Frank van Breukelen

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Development of a Model for Ischemia Tolerance
Using a Mammalian Hibernator
Networking Scientists and Resources to
Strengthen Biomedical Research in Nevada
Frank van Breukelen Department of Biological
Sciences University of Nevada, Las Vegas
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Mammalian hibernation

• Metabolic rates are as low as 1/100th of BMR
• Core body temperatures below -2 C
• Heart rate diminishes from 200 bpm to 3-4 erratic
  bpm
• Energetic savings of approximately 90 across the
  hibernation season

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Torpor bout
Additional summer active (SA) control for
seasonal variation
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Metabolic Depression in Mammals
Mammalia
Monotremata (echidna)
Eutheria
Marsupalia (opposum)
Pholidota (pangolin)
Carnivora (bear)
Edentata (anteater)
Chiroptera (bat)
Artiodactyla (deer)
Macroscelidea (elephant shrew) Lagomorpha
(rabbit) Rodentia (ground squirrel)
Scandentia (tree shrews)
Insectivora (shrew)
Cetacea (whales) Tubilendentata (aardvark) Perisso
dactyla (horses) Hyracoidea (hyraxes) Sirenia (man
atees) Proboscidea (elephants)
Dermoptera (colugo)
Primates (mouse lemur)
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Ischemia in hibernators??
Long term survivorship following cardiac arrest
in humans is poor the survival rate following
out-of-hospital arrest is less than 5 Ground
squirrels experience ischemia every time they go
into torpor - HR drops from 200-250 bpm to 3-4
bpm Hibernating ground squirrels experience 13.3
fold reduced blood flow to the brain compared to
their active counterparts (Frerichs et al.,
1994) An in vitro study on hippocampal slices
demonstrated that ground squirrel brain slices
survived aglycemia and hypoxia 2-3 fold longer
than rat counterparts (Frerichs and Hallenbeck,
1998) Tons of data suggesting an ischemic insult
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Results of NIH PULSE Meeting
The Post-Resuscitative and Initial Utility in
Life Saving Efforts (PULSE)

• Two RFAs that directly called for the production
  of an ischemia model
• based on a hibernator
• Initial funding attempt was criticized for two
  reasons
• - Ischemia during natural torpor has not been
  clearly demonstrated
• Torpor represents a steady state condition.
  Although cerebral blood
• flow may be diminished 13.3 fold, metabolic rate
  may be as low as
• 1 of BMR. Supply is sufficient for demand.
• - An assessable model is necessary
• How much ischemia is ischemic? Many ischemia
  studies are hampered by
• an inability of assessing to what degree blood
  flow has been diminished.

