The Unfolded Protein Response (UPR) & The Endoplasmic Reticulum Associated Degradation (ERAD)

1. The Unfolded Protein Response (UPR)& The Endoplasmic Reticulum Associated Degradation (ERAD) ZsuzsaBebok Department of Cell Biology bebok@uab.edu Ph: 975-5449

2. The central role of the ER in secretory and membrane protein synthesis • (1980s: Tom Rapoport: Protein translocation across and integration into membranes) 1985. Amy Lee: A Calcium ionophore induces the expression of glucose-regulated genes (GRP78). 1988. Lippincott-Schwartz & Klausner, R. D. Degradation from the endoplasmic reticulum 1991. A. Helenius’s lab: Quality control in the ER: misfolding of the VSV G UPR & ERAD 1986. John Kearney’s lab : Posttranslational association of Ig(h) chain binding protein (BiP) with nascent heavy chains in hybridomas. Sommer, & Jentsch, A protein translocation defect linked to ubiquitin conjugation at ER 1988. Hendershot, L. M.Ting, J.Lee, A. S. Identity (BiP) with (GRP78) 1996. J.Brodsky: Proteasome-dependent ERAD: an unconventional route to a familiar fate 1990. R. Kaufman’s and lab: The ER stress response. Morimoto, R.I.: Protein conformation (misfolding) and ER stress (UPR)

3. How can proteins cross and integrate into the ER membrane? Tom A. Rapoport, Nature 450, 663-669 (29 November 2007)

4. Protein translocation across the eukaryotic endoplasmic reticulum membrane Tom A. Rapoport, Nature 450, 663-669 (29 November 2007)

5. U-turn in the ER – What happens to unwanted or damaged proteins? • Klausneret al: T-cell receptor complex • non-stoichiometric synthesis of components • forward trafficking of assembled receptors only • Isolated α and μ subunits degraded in non-lysosomal compartments ER protease Retrotranslocation • A yeast vacuolar protease carboxypeptidase Y (Cpy*) • A mammalian ER resident protease (ER60) • was isolated and proposed to be the quality-control protease • Sommer & Jentsch: Sec61 mutation – ubiquitination • Disruption of UBC6 (yeast) – no degradation • CFTR degradation by the proteasome

6. Sec61 Sec61 Derlin Derlin VCP AAA ERAD ER CYTOSOL

7. The steps of ERAD Vembar & Brodsky Nat Rev Mol Cell Biol. 2008 December; 9(12): 944–957.

8. N-linked glycosylation and the degradation of glycosylated proteins

9. Hydrophobic patches: is being 'oily' sufficient for ERAD? What happens to to proteins with large cytosolic domains?

10. Damaged proteins are tagged The ubiquitylation process

11. What happens when the function of the ER is disturbed?“perturbation of ER function” STRESS

12. What is ER stress? Proteasome dysfunction Hypoxia Post-synthetic modifications Reactive Species Saturated fatty acids ER STRESS Recombinant proteins Inflammation Starvation Protein misfolding Cigarette smoke High cholesterol 2. Decrease load 1. Increase capacity UPR Membrane components Folding enzymes Chaperones ERAD Protein Synthesis mRNA decay Transcription

13. The Mammalian UPR UPR target genes NUCLEUS STRESS ATF4 ATF6 ATF6α XBP1 BiP ER LUMEN IRE1α P S2P ATP PERK sXBP1mRNA ATF4 P ATP GOLGI P P P ATP ATP eIF2α eIF2α CYTOSOL

14. The adaptive and apoptotic pathways of the UPR Micro-RNAs????

15. Adaptation to stress – chronic stress • Genetic • Insulin secreting β-cells are very sensitive to genetic defects in PERK, ATF6, Wolframin (Wolfram Syndrome) • Akita mouse– Insulin2 gene mutation prevents oxidative insulin folding – UPR - diabetes • Environmental • High cholesterol, alcohol, cigarette smoke… • Pathogenic • Hepatitis C virus – latent infection and carcinogenesis • Developmental • B cell development – antibody secretion by plasma cells

16. Diseases associated with ER stress • Obesity (leptin signaling) • Type I diabetes (insulin production) • Type II diabetes (insulin receptor signaling) • Necrotizing enterocolitis (?) • Neurological diseases (Alzheimer, Parkinson’s) • Psychiatric disorders (?) • Airway diseases (COPD, asthma, chronic bronchitis..) The pathomechanism of these diseases varies

17. Can we modulate the UPR? 2. Decrease load 1. Increase capacity Autophagy Membrane components Folding enzymes Chaperones ERAD Protein Synthesis mRNA decay Transcription Acute phase of stress - aspecific Reduce toxicity – toxic aggregates • Help to reduce aggregation • Improve trafficking • Reduce toxicity

18. Take home message You can run from the UPR – ERAD, autophagy You can hide from ERAD – modulate the UPR and enhance protein folding and rescue proteins from ERAD At the end they are still going to get you!

19. Connection of the UPR to other cellular pathways

20. Proposed models for UPR-mediated JNK and NFκ-B activation (IκB – short half life) Kezhong Zhang & Randal J. Kaufman Nature 454, 455-462(24 July 2008) doi:10.1038/nature07203

21. ER-stress-induced acute-phase response Inflammatory cytokines (TNFα) CREBH transcription  Kezhong Zhang & Randal J. Kaufman Nature 454, 455-462(24 July 2008) doi:10.1038/nature07203

22. Questions • A disease is caused by the misfolding and very efficient ERAD of a membrane protein. • How would you modulate ERAD? • A disease is caused by the formation of toxic protein aggregates that activate the UPR and apoptosis • How would you modulate the UPR and ERAD?