Download LIVING WITHOUT OXYGEN

VERTEBRATE FREEZE
TOLERANCE
ADAPTATIONS TO COLD
Below 0°C
Above 0°C
Migration Stay warm
Freeze
Freeze
Avoidance Tolerance
Hibernation
Supercool
Some reptiles
& amphibians
Invertebrates
Mammals
Others
WOOD FROG
Rana sylvatica
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
SURVIVING FREEZING
• Extracellular
freezing only
• Up to 70% of
body water frozen
• High ‘polyols’
• Acclimation
required
• Glucose
• Glycerol
• Sorbitol
[ i + e Factors]
Nucleus
mRNAs
GENES
ON/OFF
[Trans.F]
CHO
PROTEINS
PATHWAYS
ATP
K
AA
?
SAPK
P
Ca+2
PROT
SMW
FAT
ADP
ATP
KINASES (2nd)
GENES
Na
MITO
ETC
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)
• Glucose distribution
via Blood:
Liver >
Core organs >
Periphery
250
Glucose, mM
• Liver is ~12% of
body mass
Blood
200
Liver
150
Heart
100
Kidney
50
Muscle
0
0
1
2
Days frozen
3
1
3
5
7
9
Days thawed
11
GLYCOGEN
PHOSPHORYLASE
Glycogen + Pi
kinase
Phos a
Phos b
phosphatase
Glucose-1-P +
glycogen (n-1)
Liver
Phosphorylase a
Activity, U/g
0 2 5 30 60 2
3
1
2
min
hours
days
TIME OF FREEZING
3
13
1
3 4
min
hours
TIME OF THAW
PROTEIN KINASES
PROTEIN
PROTEIN-(P)n
nATP
nADP
• 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
[ i + e Factors]
Nucleus
mRNAs
GENES
ON/OFF
[Trans.F]
CHO
PROTEINS
PATHWAYS
ATP
K
AA
?
SAPK
P
Ca+2
PROT
SMW
FAT
ADP
ATP
KINASES (2nd)
GENES
Na
MITO
ETC
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%)
p38 Pathway Signaling
 Path activated by
??
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
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
TURNING OFF GENES:
Role of Epigenetics
Epigenetics:
- Stable changes in gene activity that do
not involve changes in DNA sequence
Common mechanisms:
- DNA methylation
- Histone modification / histone variants
- Regulatory non-coding RNAs
cDNA Arrays
- Methods
- Materials
- Sources
- Publications
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
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, PCR
CONTROL REGION OF A
TYPICAL EUKARYOTIC GENE
Epigenetics:
• microRNA
• RNA Polymerase-P
• Histones modified
• HDAC / HAT changes
FREEZE-INDUCED
GENES: WOOD FROGS
cDNA Library / Gene Chip
• Transcription Factors
NRF -2
• AOE
Storey KB 2004. Cryobiology 48, 134-145
NRF-2
• Increased NFR-2 protein
• Increased NFR-2 in the Nucleus
• Increased levels of co-Tf: MafG
• Downstream gene activation:
• GST, HO-1, HO-2, Peroxiredoxin
Nrf2/ARE pathway
Reactive Oxygen Species (ROS)
Actin
Keap1
Nrf2
Dissociation
Nrf2
P
Nrf2
P
Cytoplasm
Small
Maf
Nucleus
Activation
Small
Maf
Nrf2
ARE
P
Antioxidant proteins
(e.g. GSTs, HO1)
Regulation of Nrf2 Protein
Liver
C C F F
Actin
Nrf2
82 kDa
40 kDa
Nrf2
Nrf2 protein expression (~82KDa)
Nrf2
protein, 82 kDa
2.5
2
a
a
control
frozen
a
1.5
1
a
recovery
a
0.5
0
brain
gut
skin
liver
muscle
Relative protein levels
Relative protein levels
Nrf2 protein expression(~40KDa)
Nrf2
protein, 40 kDa
2
1.5
a
a
1
a
a
0.5
0
brain
gut
skin
liver
muscle
Regulation of MafG
Small
Maf
Nrf2
Antioxidant proteins
(e.g. GSTs, HO1)
P
ARE
mafG protein expression
MafG protein
a
Relative protein level
2.5
2
control
a
a
frozen
a
recovery
a
1.5
a
1
0.5
0
brain
muscle
gut
liver
skin
Glutathione S-Transferase
Pi isozyme in Wood Frogs
GST Pi mRNA expression
GST Pi mRNA
a
*
control
2.5
frozen
recovery
2
mRNA increases
- transcriptional control
a
1.5
1
0.5
GST Pi protein expression
0
brain
gut
liver
kidney
heart
GST Pi protein
muscle
12
Protein increases
- translational control
Relative protein level
Relative mRNA levels
3
control
a
10
frozen
recovery
8
6
a
a
a
4
a
a
a
2
a
0
brain
gut
liver
kidney
heart
muscle
Conclusions
Activation of the Nrf2 pathway:
 Activated in early-late torpor, along with
downstream gene protein products
 Increased GST protein
and activity
Result:
 Detoxification of H2O2,
intracellular signaling control
ANTIOXIDANT ENZYMES
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, PCR
TRANSCRIPTION FACTOR
PROFILING
GENE CHIPS
Data Leads
Confirm by
RT-PCR,
Northern blots
ELISAs in plates
Downstream genes
Confirm by EMSA
Tf
ROLE & CONTROL OF SYSTEM
Transgenics
Cell Assay
RNAi
Knock out
Epigenetics
FUNCTIONAL ASSAYS
Protein levels
- enzyme assay
- antibodies : protein
- functional analysis
e.g. HIF  EPO 
Unique Animal Stress Model
Vertebrate
whole-body
freeze tolerance
Tissue
cryopreservation
Tolerance of extreme
ischemia and
hyperglycemia
GENES
Transcription
Control by
transcriptional regulation
RNAs
Translation
No
Modification
FUNCTIONAL
ENZYMES
Inhibition
and
Activation
Control by
translational regulation
PROTEINS
(ENZYMES)
Covalent
modification
Control by
proteases
Degradation
Control by posttranslational
modification
Control at level of
enzyme function
ACTIVE
ENZYMES
www.carleton.ca/~kbstorey
INACTIVE
ENZYME