The Unfolded Protein Response (UPR) & The Endoplasmic Reticulum Associated Degradation (ERAD) Zsuzsa Bebok Department of Cell Biology Ph:

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

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

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

U-turn in the ER – What happens to unwanted or damaged proteins? Klausner et 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

ERAD Derlin VCP AAA Sec61 CYTOSOL ER

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

N-linked glycosylation and the degradation of glycosylated proteins

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

Damaged proteins are tagged The ubiquitylation process

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

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

ATF6α UPR target genes ATF6 IRE1α PERK P ATP P PS2P P BiP eIF2α P NUCLEUS ER LUMEN CYTOSOL GOLGI STRESS AT F4 sXBP1mRNA The Mammalian UPR XBP1 ATF4 ATP

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

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

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

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

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! Take home message

Connection of the UPR to other cellular pathways

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

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

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?