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Elife ; 82019 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-31873072


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.

Estresse do Retículo Endoplasmático/genética , Endorribonucleases/química , Proteínas de Choque Térmico HSP40/química , Proteínas de Membrana/química , Chaperonas Moleculares/química , Proteínas Serina-Treonina Quinases/química , Resposta a Proteínas não Dobradas/genética , Animais , Células CHO , Cricetinae , Cricetulus , Retículo Endoplasmático/genética , Endorribonucleases/genética , Escherichia coli/genética , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/genética , Humanos , Proteínas de Membrana/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Ligação Proteica/genética , Conformação Proteica , Multimerização Proteica/genética , Proteínas Serina-Treonina Quinases/genética
EMBO J ; 38(21): e102177, 2019 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-31531998


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.

Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Células HEK293 , Humanos , Conformação Proteica