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

PRESENTATION OUTLINEPRESENTATION OUTLINE1.) Introduction

– Endoplasmic Reticulum – Endoplasmic Reticulum Stress– Response

2.) Hypothesis3.) Methods4.) Results5.) Conclusions6.) 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 StressER

Results in the accumulation of unfolded/misfolded proteins which can threaten the cell.

Causes•Disrupting calcium homeostasis

•Virus

•Oxidative Stress

•Altered Glycosylation

How do you deal with stress?

CELLER Stress ER

RESPONSE

DEATH

Survival

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

ER

PERK IRE 1

eIF2α Nrf2

PhosPhos PhosPhos

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

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

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

ER

PERK IRE 1

eIF2α Nrf2

NQO1

PhosPhos

PhosPhos

PhosPhos

PhosPhos

Decreased protein load

Phase II detoxification enzymes

I

IRE 1α-CHOP Pathway Stress Conditions

ER

Cytoplasm

IRE 1α

ATF 4PhosPhos

Nucleus

ATF 4PhosPhos

CHOP

IRE 1α phosphorylates the transcription factor ATF4

ATF4 translocates to thenucleus and upregulates the expression of the transcription factor CHOP

Cytoplasm

Nucleus

Cytoplasm

CHOP

Pro-Apoptotic Genes

CHOP enters the nucleus and upregulates the expression of pro-apoptotic genes.

ER

PERK IRE 1

eIF2α Nrf2

NQO1

PhosPhos

PhosPhos

PhosPhos

PhosPhos

Decreased protein load

ATF 4PhosPhos

SURVIVAL

CHOP

DEATH

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:

ER Stress Aging

Do cells lose their ability to respond to ER stress

with age?

ER Stress Aging

HHMI 2005

HypothesisHypothesis

1.) Cells are more susceptible to ER stress with age.

2.) Signaling between Nrf2 and PERK alters with age.

Methods

QPCR

Tunicamyacin

Young Hepatocytes Old Hepatocytes

NQO1

Western Blots

Nrf2PhosPhos

eIF2α PhosPhosNucleus

CHOP

YoungTime (hrs)

OldTime (hrs)

mR

NA

NQ

O1/

B-A

ctin

YoungTime (hrs)

OldTime (hrs)

Perc

ent o

f 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

100150200250300350400450

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)

Perc

ent o

f Con

trol

0 4 12 24 0 4 12 24

100

150

200

250

300

Nrf2PhosPhos

Nucleus

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

AcknowledgementsAcknowledgements

Funding AgencyHoward 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