nd)
MITO
ETC
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PROTEIN KINASES

• Covalent modification by phosphorylation
• Families of protein kinases PKA (cAMP), PKG
  (cGMP), CaMK (Ca2), PKC (Ca2,PL,DG)
• SAPKs daisy chain phosphorylations
• Regulation is via interconversion of active
  vs subactive forms of protein substrates

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ENZYME CONTROL BY REVERSIBLE PHOSPHORYLATION
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GLYCOGEN PHOSPHORYLASE
Glycogen Pi kinase
Phos a Phos b
phosphatase Glucose-1-P glycogen (n-1)
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i e Factors
Nucleus
mRNAs
GENES ON/OFF
CHO
PROTEINS
Trans.F
Na
ATP
K
PATHWAYS
AA
SAPK
P
PROT
?
SMW
FAT
ADP
ATP
KINASES (2nd)
MITO
ETC
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SIGNAL TRANSDUCTION

• 3 MAIN PATHS
• PHOSPHORYLATION CASCADES
• REGULATORS OF TRANSCRIPTION, TRANSLATION
• INTERRELATED, UBIQUITOUS

• ANALYSIS BY ANTIBODIES (Phospho/Dephospho)

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MAP KINASES INWOOD FROG FREEZING
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p38 Pathway Signaling

• Path activated by ??

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p38 Pathway Signaling
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p38 Pathway Signaling

• P-transcription factors (active)

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FREEZE INDUCED CHANGES

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

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

Storey KB 2004. 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
• Oxidative stress

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ATF 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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ATF 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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ATF 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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ATF 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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GRPs in Wood Frog Liver
PROTEIN CHANGES UNDER OTHER STRESSES
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ATF 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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ATF pathway in Wood Frogs
Muscle
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ATF pathway

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

Conclusions

• Increased protein folding capacity via GRPs by
  ATF6
• Decreased GADD153 absence of apoptosis
• Increased EDEM reduction of folding load via
  degradation

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FREEZE-INDUCED GENES WOOD FROGS
cDNA Library / Gene Chip

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

Storey KB 2004. Cryobiology 48, 134-145
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THE UKNOWNS Li16, FR10, FR47

• Novel gene sequences discovered by cDNA library
  screening
• Complete cDNA sequences by 3 and 5 RACE
• Genomic sequences now known
• Similarities in protein folding
• In vivo regulation by second messengers

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(No Transcript)
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FUNCTION OF THE UNKNOWN PROTEINS

• Express genes in cells in culture - Li16,
  FR10 - insect or mammal cells
• Expression of Li16 FR10 protects cultured
  cells from freezing damage
• Li16 is intracellular
• FR10 is exported
• Both bind to membranes

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Unique Animal Stress Model
Vertebrate whole-body freeze tolerance
Tissue cryopreservation
Tolerance of extreme ischemia and hyperglycemia
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FREEZE TOLERANCE

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

• A. DeCROOS
• L. ZHENHONG
• C. DIENI
• Q. CAI
• F. SCHUELER
• S. BROOKS
• B. RUBINSKY
• R. BROOKS

Funded by NSERC Canada
www.carleton.ca/kbstorey
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METABOLIC RATE DEPRESSION
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CONTROL REGION OF A TYPICAL EUKARYOTIC GENE

• Epigenetics
• microRNA
• RNA Polymerase-P
• Histones modified
• HDAC / HAT changes

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GRP78
GLUCOSE REGULATED PROTEINS in Frog Liver
GRP94

• Chaperones in the ER
• Act in the UnfoldedProtein Response

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THE UNKNOWNS Li16
cGMP
PKG P-trans.
factor
mRNA
Protein
Function
In Vivo - freezing - dehydrate - anoxia
In Vitro - transfect gene - express protein
- function study
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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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