New PRA!- Due Sep 06 / Jan 07
Elucidating Natures Solutions to Heart, Lung,
and Blood Diseases and Sleep Disorder Processes
(R01)
solicits studies that elucidate the natural
molecular and cellular adaptations of mammalian
species to extreme environmental conditions that
would rapidly evoke life-threatening
cardiovascular or respiratory responses in other
species, including humans.
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Specific Aim I
Establish the physiological role of ischemia
during mammalian hibernation.
increase in vagal tone
Heart rate drops to 100 bpm before any change in
body temperature
Body temperature (C)
Time (days)
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Specific Aim I
Establish the physiological role of ischemia
during mammalian hibernation 5 golden-mantled
ground squirrels (Spermophilus lateralis) that
already have temperature-sensitive
telemeters Install chronic jugular and carotid
catheters Sample blood as they enter and arouse
from torpor (likely periods of ischemia) as well
as through the torpor bout. Measure in both
arterial and venous samples for pO2 and
pCO2 pH lactate aspartate transaminase and
lactate dehydrogenase activities Performed
preliminary experiments to show
feasibility -blood gas instrument
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Specific Aim I
Establish the physiological role of ischemia
during mammalian hibernation Squirrels will be
sacrificed at various points of the torpor cycle
Collect brain, heart, liver, kidney, and
jejunum conjugated diene production
ascorbate and glutathione redox
status ubiquitylated proteins heat shock
protein responses apoptosis
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Apoptosis
extracellular signaling event
intracellular signaling event
procaspase 8
procaspase 9
caspase 8
caspase 9
procaspase 8
caspase 3 (effector caspase)
apoptosis
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Caspase 3 is activated during IBA and LT
summer
winter
procaspase
p32
p20
active caspase
p17
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SA
LT
IBA
Results of western blots
Relative band intensity ( maximal)
caspase 3
p17
p20
p32
Relative band intensity ( maximal)
caspase 8
p23
p40
Relative band intensity ( maximal)
caspase 9
p10
p18
p34
p46
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Caspase 3/7 activity assay based on cleavage of
DEVD-AMC
Cold temperatures typical of torpor depress
caspase activity values represent means SE, n
3
Assay is linear values represent means SE, n 3
(arbitrary fluorescence units)
AMC release
Time (min)
Still to come finish temperature profiles
capacity at 0 and 37 C TUNEL assays IAP
studies (IBA??)
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Specific Aim II
Development of an assessable system for the study
of ischemia in a hibernator Squirrels and rats
will be anesthetized, ventilated, and blood flow
will be reduced by aortic clamp will include
shams (expecting an n of 5 to start) Blood flow
monitored via intermittent video monitoring of
the gut mesentery Pilot studies were done and
we can control blood flow to the desired
extent Recently acquired inverted microscope and
video monitoring capability in our own lab Should
begin next week
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Specific Aim II
Development of an assessable system for the study
of ischemia in a hibernator 10 Blood flow for
1 h followed by reperfusion 0 blood flow for 10
min followed by reperfusion Collect brain,
heart, liver, kidney, and jejunum conjugated
diene production ascorbate and glutathione
redox status ubiquitylated proteins heat
shock protein responses apoptosis
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RO1 Development
Exploitation of the assessable system for the
study of ischemia in a hibernator Collaboration
with S. L. Martin (UCHSC) and A. Gracey
(USC) proteomics and microarray use winter vs
summer animals (seasonality issues) summer
animals are intermediate to rats and ground
squirrels in studies of organ preservation LT
vs IBA monocyte adhesion polyunsaturated
fats New PRA- I. Efimov (WUSTL) Current NSF
Careeer and NSF MRI grants
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Hypotheses and Rationale(reasons why I am
interested in protein metabolism)

• Hibernators must balance need to maintain
  cellular homeostasis with low energetic
  availability- basic processes must be depressed
  but how?
• Protein synthesis/metabolism is a basic process
  that can account for as much as 30 of SMR
• Hibernators must exploit the low body
  temperatures of torpor to downregulate basic
  processes i.e. hibernators are not as specialized
  to function at low Tb as was previously thought
• Function of interbout arousal is to allow
  expression of gene products and metabolism of
  protein pools
• Predicts that protein turnover will be inhibited
  during torpor and activated during the interbout
  arousal
• The potential for torpor is not restricted to a
  few unique mammals
• The genetic wiring is inherent to mammalia
• Predicts that the processes that underlie torpor
  are the result of differential gene expression
  rather than expression of de novo genes

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Status of Transcription

• Transcription accounts for as much as 10 of a
  cellular energy budget
• Given the severe reduction in metabolic rate
  inherent with hibernation, hepatic transcription
  should be downregulated
• Bocharova et al., (1992) found that cerebral
  incorporation of uridine was 8.8X lower during
  torpor- problems with experimental method

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Estimation of in vivo transcription

• Mechanistic analysis
• Isolate nuclei and perform run-on assays with
  32P-UTP
• very little in vitro initiation (estimates of
  1-2)
• measures elongation of in vivo-initiated
  transcripts
• Provides a portrait of transcription at the
  moment of isolation
• Allows for assessment of in vivo-relevant
  initiation when performed under optimal
  conditions and can also allow for an examination
  of elongation when assay conditions are altered

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Transcriptional initiation is reduced during
torpor(assay conditions were optimal and linear
rates of incorporation were normalized for
nuclear concentration rates are proportional to
the amount of initiated transcript)
120
100
80
( of interbout aroused rate)
P-UTP incorporation
60
40
20
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0
SA
IBA
ET
LT
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Transcriptional elongation is depressed during
torpor (assay temperature was varied batches of
nuclei contained the same amount of initiated
transcript)
P-UTP incorporation rate
(expressed as 25 C rate)
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Assay temperature (C)
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Conclusions

• Transcriptional initiation is reduced about 2X
  during torpor
• Elongation is arrested at the low temperatures of
  torpor
• No differences between ET and LT initiation
  levels
• Specialized adaptations to permit transcription
  at low temperatures do not appear to be present
  in hibernators
• Hibernators exploit the low Tb of torpor to
  depress transcription and resume gene expression
  during the arousal when Tb is high

