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Loss Of Nrf2 Dependent Signaling Following Induction of Endoplasmic Reticulum Stress

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. 1.) Introduction Endoplasmic Reticulum Endoplasmic Reticulum Stress

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Loss Of Nrf2 Dependent Signaling Following Induction of Endoplasmic Reticulum Stress

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

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

  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 Results in the accumulation of unfolded/misfolded proteins which can threaten the cell. • Causes • Disrupting calcium homeostasis • Virus • Oxidative Stress • Altered Glycosylation ER

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

  6. BiP Activates Kinase PERK ER 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. eIF2α PERK ER IRE 1 Phos Phos Nrf2

  8. eIF2α eIF2α PERK-eIF2α Pathway Stress Conditions ER Normal Conditions PERK Phos 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. Phos Nrf2 Keap 1 Keap 1 PERK-Nrf2 Pathway Stress Conditions Nucleus ER Normal Conditions PERK Phos Phos Nrf2 Nrf2 Nrf2 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. Phos Nrf2 Nrf2 Turns on Detoxification Genes Cytoplasm Free Nrf2 enters nucleus and binds to ARE sequence (Antioxidant Response Element) NQO1 ARE GSH Promotes expression of phase 2 detoxification enzymes such as NQO1 and GSH that promote cell survival. Nucleus Cytoplasm

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

  12. ATF 4 Phos IRE 1α-CHOP Pathway I Stress Conditions ER 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. Cytoplasm CHOP CHOP enters the nucleus and upregulates the expression of pro-apoptotic genes. Pro-Apoptotic Genes Nucleus Cytoplasm

  14. ATF 4 eIF2α Phos PERK ER IRE 1 Phos Phos SURVIVAL DEATH 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. ER Stress Aging Do cells lose their ability to respond to ER stress with age?

  17. Aging ER Stress HHMI 2005

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

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

  20. 0.3 450 400 350 0.2 300 250 200 0.1 150 100 0.0 0 4 12 24 0 4 12 24 0 4 12 24 0 4 12 24 NQO1 mRNA Levels ** 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. Nucleus 50000 * 40000 N.S. N.S. N.S. Densitometry 30000 Percent of Control N.S. 20000 N.S. 10000 0 0 4 12 24 0 4 12 24 Young Time (hrs) Old Time (hrs) Young Time (hrs) Old Time (hrs) 300 Phos Nrf2 250 200 150 100 0 4 12 24 0 4 12 24 Nrf2 Nuclear Localization Nrf2 N=3;*p<0.05 vs. Control; **p<0.01 vs. Control; N.S. (not significant) vs. Control

  22. ATF 4 eIF2α Phos Conclusion PERK ER IRE 1 Phos Phos Still needs to be looked at Nrf2 Decreases with age Does not alter with age. Phos 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 Funding Agency Howard Hughes Medical Institute (HHMI) Oregon State University Dept. Biochemistry/Biophysics

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