RESUMEN
Intestinal goblet cells are secretory cells specialized in the production of mucins, and as such are challenged by the need for efficient protein folding. Goblet cells express Inositol-Requiring Enzyme-1ß (IRE1ß), a unique sensor in the unfolded protein response (UPR), which is part of an adaptive mechanism that regulates the demands of mucin production and secretion. However, how IRE1ß activity is tuned to mucus folding load remains unknown. We identified the disulfide isomerase and mucin chaperone AGR2 as a goblet cell-specific protein that crucially regulates IRE1ß-, but not IRE1α-mediated signaling. AGR2 binding to IRE1ß disrupts IRE1ß oligomerization, thereby blocking its downstream endonuclease activity. Depletion of endogenous AGR2 from goblet cells induces spontaneous IRE1ß activation, suggesting that alterations in AGR2 availability in the endoplasmic reticulum set the threshold for IRE1ß activation. We found that AGR2 mutants lacking their catalytic cysteine, or displaying the disease-associated mutation H117Y, were no longer able to dampen IRE1ß activity. Collectively, these results demonstrate that AGR2 is a central chaperone regulating the goblet cell UPR by acting as a rheostat of IRE1ß endonuclease activity.
Asunto(s)
Células Caliciformes , Chaperonas Moleculares , Mucinas , Endonucleasas , Células Caliciformes/metabolismo , Chaperonas Moleculares/genética , Mucinas/genética , Proteína Disulfuro Isomerasas , Humanos , Línea Celular TumoralRESUMEN
Effector mechanisms of the unfolded protein response (UPR) in the endoplasmic reticulum (ER) are well-characterised, but how ER proteostasis is sensed is less well understood. Here, we exploited the beta isoform of the UPR transducer IRE1, that is specific to mucin-producing cells in order to gauge the relative regulatory roles of activating ligands and repressing chaperones of the specialised ER of goblet cells. Replacement of the stress-sensing luminal domain of endogenous IRE1α in CHO cells (normally expressing neither mucin nor IRE1ß) with the luminal domain of IRE1ß deregulated basal IRE1 activity. The mucin-specific chaperone AGR2 repressed IRE1 activity in cells expressing the domain-swapped IRE1ß/α chimera, but had no effect on IRE1α. Introduction of the goblet cell-specific client MUC2 reversed AGR2-mediated repression of the IRE1ß/α chimera. In vitro, AGR2 actively de-stabilised the IRE1ß luminal domain dimer and formed a reversible complex with the inactive monomer. These features of the IRE1ß-AGR2 couple suggest that active repression of IRE1ß by a specialised mucin chaperone subordinates IRE1 activity to a proteostatic challenge unique to goblet cells, a challenge that is otherwise poorly recognised by the pervasive UPR transducers.
Asunto(s)
Endorribonucleasas , Células Caliciformes , Mucinas , Animales , Cricetinae , Humanos , Cricetulus , Células Caliciformes/metabolismo , Chaperonas Moleculares/genética , Mucinas/genética , Mucoproteínas/genética , Proteínas Oncogénicas , Proteínas Serina-Treonina Quinasas/genética , Células CHORESUMEN
Coupling of endoplasmic reticulum (ER) stress to dimerisation-dependent activation of the UPR transducer IRE1 is incompletely understood. Whilst the luminal co-chaperone ERdj4 promotes a complex between the Hsp70 BiP and IRE1's stress-sensing luminal domain (IRE1LD) that favours the latter's monomeric inactive state and loss of ERdj4 de-represses IRE1, evidence linking these cellular and in vitro observations is presently lacking. We report that enforced loading of endogenous BiP onto endogenous IRE1α repressed UPR signalling in CHO cells and deletions in the IRE1α locus that de-repressed the UPR in cells, encode flexible regions of IRE1LD that mediated BiP-induced monomerisation in vitro. Changes in the hydrogen exchange mass spectrometry profile of IRE1LD induced by ERdj4 and BiP confirmed monomerisation and were consistent with active destabilisation of the IRE1LD dimer. Together, these observations support a competition model whereby waning ER stress passively partitions ERdj4 and BiP to IRE1LD to initiate active repression of UPR signalling.
Asunto(s)
Estrés del Retículo Endoplásmico/genética , Endorribonucleasas/química , Proteínas del Choque Térmico HSP40/química , Proteínas de la Membrana/química , Chaperonas Moleculares/química , Proteínas Serina-Treonina Quinasas/química , Respuesta de Proteína Desplegada/genética , Animales , Células CHO , Cricetinae , Cricetulus , Retículo Endoplásmico/genética , Chaperón BiP del Retículo Endoplásmico , Endorribonucleasas/genética , Escherichia coli/genética , Proteínas del Choque Térmico HSP40/genética , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/genética , Humanos , Proteínas de la Membrana/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Unión Proteica/genética , Conformación Proteica , Multimerización de Proteína/genética , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
AMPylation is an inactivating modification that alters the activity of the major endoplasmic reticulum (ER) chaperone BiP to match the burden of unfolded proteins. A single ER-localised Fic protein, FICD (HYPE), catalyses both AMPylation and deAMPylation of BiP. However, the basis for the switch in FICD's activity is unknown. We report on the transition of FICD from a dimeric enzyme, that deAMPylates BiP, to a monomer with potent AMPylation activity. Mutations in the dimer interface, or of residues along an inhibitory pathway linking the dimer interface to the enzyme's active site, favour BiP AMPylation in vitro and in cells. Mechanistically, monomerisation relieves a repressive effect allosterically propagated from the dimer interface to the inhibitory Glu234, thereby permitting AMPylation-competent binding of MgATP. Moreover, a reciprocal signal, propagated from the nucleotide-binding site, provides a mechanism for coupling the oligomeric state and enzymatic activity of FICD to the energy status of the ER.
