title: Investigating the role of ERp18 during activation of UPR sensor ATF6α
Creators: name: Pierre, Arvin Shedrach
Publisher: name: University of Glasgow
Year: 2023

description: Proteins destined for the cell surface membrane or secretion enter the secretory pathway via the ER. As the gateway to the secretory pathway, the endoplasmic reticulum is responsible for the folding and maturation of proteins to the native functional conformation. Physiological or pathological changes can create an imbalance between the protein folding load and capacity of the ER, a condition known as ER stress. The ER hosts stringent quality control mechanisms to maintain a suitable environment for the faithful folding of proteins. One such mechanism, the unfolded protein response (UPR), is primarily adaptive and maintains homeostasis by regulating translational and transcriptional networks that increase the folding capacity of the ER while reducing the burden. However, when ER stress persists the UPR can engage apoptotic mechanisms. Thus, unresolved ER stress can be deleterious and contributes to pathogenesis of diseases including neurodegenerative diseases, diabetes and cancers. Transmembrane proteins Ire1α, PERK, and ATF6α mediate the UPR networks by detecting and responding to ER stress. These parallel pathways regulate prosurvival outcomes during acute ER stress. However, during chronic stress, Ire1α and PERK direct apoptotic outcomes frequently associated with disease. Presently, our understanding of the mechanisms of activation of Ire1α and PERK exceed that of ATF6α. Thus, there is ample room to increase our understanding of the mechanisms of ATF6α activation and function to exploit the pro-survival outcomes in the context of disease pathology. ATF6α is repressed by interaction with BiP as part of regulatory complex. In response to ER stress, release from BiP and reduction of lumenal cysteine residues prelude its exit from the ER. Activation is achieved through trafficking to the Golgi where intramembrane proteolysis by S1P and S2P releases an active transcription factor that regulates transcription of target genes at the nucleus. Some aspects of ATF6α regulation remain unclear. There are three oligomeric states of ATF6α; a monomer and two dimers, but their contributions toward regulation or activation are yet to be elucidated. Modulation of ATF6α redox status is important for activation however, the exact role it plays remains unclear. Also unclear, is the role of ERp18, a small PDI-like protein which can reduce ATF6α and regulates trafficking by an unknown mechanism. This thesis aimed to investigate how redox modulation contributes to the activation of ATF6α and identify roles for the different oligomeric states. In doing so, we interrogate ERp18 reductase activity to understand the mechanism by which it regulates trafficking. Finally, we investigate the contributions of putative binding residues S136 and T137 to ERp18 reductase activity. Our results showed that, during the initial stages of activation, the oligomeric status of ATF6α shifts from monomer to a disulfide stabilised dimer, designated 467D, in response to ER stress. This change was evaluated based on the relative abundance of the two redox forms in stressed compared to unstressed cells. We also showed that this dimer trafficked to the Golgi where S1P cleavage liberated dimeric lumenal domain. By overexpressing ERp18, we showed that it antagonised the formation of the dimer by reducing the interchain disulfide thereby regulating ATF6α trafficking to the Golgi. Mutation of Tyrosine-137 to Threonine (Y137T) within the proposed binding peptide of ERp18 affected its activity reductase activity in vitro but not in vivo. The S136D mutation had no discernible effect on ERp18 activity. Our findings revealed that, following reduction of ATF6α cysteines, redox dependent dimerisation and ERp18 activity function as additional regulatory mechanisms in the early stages of ATF6α activation. From these findings we proposed a model in which the monomer is engaged during retention while 467D is involved in trafficking which is policed by ERp18 reduction. These findings have contributed to the understanding of ATF6α activation but also allude to further aspects of regulation that are yet to be elucidated.