Abstract
Upon ER stress cells activate the unfolded protein response through PERK, IRE1 and ATF6. Remarkable effort has been made to delineate the downstream signaling of these three ER stress sensors after activation, but upstream regulation at the ER luminal site still remains mostly undefined. Here we report that the thiol oxidoreductase PDI is mandatory for activation of the PERK pathway in HEK293T as well as in human pancreatic, lung and colon cancer cells. Under ER stress, depletion of PDI selectively abrogated eIF2α phosphorylation, induction of ATF4, CHOP and even BiP. Furthermore, we could demonstrate that PDI prevented degradation of activated PERK by the 26S proteasome and therefore contributes to maintained PERK signaling. As a result of decreased PERK activity, PDI depleted cells showed an increased vulnerability to ER stress induced by chemicals or ionizing radiation in 2D as well as in 3D culture models. We conclude that PDI is an obligatory regulator of the PERK pathway with future therapy implications.
Highlights
Abbreviations activating transcription factor 4 (ATF4) Activating transcription factor 4 activating transcription factor 6 (ATF6) Activating transcription factor 6 binding immunoglobulin protein (BiP) Binding immunoglobulin protein C/EBPhomologous protein (CHOP) C/EBP-homologous protein CNPY2 Protein canopy homolog 2 DMSO Dimethyl sulfoxide Dox Doxycycline DTT Dithiothreitol eIF2α Eukaryotic initiation factor 2 ER Endoplasmic reticulum ERp46 ER protein 46 ERp57 ER protein 57 GAPDH Glyceraldehyde 3-phosphate dehydrogenase GCN2 General control nonderepressible 2 inositol-requiring enzyme 1 (IRE1) Inositol-requiring enzyme 1 KD Knockdown protein disulfide isomerase family (PDIs) Protein disulfide isomerase PERK Protein kinase R (PKR)-like endoplasmic reticulum kinase ROS Reactive oxygen species RT-PCR Reverse transcription polymerase chain reaction Thaps Thapsigargin TM Tunicamycin unfolded protein response (UPR) Unfolded protein response
The most common theory how ATF6, IRE1 and PERK are activated in response to ER stress to date is the dissociation of BiP, which allows dimerization and autophosphorylation for IRE1 and PERK or cleavage of ATF6 respectively
In this study we were able to almost completely abrogate PERK signaling by depletion of PDI in HEK293T, BxPC3, A549 and HCT116 cells
Summary
Abbreviations ATF4 Activating transcription factor 4 ATF6 Activating transcription factor 6 BiP Binding immunoglobulin protein CHOP C/EBP-homologous protein CNPY2 Protein canopy homolog 2 DMSO Dimethyl sulfoxide Dox Doxycycline DTT Dithiothreitol eIF2α Eukaryotic initiation factor 2 ER Endoplasmic reticulum ERp46 ER protein 46 ERp57 ER protein 57 GAPDH Glyceraldehyde 3-phosphate dehydrogenase GCN2 General control nonderepressible 2 IRE1 Inositol-requiring enzyme 1 KD Knockdown PDI Protein disulfide isomerase PERK Protein kinase R (PKR)-like endoplasmic reticulum kinase ROS Reactive oxygen species RT-PCR Reverse transcription polymerase chain reaction Thaps Thapsigargin TM Tunicamycin UPR Unfolded protein response. Depending on the origin of the cell and its specific function, 30 up to 50% of all cellular proteins pass this organelle to achieve disulfide bonding and proper folding[2,3] This is executed through the highly oxidative environment in the ER and supported by classes of specialized enzymes like chaperones and ER oxidoreductases[4]. The capacities of this organelle are exceeded by intrinsic (genetic aberrations, ROS, high demand of secretory proteins, cell division etc.) and extrinsic factors (hypoxia, chemoand radiotherapy, lack of nutrients etc.) which result in unfolded protein burden and ER s tress[5]. We expand PDI’s role as a PERK activator to that of a maintainer of PERK signaling and offer a new therapeutic strategy to inhibit PERK signaling in tumor cells
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