Multiple roles of the ER stress sensor IRE1 demonstrated by gene targeting in rice.
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ABSTRACT:
SUBMITTER: Wakasa Y
PROVIDER: S-EPMC3517978 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:IRE1, an ER-localized transmembrane protein, plays a central role in the unfolded protein response (UPR). IRE1 senses the accumulation of unfolded proteins in its luminal domain and transmits a signal to the cytosolic side through its kinase and RNase domains. Although the downstream pathways mediated by two mammalian IRE1s, IRE1? and IRE1?, are well documented, their luminal events have not been fully elucidated. In particular, there have been no reports on how IRE1? senses the unfolded proteins. In this study, we performed a comparative analysis to clarify the luminal event mediated by the mammalian IRE1s. Confocal fluorescent microscopy using GFP-fused IRE1s revealed that IRE1? clustered into discrete foci upon ER stress. Also, fluorescence correlation spectroscopy (FCS) analysis in living cells indicated that the size of the IRE1? complex is robustly increased upon ER stress. Moreover, unlike IRE1?, the luminal domain of IRE1? showed anti-aggregation activity in vitro, and IRE1? was coprecipitated with the model unfolded proteins in cells. Strikingly, association with BiP was drastically reduced in IRE1?, while IRE1? was associated with BiP and dissociated upon ER stress. This is the first report indicating that, differently from IRE1?, the luminal event mediated by IRE1? involves direct interaction with unfolded proteins rather than association/dissociation with BiP, implying an intrinsic diversity in the sensing mechanism of mammalian sensors.
| S-EPMC3517461 | biostudies-literature
Project description:The complex multistep activation cascade of Ire1 involves changes in the Ire1 conformation and oligomeric state. Ire1 activation enhances ER folding capacity, in part by overexpressing the ER Hsp70 molecular chaperone BiP; in turn, BiP provides tight negative control of Ire1 activation. This study demonstrates that BiP regulates Ire1 activation through a direct interaction with Ire1 oligomers. Particularly, we demonstrated that the binding of Ire1 luminal domain (LD) to unfolded protein substrates not only trigger conformational changes in Ire1-LD that favour the formation of Ire1-LD oligomers but also exposes BiP binding motifs, enabling the molecular chaperone BiP to directly bind to Ire1-LD in an ATP-dependent manner. These transient interactions between BiP and two short motifs in the disordered region of Ire1-LD are reminiscent of interactions between clathrin and another Hsp70, cytoplasmic Hsc70. BiP binding to substrate-bound Ire1-LD oligomers enables unfolded protein substrates and BiP to synergistically and dynamically control Ire1-LD oligomerisation, helping to return Ire1 to its deactivated state when an ER stress response is no longer required.
| S-EPMC11300964 | biostudies-literature
Project description:Conditions within the endoplasmic reticulum (ER) influence most secretory proteins that pass through the ER. Therefore, eukaryotic cells must strike a balance between the ER stress response, which changes the conditions in the ER, and other considerations associated with protein secretion. Here, an interaction between the ER stress and defence responses in rice is described. Expression of OsWRKY45, which encodes a transcription factor that plays a central role in defence mediated by salicylic acid (SA), is induced by ER stress. Additionally, expression of some genes encoding pathogenesis-related (PR) secretory proteins is reduced by the ER stress response mediated by the stress sensor IRE1. Concomitant activation of the SA and ER stress responses suppresses the induction of ER stress-responsive genes, with the exception of OsWRKY45, and the reduction of PR gene expression. These findings demonstrate a functional integration between the defence and ER stress responses in plants.
| S-EPMC3445914 | biostudies-literature
Project description:BiP is a major endoplasmic reticulum (ER) chaperone and is suggested to act as primary sensor in the activation of the unfolded protein response (UPR). How BiP operates as a molecular chaperone and as an ER stress sensor is unknown. Here, by reconstituting components of human UPR, ER stress and BiP chaperone systems, we discover that the interaction of BiP with the luminal domains of UPR proteins IRE1 and PERK switch BiP from its chaperone cycle into an ER stress sensor cycle by preventing the binding of its co-chaperones, with loss of ATPase stimulation. Furthermore, misfolded protein-dependent dissociation of BiP from IRE1 is primed by ATP but not ADP. Our data elucidate a previously unidentified mechanistic cycle of BiP function that explains its ability to act as an Hsp70 chaperone and ER stress sensor.
| S-EPMC6858872 | biostudies-literature
Project description:The unfolded protein response (UPR) is an intracellular signaling pathway that counteracts variable stresses that impair protein folding in the endoplasmic reticulum (ER). As such, the UPR is thought to be a homeostat that finely tunes ER protein folding capacity and ER abundance according to need. The mechanism by which the ER stress sensor Ire1 is activated by unfolded proteins and the role that the ER chaperone protein BiP plays in Ire1 regulation have remained unclear. Here we show that the UPR matches its output to the magnitude of the stress by regulating the duration of Ire1 signaling. BiP binding to Ire1 serves to desensitize Ire1 to low levels of stress and promotes its deactivation when favorable folding conditions are restored to the ER. We propose that, mechanistically, BiP achieves these functions by sequestering inactive Ire1 molecules, thereby providing a barrier to oligomerization and activation, and a stabilizing interaction that facilitates de-oligomerization and deactivation. Thus BiP binding to or release from Ire1 is not instrumental for switching the UPR on and off as previously posed. By contrast, BiP provides a buffer for inactive Ire1 molecules that ensures an appropriate response to restore protein folding homeostasis to the ER by modulating the sensitivity and dynamics of Ire1 activity.
