1. Loss Of Nrf2 Dependent Signaling Following Induction of Endoplasmic Reticulum Stress
Anahita Fallahi Brian Dixon Tory Hagen, Ph.D. Dept. of Biochemistry/Biophysics The Linus Pauling Institute Oregon State University

2. PRESENTATION OUTLINE 1.) Introduction 2.) Hypothesis 3.) Methods
Endoplasmic Reticulum
Endoplasmic Reticulum Stress
Response
2.) Hypothesis
3.) Methods
4.) Results
5.) Conclusions
6.) Future Research

3. 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.

4. ER Stress Causes Disrupting calcium homeostasis Virus Oxidative Stress
Results in the accumulation of unfolded/misfolded proteins which can threaten the cell.
Causes
Disrupting calcium homeostasis
Virus
Oxidative Stress
Altered Glycosylation ER
After altered glycosylation talk about how certain antibiotics work by preventing the maturation of proteins in bacteria.

5. How do you deal with stress?
CELL
ER Stress ER
DEATH
RESPONSE
Survival

6. BiP Activates Kinase PERK
Normal Conditions
Stress Conditions
BiP is released from PERK
Bip binds to
unfolded/misfolded
proteins
BiP
BiP
IRE 1 α
IRE 1 α
PERK
PERK
Phos
Phos
Phos
Phos
Perk dimerizes and becomes phosphorylated.
Cytoplasm

7. ER
IRE 1
PERK
Phos
Phos
eIF2α
Nrf2

8. PERK-eIF2α Pathway Stress Conditions ER Normal Conditions PERK
Phos
eIF2α
eIF2α is phosphorylated preventing the formation of the translation complex.
Decreases the amount of total mRNA translation of proteins and reduces workload on stressed ER. It can now selectively translate specific mRNA.
eIF2-GTP-tRNA
Initiates Translation
Cytoplasm

9. PERK-Nrf2 Pathway Cytoplasm Stress Conditions Nucleus ER
Normal Conditions
PERK
Phos
Phos
Nrf2
Phos
Nrf2
Nrf2
Nrf2
Keap 1
Keap 1
Nrf2 is phosphorylated and dissociates from Keap 1
Keap 1
Nrf2 translocates to nucleus
Nrf2 is bounded to the
cytoskeleton anchor Keap 1
leaving Nrf2 inactive
Cytoplasm

10. Nrf2 Turns on Detoxification Genes
Cytoplasm
Free Nrf2 enters nucleus and binds to ARE sequence
(Antioxidant Response Element)
Nrf2
Phos
NQO1
ARE
GSH
Promotes expression
of phase 2 detoxification enzymes such as NQO1 and GSH that promote cell survival.
Nucleus
Cytoplasm

11. PERK ER IRE 1 Nrf2 NQO1 eIF2α Phase II detoxification enzymes
Phos
Phos
eIF2α
Nrf2
Phos
Phos
Phase II
detoxification
enzymes
Decreased protein load
NQO1

12. IRE 1α-CHOP Pathway CHOP I ATF 4 ATF 4 Stress Conditions ER Nucleus
Phos
ATF 4
ATF4 translocates to the
nucleus and upregulates the
expression of the transcription factor CHOP
IRE 1α
Phos
IRE 1α phosphorylates the transcription factor ATF4
Nucleus
CHOP
Cytoplasm

13. CHOP Nucleus Cytoplasm Pro-Apoptotic Genes Cytoplasm
CHOP enters the nucleus and upregulates the expression of pro-apoptotic genes.
Pro-Apoptotic Genes
Nucleus
Cytoplasm

14. PERK ER SURVIVAL DEATH IRE 1 ATF 4 CHOP Nrf2 NQO1 eIF2α
Phos
Phos
SURVIVAL
DEATH
eIF2α
ATF 4
Phos
Nrf2
Phos
Phos
Decreased protein load
NQO1
CHOP

15. ER stress is worth stressing over…
ER stress has been linked to the following medical conditions: A G E
Parkinson’s
Huntington’s
Alzheimer's
Heart Disease

16. Do cells lose their ability to respond to ER stress with age?
Aging
Do cells lose their ability to respond to ER stress with age?

17. ER Stress
Aging
HHMI 2005

18. Hypothesis 1.) Cells are more susceptible to ER stress with age.
2.) Signaling between Nrf2
and PERK alters with age.

19. Methods CHOP Tunicamyacin QPCR Western Blots Young Hepatocytes
Old Hepatocytes
Tunicamyacin
QPCR
Western Blots
Nrf2
Phos
eIF2α
Nucleus
NQO1
CHOP

20. NQO1 mRNA Levels Percent of Control mRNA NQO1/B-Actin Young Old Young4 12 24
0.0
0.1
0.2
0.3 4 12 24
100
150
200
250
300
350
400
450 **
N.S.
N.S.
N.S. *
Percent of Control
mRNA NQO1/B-Actin
N.S.
Young
Time (hrs)
Old
Time (hrs)
Young
Time (hrs)
Old
Time (hrs)
N=3;*p<0.05 vs. Control; **p<0.01 vs. Control; N.S. (not significant) vs. Control

21. Nrf2 Nuclear Localization
Phos
Nucleus
Nrf2
Young
Time (hrs)
Old
Percent of Control 4 12 24
100
150
200
250
300
Young
Time (hrs)
Old
N.S. * 4 12 24
10000
20000
30000
40000
50000
Densitometry
N=3;*p<0.05 vs. Control; **p<0.01 vs. Control; N.S. (not significant) vs. Control

22. PERK ER Conclusion IRE 1 ATF 4 CHOP Nrf2 NQO1 Still needs to be
Phos
Phos
Still needs
to be
looked at
eIF2α
Nrf2
ATF 4
Phos
Phos
Decreases
with age
Does not alter with age.
Phos
Decreased protein load
NQO1
CHOP

23. Acknowledgements Special Thanks Dr. Tory Hagen Brian Dixon
Dr. Kevin Ahern
The Hagen Lab
Brian Dixon
Sesha Duvvuri
Tory Hagen
Du Heath
Alex Michels
Jeff Monette
Regis Moreau
Kate Peterson-Shay
Swapna Shenvi
Oregon State University
Dept. Biochemistry/Biophysics
Funding Agency
Howard Hughes Medical Institute (HHMI)