The Unfolded Protein Response (UPR) & The Endoplasmic Reticulum Associated Degradation (ERAD) Zsuzsa Bebok Department of Cell Biology [email protected] Ph: 975-5449 The central role of the ER in secretory and membrane protein synthesis (1980s: Tom Rapoport: Protein translocation across and integration into membranes) 1988. Lippincott-Schwartz & Klausner, R. D. Degradation from the endoplasmic reticulum 1985. Amy Lee: A Calcium ionophore induces the expression of glucoseregulated genes (GRP78). 1986. John Kearney’s lab : Posttranslational association of Ig(h) chain binding protein (BiP) with nascent heavy chains in hybridomas. 1988. Hendershot, L. M.Ting, J.Lee, A. S. Identity (BiP) with (GRP78) UPR & ERAD 1991. A. Helenius’s lab: Quality control in the ER: misfolding of the VSV G Sommer, & Jentsch, A protein translocation defect linked to ubiquitin conjugation at ER 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) How can proteins cross and integrate into the ER membrane? Tom A. Rapoport, Nature 450, 663-669 (29 November 2007) Protein translocation across the eukaryotic endoplasmic reticulum membrane Tom A. Rapoport, Nature 450, 663-669 (29 November 2007) 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*) •Sommer & Jentsch: Sec61 mutation – ubiquitination •A mammalian ER resident protease (ER60) was isolated and proposed to be the quality-control protease •Disruption of UBC6 (yeast) – no degradation •CFTR degradation by the proteasome ERAD Derlin AAA CYTOSOL VCP Derlin Sec61 Sec61 ER The steps of ERAD Vembar & Brodsky Nat Rev Mol Cell Biol. 2008 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? Proteasome dysfunction Hypoxia Post-synthetic modifications Reactive Species Saturated fatty acids ER STRESS Recombinant proteins Inflammation Starvation Cigarette smoke 1. Increase capacity Membrane components Folding enzymes Chaperones High cholesterol UPR ERAD Protein misfolding 2. Decrease load Protein Synthesis mRNA decay Transcription The Mammalian UPR NUCLEUS UPR target genes ATF4 ATF6α XBP1 STRESS BiP ER LUMEN P ATP P ATP GOLGI PeIF2α CYTOSOL eIF2α P P ATP 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? 2. Decrease load 1. Increase capacity Autophagy Membrane components Folding enzymes Chaperones ERAD Protein Synthesis mRNA decay Transcription Acute phase of stress - aspecific •Help to reduce aggregation •Improve trafficking •Reduce toxicity Reduce toxicity – toxic aggregates 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! Connection of the UPR to other cellular pathways 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 ER-stressinduced acute-phase response Inflammatory cytokines (TNFα) CREBH transcription Kezhong Zhang & Randal J. Kaufman Nature 454, 455-462(24 July 2008) doi:10.1038/nature07203 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?