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
PRESENTATION OUTLINE
1.) Introduction
–
–
–
2.)
3.)
4.)
5.)
6.)
Endoplasmic Reticulum
Endoplasmic Reticulum Stress
Response
Hypothesis
Methods
Results
Conclusions
Future Research
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.
ER Stress
Results in the accumulation of unfolded/misfolded proteins which can threaten the cell.
Causes
•Disrupting calcium
homeostasis
•Virus
•Oxidative Stress
•Altered Glycosylation
ER
How do you deal with stress?
ER Stress
ER
DEATH
CELL
RESPONSE
Survival
BiP Activates Kinase PERK
Normal Conditions
ER
Stress Conditions
BiP is released
from PERK
Bip binds to
unfolded/misfolded
proteins
BiP
BiP
PERK
PERK
Phos
Phos
IRE 1 α IRE 1 α
Phos
Perk dimerizes and becomes phosphorylated.
Cytoplasm
Phos
ER
PERK
eIF2α
Nrf2
IRE 1
Phos
Phos
PERK-eIF2α Pathway
Stress Conditions
ER
Normal Conditions
eIF2α
PERK
Phos
eIF2α
eIF2α is phosphorylated
preventing the formation of
the translation complex.
eIF2-GTP-tRNA
Initiates Translation
Cytoplasm
Decreases the amount of
total mRNA translation of
proteins and reduces
workload on stressed ER. It
can now selectively
translate specific mRNA.
PERK-Nrf2 Pathway
Stress Conditions
ER
Normal Conditions
Nucleus
PERK
Phos
Phos
Nrf2
Keap 1
Nrf2
Nrf2 is phosphorylated
and dissociates from
Keap 1
Nrf2 is bounded to the
cytoskeleton anchor Keap 1
leaving Nrf2 inactive
Cytoplasm
Keap 1
Nrf2 translocates to nucleus
Nrf2 Turns on Detoxification Genes
Free Nrf2 enters nucleus and
binds to ARE sequence
(Antioxidant Response
Element)
Phos
Nrf2
NQO1
ARE
Cytoplasm
GSH
Nucleus
Cytoplasm
Promotes expression
of phase 2 detoxification
enzymes such as NQO1
and GSH that promote
cell survival.
ER
PERK
eIF2α
Nrf2
IRE 1
Phos
Phos
Phos
Phase II
detoxification
enzymes
Decreased
protein load
NQO1
Phos
IRE 1α-CHOP Pathway
Stress Conditions
ER
ATF 4
IRE 1α
Phos
Phos
IRE 1α phosphorylates
the transcription factor
ATF4
ATF4 translocates
to the
nucleus and
upregulates the
expression of the
transcription
factor CHOP
I
CHOP
Nucleus
Cytoplasm
CHOP
CHOP enters the nucleus and
upregulates the expression of proapoptotic genes.
Cytoplasm
Nucleus
Cytoplasm
Pro-Apoptotic
Genes
ER
PERK
IRE 1
Phos
Nrf2
SURVIVAL
eIF2α
Phos
Phos
Phos
DEATH
ATF 4
Phos
Decreased
protein load
NQO1
CHOP
ER stress is worth stressing over…
ER stress has been linked to the following medical conditions:
Parkinson’s
Huntington’s
Alzheimer's
Heart Disease
A
G
E
ER Stress
Aging
Do cells lose their ability
to respond to ER stress
with age?
HHMI 2005
ER Stress
Aging
Hypothesis
1.) Cells are more susceptible
to ER stress with age.
2.) Signaling between Nrf2
and PERK alters with age.
Methods
Old Hepatocytes
Young Hepatocytes
Tunicamyacin
Western Blots
QPCR
NQO1
Phos
Nrf2
eIF2α
Nucleus
Phos
CHOP
NQO1 mRNA Levels
N.S.
**
0.3
450
400
N.S.
350
0.2
N.S.
*
300
N.S.
250
200
0.1
150
100
0.0
0
4
12
24
0
4
12
24
0
4
12
24
0
4
12
Young
Old
Young
Old
Time (hrs)
Time (hrs)
Time (hrs)
Time (hrs)
N=3;*p<0.05 vs. Control; **p<0.01 vs. Control; N.S. (not significant) vs. Control
24
Phos
Nrf2
Nrf2 Nuclear Localization
Nucleus
Nrf2
50000
300
*
40000
N.S.
N.S.
30000
N.S.
250
200
N.S.
20000
150
N.S.
10000
100
0
0
4
12
24
0
4
12
24
0
4
12
24
0
4
12
Young
Old
Young
Old
Time (hrs)
Time (hrs)
Time (hrs)
Time (hrs)
N=3;*p<0.05 vs. Control; **p<0.01 vs. Control; N.S. (not significant) vs. Control
24
Conclusion
ER
PERK
Still needs
to be
looked at
eIF2α
Nrf2
IRE 1
Phos
Phos
Phos
Phos
Decreases
with age
ATF 4
Phos
Decreased
protein load
NQO1
CHOP
Does not alter with
age.
Acknowledgements
Special Thanks
Dr. Tory Hagen
Brian Dixon
Dr. Kevin Ahern
Oregon State University
Dept. Biochemistry/Biophysics
The Hagen Lab
Brian Dixon
Sesha Duvvuri
Tory Hagen
Du Heath
Alex Michels
Jeff Monette
Regis Moreau
Kate Peterson-Shay
Swapna Shenvi
Funding Agency
Howard Hughes Medical Institute (HHMI)