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Anahita Fallahi Brian Dixon
Tory Hagen, Ph.D.Dept. of Biochemistry/Biophysics
The Linus Pauling InstituteOregon State University
Loss Of Nrf2 Dependent Loss Of Nrf2 Dependent Signaling Following Induction of Signaling Following Induction of Endoplasmic Reticulum Stress Endoplasmic Reticulum Stress
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PRESENTATION OUTLINEPRESENTATION OUTLINE1.) Introduction
– Endoplasmic Reticulum – Endoplasmic Reticulum Stress– Response
2.) Hypothesis
3.) Methods
4.) Results
5.) Conclusions
6.) Future Research
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Endoplasmic Reticulum (ER)
• Membranous network within the cell that processes and folds 1/3 of all proteins.
• The ER makes up approximately 12% of the cell’s volume.
• The ER plays an important role in maintaining calcium homeostasis.
• Anything that affects the ability of the ER to mature proteins can potentially damage the cell.
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ER Stress
ER
Results in the accumulation of unfolded/misfolded proteins which can threaten the cell.
Causes•Disrupting calcium homeostasis
•Virus
•Oxidative Stress
•Altered Glycosylation
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How do you deal with stress?
CELL
ER Stress ER
RESPONSE
DEATH
Survival
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BiP Activates Kinase PERK
Normal Conditions
PERK
Stress Conditions
BiP is released from PERK Bip binds to
unfolded/misfolded proteins
Perk dimerizes and becomes phosphorylated.
ER
BiP
PERK
BiP
PhosPhos PhosPhos
Cytoplasm
IRE 1 α IRE 1 αPhosPhos PhosPhos
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ER
PERK IRE 1
eIF2α Nrf2
PhosPhos PhosPhos
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PERK-eIF2α Pathway
eIF2α
Normal Conditions
eIF2-GTP-tRNA Initiates Translation
eIF2α
Stress Conditions
Decreases the amount of total mRNA translation of proteins and reduces workload on stressed ER. It can now selectively translate specific mRNA.
eIF2α is phosphorylated preventing the formation of the translation complex.
PERK
ER
PhosPhos
Cytoplasm
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Nrf2PhosPhos
PERK-Nrf2 Pathway
Normal Conditions
Nrf2
Keap 1
Nrf2 is bounded to the cytoskeleton anchor Keap 1 leaving Nrf2 inactive
Stress Conditions
Nrf2 translocates to nucleus
Nrf2 is phosphorylated and dissociates from Keap 1
Cytoplasm
PERK
ER
Nucleus
Nrf2
Keap 1
PhosPhos
Nrf2PhosPhos
Keap 1
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Cytoplasm
Nrf2 Turns on Detoxification Genes
ARE
Nucleus
Free Nrf2 enters nucleus and binds to ARE sequence(Antioxidant Response Element)
NQO1
GSH
Promotes expressionof phase 2 detoxification enzymes such as NQO1 and GSH that promote cell survival.
Nrf2PhosPhos
Cytoplasm
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ER
PERK IRE 1
eIF2α Nrf2
NQO1
PhosPhos
PhosPhos
PhosPhos
PhosPhos
Decreased protein load
Phase II detoxification enzymes
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I
IRE 1α-CHOP Pathway Stress Conditions
ER
Cytoplasm
IRE 1α
ATF 4
PhosPhos
Nucleus
ATF 4
PhosPhos
CHOP
IRE 1α phosphorylates the transcription factor ATF4
ATF4 translocates to thenucleus and upregulates the expression of the transcription factor CHOP
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Cytoplasm
Nucleus
Cytoplasm
CHOP
Pro-Apoptotic Genes
CHOP enters the nucleus and upregulates the expression of pro-apoptotic genes.
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ER
PERK IRE 1
eIF2α Nrf2
NQO1
PhosPhos
PhosPhos
PhosPhos
PhosPhos
Decreased protein load
ATF 4PhosPhos
SURVIVAL
CHOP
DEATH
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ER stress is worth stressing over…
Parkinson’s
AGE
Huntington’s
Alzheimer's
Heart Disease
ER stress has been linked to the following medical conditions:
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ER Stress Aging
Do cells lose their ability to respond to ER stress
with age?
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ER Stress Aging
HHMI 2005
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HypothesisHypothesis
1.) Cells are more susceptible to ER stress with age.
2.) Signaling between Nrf2 and PERK alters with age.
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Methods
QPCR
Tunicamyacin
Young Hepatocytes Old Hepatocytes
NQO1
Western Blots
Nrf2PhosPhos
eIF2α PhosPhosNucleus
CHOP
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YoungTime (hrs)
OldTime (hrs)
mR
NA
NQ
O1/
B-A
ctin
YoungTime (hrs)
OldTime (hrs)
Per
cen
t of
Con
trol
NQO1 mRNA Levels
N.S.
N.S.
N.S.
N.S.**
*
N=3;*p<0.05 vs. Control; **p<0.01 vs. Control; N.S. (not significant) vs. Control
0 4 12 24 0 4 12 240.0
0.1
0.2
0.3
0 4 12 24 0 4 12 24
100
150
200
250
300
350
400
450
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Nrf2 Nuclear Localization
N=3;*p<0.05 vs. Control; **p<0.01 vs. Control; N.S. (not significant) vs. Control
Nrf2
YoungTime (hrs)
OldTime (hrs)
N.S.
*
N.S.
N.S.
N.S.N.S.
0 4 12 24 0 4 12 240
10000
20000
30000
40000
50000
Den
sito
met
ry
YoungTime (hrs)
OldTime (hrs)
Per
cen
t of
Con
trol
0 4 12 24 0 4 12 24
100
150
200
250
300
Nrf2PhosPhos
Nucleus
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ER
PERK IRE 1
eIF2α Nrf2
NQO1
PhosPhos
PhosPhos
PhosPhos
PhosPhos
Decreased protein load
ATF 4PhosPhos
CHOP
Does not alter with age.
Decreases with age
Conclusion
Still needs to be looked at
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AcknowledgementsAcknowledgements
Funding Agency
Howard Hughes Medical Institute (HHMI)
Special ThanksDr. Tory Hagen
Brian DixonDr. Kevin Ahern
The Hagen LabBrian Dixon
Sesha DuvvuriTory HagenDu Heath
Alex MichelsJeff Monette
Regis MoreauKate Peterson-Shay
Swapna ShenviOregon State UniversityDept. Biochemistry/Biophysics