| S-EPMC2897766 | biostudies-literature
Project description:The protein folding capacity of the endoplasmic reticulum (ER) is tightly regulated by a network of signaling pathways, known as the unfolded protein response (UPR). UPR sensors monitor the ER folding status to adjust ER folding capacity according to need. To understand how the UPR sensor IRE1 maintains ER homeostasis, we identified zero-length crosslinks of RNA to IRE1 with single nucleotide precision in vivo. We found that IRE1 specifically crosslinks to a subset of ER-targeted mRNAs, SRP RNA, ribosomal and transfer RNAs. Crosslink sites cluster in a discrete region of the ribosome surface spanning from the A-site to the polypeptide exit tunnel. Moreover, IRE1 binds to purified 80S ribosomes with high affinity, indicating association with ER-bound ribosomes. Our results suggest that the ER protein translocation and targeting machineries work together with the UPR to tune the ER's protein folding load.
| S-EPMC6336407 | biostudies-literature
Project description:Accumulation of misfolded proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), an intracellular signaling pathway that adjusts the protein folding capacity of the ER according to need. If homeostasis in the ER protein folding environment cannot be reestablished, cells commit to apoptosis. The ER-resident transmembrane kinase-endoribonuclease inositol-requiring enzyme 1 (IRE1) is the best characterized UPR signal transduction molecule. In yeast, Ire1 oligomerizes upon activation in response to an accumulation of misfolded proteins in the ER. Here we show that the salient mechanistic features of IRE1 activation are conserved: mammalian IRE1 oligomerizes in the ER membrane and oligomerization correlates with the onset of IRE1 phosphorylation and RNase activity. Moreover, the kinase/RNase module of human IRE1 activates cooperatively in vitro, indicating that formation of oligomers larger than four IRE1 molecules takes place upon activation. High-order IRE1 oligomerization thus emerges as a conserved mechanism of IRE1 signaling. IRE1 signaling attenuates after prolonged ER stress. IRE1 then enters a refractive state even if ER stress remains unmitigated. Attenuation includes dissolution of IRE1 clusters, IRE1 dephosphorylation, and decline in endoribonuclease activity. Thus IRE1 activity is governed by a timer that may be important in switching the UPR from the initially cytoprotective phase to the apoptotic mode.
| S-EPMC2941319 | biostudies-literature
Project description:Here we identify and characterize a cytoskeletal myosin protein required for IRE1? oligomerization, activation, and signaling. Proteomic screening identified nonmuscle myosin heavy chain IIB (NMHCIIB), a subunit of nonmuscle myosin IIB (NMIIB), as an ER stress-dependent interacting protein specific to IRE1?. Loss of NMIIB compromises XBP1s and UPR target gene expression with no effect on the PERK pathway. Mechanistically, NMIIB is required for IRE1? aggregation and foci formation under ER stress. The NMIIB-mediated effect on IRE1? signaling is in part dependent on the phosphorylation of myosin regulatory light chain and the actomyosin contractility of NMIIB. Biologically, the function of NMIIB in ER stress response is conserved as both mammalian cells and C. elegans lacking NMIIB exhibit hypersensitivity to ER stress. Thus, optimal IRE1? activation and signaling require concerted coordination between the ER and cytoskeleton.
| S-EPMC3547290 | biostudies-literature
Project description:Cells modify their gene expression pattern in response to stress signals emanating from the endoplasmic reticulum (ER). The well-characterized aspect of this response consists of the activation of genes that encode protein chaperones and other ER resident proteins, and is conserved between mammals and yeast. In mammalian cells, however, ER stress also activates other pathways, including the expression of the transcription factor CHOP/GADD153 and its downstream target genes. ER stress is also linked to the development of programmed cell death, a phenomenon in which CHOP plays an important role. Here we report on the cloning of a murine homolog of yeast IRE1, an essential upstream component of the ER stress-response in yeast. The mammalian Ire1 is located in the ER membrane and its over-expression in mammalian cells activates both the endogenous ER chaperone GRP78/BiP and CHOP-encoding genes. Over-expression of a dominant-negative form of Ire1 blocks the induction of GRP78/BiP and CHOP in response to the ER stress induced by tunicamycin treatment. Over-expression of murine Ire1 also leads to the development of programmed cell death in transfected cells. These results indicate that a single upstream component, Ire1, plays a role in multiple facets of the ER stress-response in mammalian cells.
| S-EPMC1170899 | biostudies-other
Project description:Inflammation of human bronchial epithelia (HBE) activates the endoplasmic reticulum (ER) stress transducer inositol-requiring enzyme 1 (IRE1)?, resulting in IRE1?-mediated cytokine production. Previous studies demonstrated ubiquitous expression of IRE1? and gut-restricted expression of IRE1?. We found that IRE1? is also expressed in HBE, is absent in human alveolar cells, and is upregulated in cystic fibrosis and asthmatic HBE. Studies with Ire1?(-/-) mice and Calu-3 airway epithelia exhibiting IRE1? knockdown or overexpression revealed that IRE1? is expressed in airway mucous cells, is functionally required for airway mucin production, and this function is specific for IRE1? vs. IRE1?. IRE1?-dependent mucin production is mediated, at least in part, by activation of the transcription factor X-box binding protein-1 (XBP-1) and the resulting XBP-1-dependent transcription of anterior gradient homolog 2, a gene implicated in airway and intestinal epithelial mucin production. These novel findings suggest that IRE1? is a potential mucous cell-specific therapeutic target for airway diseases characterized by mucin overproduction.
| S-EPMC4031691 | biostudies-literature