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.) Hypothesis 3.) Methods Endoplasmic Reticulum Endoplasmic Reticulum Stress Response 2.) Hypothesis 3.) Methods 4.) Results 5.) Conclusions 6.) 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 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.

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

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

ER IRE 1 PERK Phos Phos eIF2α Nrf2

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

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

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

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

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

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

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

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

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?

ER Stress Aging HHMI 2005

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

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

NQO1 mRNA Levels Percent of Control mRNA NQO1/B-Actin Young Old Young 4 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

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

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

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)