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Translation

• in vivo radiolabel incorporation studies
• Depressed to 0.13 to 0.5 of euthermic rates
  during torpor
• Hyperactivated approximately 1.5 - 2 fold during
  the interbout arousal
• Few data for arousal and entrance phases of the
  torpor cycle- incorporation studies don't work
  well
• Utilized polysome distributions to assess
  translation
• Increased association of ribosomes with an mRNA
  (polysomes) is indicative of increased translation

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Northern blots of gradient fractions
Density
monosomes polysomes (light)
(heavy)
ethidium bromide
actin mRNA
28 S rRNA
18 S rRNA
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Translational initiation is inhibited during
torpor
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Translational initiation is inhibited during
torpor
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Translational initiation is inhibited during
torpor
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Initiation is inhibited at 18 C during entrance
into torpor
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eIF4E is constitutively more active in winter
eIF4E binds the 5 mRNA cap and recruits the mRNA
to the 40S subunit
4E
4E
PS
PS
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Expression of 4E-BP1 is seasonal and its activity
is regulated during torpor
Cap-dependent initiation
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Translation lysates

• Direct assessment of temperature effects on
  translational initiation and elongation

• Elongation- endogenous mRNA serves as template
  for translation. Linear rates are compared to
  assess temperature effects on elongation.

-1
S methionine incorporation
CPM 10 µl lysate
35
Time (min)

• Initiation- exogenous mRNA serves as template for
  translation. Endogenous mRNA is degraded with a
  Ca dependent nuclease. Ca is removed. mRNA
  is added. Linear rates are compared to assess
  temperature effects on initiation. Rate is also
  dependent on elongation.

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Translational elongation is passively depressed
S-methionine incorporation
( maximum elongation rate)
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Temperature (C)
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Future directions
Initiation in lysates and isolated hepatocytes
IRES mediated initiation Internal ribosome
entry site mediated initiation is cap-independent
and favors productions of stress proteins May
be critical during arousal Microarray analyses
of polysome and nuclear run-on material- IRES
activity
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Conclusions

• Translational initiation is uncoupled from
  elongation below 18 C
• Translation is not static during torpor. Reduced
  levels of elongation occur during torpor. By the
  end of torpor, there are few polysomes
• Passive and active mechanisms appear to work in
  concert to depress translation during torpor
• Active mechanisms likely favor cap-independent
  translation (IRES) Possible mechanism for global
  differential gene expression Invocation of IRES
  mediated initiation during arousal from torpor
  may facilitate translation of proteins geared
  towards survivorship
• Hibernators exploit the low Tb of torpor to
  depress the bulk of translation and resume gene
  expression during the arousal when Tb is high

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Protein degradation?

• protein synthesis - degradation
• Negligible protein synthesis during torpor
• Average protein half life of 20-30 h
• In the presence of normal degradation, it would
  take 45-70 h to reduce the protein to 20 of its
  original level -- Torpor can last up to 3 weeks!
• Ubiquitin-mediated proteolysis
• Ubiquitin is a highly conserved 76 amino acid
  polypeptide tag that signals proteins for
  degradation
• Most short lived regulatory proteins are degraded
  through this pathway
• Estimates are as high as 90

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Conjugation
E1, E2, E3
AMP PPi
ATP

Ubiquitylated protein
Free ubiquitin
Target protein to be degraded
Peptides and amino acids

Poly-ubiquitylated protein
26S Proteasome
Ubiquitin is recycled
Degradation
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Ubiquitin conjugate levels increase 2-3 X during
torpor
SA
IBA
LT
ubiquitin conjugates
(pmol/µg total protein)
SP
ENT
ARO
IBA
LT
Problem ubiquitin conjugates usually
correlate with increased degradation!
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Assay for proteolysis(note assay is independent
of ubiquitylation status)
suc-LY

