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FREEZE TOLERANCE: ITS ALL IN THE GENES

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Strategies for exploration of freeze responsive gene. expression: advances in vertebrate freeze tolerance. Cryobiology 48, 134-145 ... – PowerPoint PPT presentation

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Title: FREEZE TOLERANCE: ITS ALL IN THE GENES


1
FREEZE TOLERANCE ITS ALL IN THE GENES
www.carleton.ca/kbstorey
2
ADAPTATIONS TO COLD
Below 0C
Above 0C
Migration
Stay warm
Freeze Avoidance
Freeze Tolerance
Hibernation
Supercool
Mammals
Some reptiles amphibians
Others
Invertebrates
3
VERTEBRATE FREEZE TOLERANCE
4
WOOD FROGRana sylvatica
5
WOOD FROGRana sylvatica
6
Painted turtle hatchlings Chrysemys
picta marginata
7
SURVIVING FREEZING
  • Extracellular freezing only
  • Up to 70 ofbody water frozen
  • High polyols
  • Acclimation required
  • Glucose
  • Glycerol
  • Sorbitol

8
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

9
i e Factors
mRNAs
CHO
PROTEINS
Na
ATP
K
PATHWAYS
AA
PROT
?
SMW
FAT
ADP
ATP
KINASES (2nd)
MITO
ETC
10
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

11
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

12
Frozen
Control
13
  • cDNA Arrays- Methods
  • Materials
  • Sources- Publications

14
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
15
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

16
CONTROL REGION OF A TYPICAL EUKARYOTIC GENE
17
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
18
GRP78 structure and mechanism of action
  • ER-related protein
  • Member of HSP70 family
  • Functions
  • Protein folding / chaperone
  • Protein stabilization
  • Anti-apoptotic function

19
GLUCOSE-REGULATED PROTEIN 78 WOOD FROG FREEZING
Relative to Control
PROTEIN ANOXIA, 24 h DEHYDRATE, 40
Heart Others
20
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
21
ATF6 Pathway
  • During stress
  • Increase protein folding capacity via GRP78/94
  • Reduce folding load via EDEM up-regulation
  • Induce apoptosis via GADD153

22
ATF6 pathway in Wood Frogs
Muscle
23
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

24
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

25
The TURTLE
26
Hatchling painted turtles Chrysemys picta
marginata
  • Overwinter in natal nests, less
  • than 10 cm underground

27
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

28
OXYRADICAL DAMAGE TO PROTEINS
29
ANTIOXIDANT ENZYMES
30
SERPINS
  • Serine Proteinase Inhibitors
  • irreversible inhibitors
  • intra- extracellular forms
  • 40-50 kD (large family)
  • 2 of plasma proteins

31
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.

32
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

33
TURTLE HYPOXIA GENE RESPONSE
  • Ferritin light heavy chain (HIF)
  • Antioxidant enzymes Glutathione peroxidase 1 /
    4 Glutathione S-transferase M5 / A2
    Peroxiredoxin
  • Serpins

34
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)

35
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
www.carleton.ca/kbstorey
36
(No Transcript)
37
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
38
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

39
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).

40
IRE1/XBP1 pathway Data
Control
Thawed
Freezing
Muscle
Liver
41
XBP-1 pathway
42
ERAD
43
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

44
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

45
PERK/eIF2a/ATF4 pathwayData
Thawed
Freezing
Control
Muscle
Liver
46
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.

47
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.
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