The unfolded protein response (UPR) maintains protein folding homeostasis in the endoplasmic reticulum (ER) by adjusting its folding capacity to the load of unfolded proteins in the compartment. This critical feedback mechanism governs the functioning of the secretory pathway, impacting various proteins such as receptors, extracellular matrix components, and signalling molecules (Kelly, 1985). Malfunctions in this pathway are linked to diseases like cancer, diabetes, and neurodegeneration (Sherwood, L., 2015; Uhlen et al, 2015; Pohlschroder et al, 2005; Wang & Kaufman, 2016).

Whilst effector mechanisms in the UPR are well characterised, sensing of the unfolded protein load in the ER is incompletely understood. It is widely accepted that the key UPR transducer – IRE1 – responds to the unfolded protein burden by dimerisation/oligomerisation-dependent activation, however, the molecular basis of this upstream event in the UPR remains elusive. This thesis provides insight into regulatory mechanisms acting on the two IRE1 paralogues IRE1α and IRE1β to understand their unique physiological function and infer general principles of UPR regulation.

For IRE1α, it has been proposed that the Hsp70 chaperone BiP, a major component of the ER protein folding machinery, couples IRE1α signalling to the folding state of the compartment. This is supported by data showing that the luminal J-domain co-chaperone ERdj4 promotes the formation of a complex between BiP and IRE1's stress-sensing luminal domain (LD) in vitro using recombinantly expressed proteins. This observation suggests that the interaction with BiP favours IRE1α LD’s monomeric, inactive state. In line with this concept, loss of ERdj4 derepresses IRE1α activity in cells (Amin-Wetzel et al, 2017). However, evidence linking these in vitro and cellular observations is sparse. Data reported here show that enforced loading of endogenous BiP onto endogenous IRE1α represses UPR signalling in cells. Furthermore, deletions in the IRE1α-encoding ERN1 locus that de-repress the UPR in cells, encode flexible regions in IRE1α LD that are required for BiP binding and BiP-induced monomerisation in vitro.

In contrast to IRE1α, little is known about the regulation of IRE1β, a tissue-restricted IRE1 paralogue expressed in mucin producing cells. The evidence presented here identifies the mucin chaperone AGR2 as a repressor for UPR signalling mediated by IRE1β. In cells, AGR2 is a selective repressor of IRE1β signalling without affecting IRE1α activity. In vitro, AGR2 binds IRE1β’s LD and promotes monomerisation. In summary, these findings support the concept that the physiological regulation of IRE1 paralogues is governed by ER chaperones with a dual function: Firstly, as UPR repressors, and secondly, as effectors directly involved in maintaining protein folding homeostasis in the compartment.