26S proteasome
suc-LY-AMC
(non-fluorogenic)
AMC (fluorogenic)
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Proteolytic capacity is unchanged during
torpor(would have expected a difference if
active mechanisms were in place)
IBA
SA
LT
AMC release
Time (min)
substrate suc-Leu-Tyr-AMC assay performed at 37
C with identical amounts of protein data are
means SE
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Proteolysis is depressed at low temperatures
(substrate suc-Leu-Tyr-AMC)
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19S regulatory cap
26S Proteasome
20S core protease
3 distinct major protease activities
peptidyl-glutamyl peptidase-like (caspase-like)
measured with Z-Leu-Leu-glu-?
naphthylamide chymotrypsin-like
(rate-limiting under steady-state)
measured with Suc-Leu-Leu-Val-Tyr-AMC
trypsin-like measured with
Boc-Leu-Arg-Arg-AMC
Are all activities equally depressed by
temperature? Who cares? Estimated 200,000 to 2
million peptides per cell
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Caspase-like activity is depressed
(Z-Leu-Leu-glu-? naphthylamide)
( of maximum)
AMC release
AMC release
( of maximum)
assay temperature (C)
assay temperature (C)
120
RAT lysate
RAT lysate MG115
100
80
60
AMC release
( of maximum)
AMC release
( of maximum)
40
20
0
0
10
20
30
40
assay temperature (C)
assay temperature (C)
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Chymotryptic-like activity is depressed
(Suc-Leu-Leu-Val-Tyr-AMC)
LT lysate
SA lysate
LT lysate MG115
SA lysate MG115
7-amido-4-methylcoumarin (AMC) release
(arbritarary fluorescence units)
(arbritarary fluorescence units)
7-amido-4-methylcoumarin (AMC) release
Assay Temperature (C)
Assay Temperature (C)
IBA lysate
rat lysate
rat lysate MG115
IBA lysate MG115
7-amido-4-methylcoumarin (AMC) release
(arbritarary fluorescence units)
(arbritarary fluorescence units)
7-amido-4-methylcoumarin (AMC) release
A
Assay Temperature (C)
Assay Temperature (C)
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Tryptic-like activity is depressed
(Boc-Leu-Arg-Arg-AMC)
LT lysate
SA lysate
LT lysate MG115
SA lysate MG115
7-amido-4-methylcoumarin (AMC) release
(arbritarary fluorescence units)
(arbritarary fluorescence units)
7-amido-4-methylcoumarin (AMC) release
Assay Temperature (C)
Assay Temperature (C)
IBA lysate
IBA lysate MG115
rat lysate
rat lysate MG115
7-amido-4-methylcoumarin (AMC) release
(arbritarary fluorescence units)
7-amido-4-methylcoumarin (AMC) release
(arbritarary fluorescence units)
Assay Temperature (C)
Assay Temperature (C)
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What about ubiquitylation?
Implication of continued ubiquitylation at low
temperatures would be elevated ubiquitin
conjugates
Assay for ubiquitylation

liver lysate depleted of free ubiquitin ATP
contains target proteins and ubiquitylation
machinery
biotinylated free ubiquitin ATP
poly-ubiquitylated protein
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Ubiquitylation continues at low temperatures
SA
IBA
LT
RAT
100
0 5 10 15 20 25 30 37
80
Temperature (C)
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Ubiquitylation rate
( of maximal)
Ubiquitylation is gt 30 of maximal at low
temperatures
40
20
0
Temperature (C)
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Conclusions

• Ubiquitin conjugate levels are elevated 2-3 X
  during torpor
• High levels of ubiquitin conjugates most likely
  reflect an accumulation of ubiquitylated proteins
• Proteolytic capacity is unchanged by state which
  does not support an active mechanism of
  inhibition
• Proteolysis is depressed at cold temperatures
  typical of torpor
• Actual rate of protein degradation is probably
  even lower since a ubiquitylated protein will be
  harder to degrade than these small peptides
• All activities associated with the proteasome are
  affected similarly
• Ubiquitylation continues at cold temperatures
• Proteolysis regulated at level of proteasome
  rather than ubiquitylation
• Hibernators depress protein degradation during
  torpor but exploit the warm Tb of IBA to degrade
  proteins

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Future directions
ubiquitylation mono vs poly ubiquitylation in
the cold isopeptidase T and other dUB
activities loss of ub conjugates upon arousal
ubiquitylated protein
iso-T
26S proteasome
de-ubiquitylated protein
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Conclusions
Transcription Initiation is moderately depressed
but elongation is arrested at cold temperatures
typical of torpor. Translation The bulk of
initiation is inhibited at 18 C during
entrance into torpor. Regulation of the
initiation factor eIF4E may promote gene
expression geared towards survivorship. Proteolysi
s Ubiquitin-mediated proteolysis is regulated
primarily at the level of 26S proteasome
activity through passive temperature effects.
All data support the hypothesis that a return to
euthermy allows for turnover of proteins
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Acknowledgements

• NSF
• NIH
• American Physiological Society
• Army Research Office

• Sandy Martin
• Hannah Carey , Nahum Sonenberg, Art Haas, Les
  Krushel, Andy Cossins
• My Laboratory
• Vanja Velickovska, Jen Utz, Candice Rausch,
  PeiPei Pan
• Bryan Lloyd, Sarah Dannan, Safdar Qureshi, Julie
  Baker, Amy Tongsiri, Anastacia Shmereva, Rose
  Gentles, Monica Modi

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(CPM)
-1
S methionine incorporation 10 µl lysate
35
Time (min)
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(CPM)
-1
S methionine incorporation 10 µl lysate
35
Time (min)
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E1
26S Proteasome
Ub

ATP
Ub
Ub
ATPase
Ub
ADP
Target Protein
E1
Ub
S
E2
/ -
E2
E1
Ub
S
E3
E3
Ub
Ub
Amino Acids

Ub
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Polysome profiles
Spermophilus lateralis
Frozen livers pulverized, cell debris removed
Sucrose gradient to fractionate
6 Groups ET-early torpor hibernators LT- late
torpor hibernators IBA- interbout aroused SA-
summer active ENT- entrance ARO-arousal
254 nM
Analyze RNA distribution
Light heavy
ISCO flow cell
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Proteomics
Problem Rat or human livers destined for
transplantation can withstand 24-48 h of cold
storage. Ground squirrel livers withstand cold
storage much better.
rat
ground squirrel
liver)
-1
g
-1
Goal Identify those proteins that are associated
with the difference in survivorship of rat and
ground squirrel livers.
LDH release (unitsl
cold storage period (h)
Susanne Lindell, Jim Southard and Hannah Carey
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Conservation of mechanism?
Artemia franciscana embryos
Great Salt Lake
diapause embryo
environmental cues
activating conditions
non-activating conditions
activated embryo
unfavorable conditions
favorable conditions
quiescent embryo
nauplius
diapause embryo
redrawn after Drinkwater and Clegg, 1991
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Quiescence in Artemia franciscana embryos
Very low metabolic rates 0.02 of aerobic heat
dissipation Rapid fall in pHi pH 7.7-7.9 in
normoxic embryo pH 6.8-6.3 in anoxic
embryo Associated decrease in ATP Translation
is depressed gt 90 Survivorship after 5 years is
65 What happens to protein
degradation? only really have ubiquitin-dependent
proteolysis available to them
no temperature change associated with metabolic
depression!
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Total ubiquitin conjugates decline under anoxia
pmol conjugated ubiquitin mg protein-1
Period of anoxic exposure (h)
Anchordoguy and Hand, 1994
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Mitochondrial ubiquitylation decreases during
anoxia
ubiquitin conjugates arbitrary phosphorimager
units
Time (minutes)
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Ubiquitin-dependent proteolysis is reduced when
ATP is being actively hydrolyzed
ATP is being regenerated
ATP is being hydrolyzed but is still in
saturating levels (no ? in pH)
CPM released 0.1 ml supernatant-1
Control
Time (h)
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pH dependence of ubiquitin-dependent proteolysis
ATP/ubiquitin dependent proteolysis ( pH 7.7
rate)
pH
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Conclusions
Ubiquitin conjugate concentrations drop in both
whole cell and mitochondrial fractions during
quiescence Proteolysis is also reduced under the
conditions of quiescence (low pH and hydrolyzed
ATP) Artemia franciscana embryos downregulate
protein degradation at the level of
ubiquitylation and to a lesser degree proteolysis
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The mother of all two species comparisons!

• 35/36 major phyla have metabolic depressions-
  should a mechanism that is
• fundamental towards effecting a metabolic
  depression be conserved?
• Mechanisms of proteolytic block are fundamentally
  different
• between hibernators and quiescent Artemia spp.
  embryos
• Hibernators regulate at level of proteolysis not
  ubiquitylation
• Artemia embryos regulate primarily at level of
  ubiquitylation and
• somewhat at level of proteolysis
• Reflects both availability of cues and efficacy
  requirement?
• degree and sustainability of metabolic
  depression
• Role of key processes in metabolic depression
• consequence vs causative mechanism?
• passive vs active
• evolution of metabolic depression

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