A J-Protein Co-chaperone Recruits BiP to Monomerize IRE1 and Repress the Unfolded Protein Response.

When unfolded proteins accumulate in the endoplasmic reticulum (ER), the unfolded protein response (UPR) increases ER-protein-folding capacity to restore protein-folding homeostasis. Unfolded proteins activate UPR signaling across the ER membrane to the nucleus by promoting oligomerization of IRE1, a conserved transmembrane ER stress receptor. However, the coupling of ER stress to IRE1 oligomerization and activation has remained obscure. Here, we report that the ER luminal co-chaperone ERdj4/DNAJB9 is a selective IRE1 repressor that promotes a complex between the luminal Hsp70 BiP and the luminal stress-sensing domain of IRE1α (IRE1LD). In vitro, ERdj4 is required for complex formation between BiP and IRE1LD. ERdj4 associates with IRE1LD and recruits BiP through the stimulation of ATP hydrolysis, forcibly disrupting IRE1 dimers. Unfolded proteins compete for BiP and restore IRE1LD to its default, dimeric, and active state. These observations establish BiP and its J domain co-chaperones as key regulators of the UPR

Early Events in the Endoplasmic Reticulum Unfolded Protein Response.

The physiological consequences of the unfolded protein response (UPR) are mediated by changes in gene expression. Underlying them are rapid processes involving preexisting components. We review recent insights gained into the regulation of the endoplasmic reticulum (ER) Hsp70 chaperone BiP, whose incorporation into inactive oligomers and reversible AMPylation and de-AMPylation present a first line of response to fluctuating levels of unfolded proteins. BiP activity is tied to the regulation of the UPR transducers by a recently discovered cycle of ER-localized, J protein-mediated formation of a repressive IRE1-BiP complex, whose working we contrast to an alternative model for UPR regulation that relies on direct recognition of unfolded proteins. We conclude with a discussion of mechanisms that repress messenger RNA (mRNA) translation to limit the flux of newly synthesized proteins into the ER, a rapid adaptation that does not rely on new macromolecule biosynthesis.Wellcome Trus

Evolution and function of the epithelial cell-specific ER stress sensor IRE1β

Barrier epithelial cells lining the mucosal surfaces of the gastrointestinal and respiratory tracts interface directly with the environment. As such, these tissues are continuously challenged to maintain a healthy equilibrium between immunity and tolerance against environmental toxins, food components, and microbes. An extracellular mucus barrier, produced and secreted by the underlying epithelium plays a central role in this host defense response. Several dedicated molecules with a unique tissue-specific expression in mucosal epithelia govern mucosal homeostasis. Here, we review the biology of Inositol-requiring enzyme 1β (IRE1β), an ER-resident endonuclease and paralogue of the most evolutionarily conserved ER stress sensor IRE1α. IRE1β arose through gene duplication in early vertebrates and adopted functions unique from IRE1α which appear to underlie the basic development and physiology of mucosal tissues

Recent advances in signal integration mechanisms in the unfolded protein response [version 1; peer review: 2 approved]

Since its discovery more than 25 years ago, great progress has been made in our understanding of the unfolded protein response (UPR), a homeostatic mechanism that adjusts endoplasmic reticulum (ER) function to satisfy the physiological demands of the cell. However, if ER homeostasis is unattainable, the UPR switches to drive cell death to remove defective cells in an effort to protect the health of the organism. This functional dichotomy places the UPR at the crossroads of the adaptation versus apoptosis decision. Here, we focus on new developments in UPR signaling mechanisms, in the interconnectivity among the signaling pathways that make up the UPR in higher eukaryotes, and in the coordination between the UPR and other fundamental cellular processes

Structure and Molecular Mechanism of ER Stress Signaling by the Unfolded Protein Response Signal Activator IRE1

The endoplasmic reticulum (ER) is an important site for protein folding and maturation in eukaryotes. The cellular requirement to synthesize proteins within the ER is matched by its folding capacity. However, the physiological demands or aberrations in folding may result in an imbalance which can lead to the accumulation of misfolded protein, also known as “ER stress.” The unfolded protein response (UPR) is a cell-signaling system that readjusts ER folding capacity to restore protein homeostasis. The key UPR signal activator, IRE1, responds to stress by propagating the UPR signal from the ER to the cytosol. Here, we discuss the structural and molecular basis of IRE1 stress signaling, with particular focus on novel mechanistic advances. We draw a comparison between the recently proposed allosteric model for UPR induction and the role of Hsp70 during polypeptide import to the mitochondrial matrix

Unstructured regions in IRE1α specify BiP-mediated destabilisation of the luminal domain dimer and repression of the UPR

Funder: Medical Research Council; FundRef: http://dx.doi.org/10.13039/501100000265Funder: European Molecular Biology Organization; FundRef: http://dx.doi.org/10.13039/100004410Coupling 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

Unstructured regions in IRE1α specify BiP-mediated destabilisation of the luminal domain dimer and repression of the UPR

Coupling of endoplasmic reticulum 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

Negative regulation of Ire1 during the unfolded protein response

When cells undergo endoplasmic reticulum stress due to a build-up of unfolded or misfolded proteins, the cell must adapt to this stress and does so through the unfolded protein response (UPR). Ire1, a protein kinase endoribonuclease, is a protein found in the endoplasmic reticulum (ER) of Saccharomyces cerevisiae and plays a major role in the cell’s adaptive response to ER stress. Upon accumulation of unfolded proteins in the ER, Ire1 becomes active and splices HAC1 mRNA. After splicing the HAC1 mRNA is translated to produce the Hac1i protein, the Hac1i protein contains a bZIP transcription factor which leads to alleviation of ER stress by promoting inducing expression of UPR-associated genes. Previous work has shown that although phosphorylation is not essential to RNase activation, it still plays a critical role. Therefore, this study investigates previously identified phosphatases, Dcr2 and Ptc2, which were proposed to be negative regulators of Ire1. This study shows that out of the two investigated phosphatases, only Ptc2 was observed to negatively regulate the UPR. The mechanism of activation for the way in which the UPR was inactivated determined that interference of IRE1 clustering was affected by overexpression of either phosphatase, which suggests an alternative mechanism

Expression and purification of inositol requiring enzyme 1 (IRE1)

Endoplasminen retikkeli (ER) on tärkeä organelli solulle, missä suuri määrä proteiineja syntetisoidaan ja muokataan lopulliseen toiminnalliseen muotoonsa. Tämän takia ER:in stressiä, joka johtuu väärin laskostuneiden proteiinien kerääntymisestä ER:iin, ei pidä ottaa kevyesti, koska se voi vaikuttaa moniin sairauksiin, kuten syöpään ja eri neurodegeneratiivisiin sairauksiin. Täten laskostumattomien proteiinienvaste (UPR) on mukautuva järjestelmä, joka auttaa ER:ia sopeutumaan lisääntyneeseen laskostumistarpeeseen. Yksi vasteen pääproteiineista on inositolia vaativa entsyymi 1 (IRE1). IRE1 havaitsee proteiinien laskostumisen tilan ER:issa ja käynnistää UPR-signalointireitin, jotta saavutetaan joko normaali laskostumistila tai solukuolema. Tämän tutkimuksen tarkoituksena oli tuottaa hiivan IRE1 proteiinia E.colissa ja ihmisen IRE1 hyönteissoluissa, puhdistaa proteiini affiniteettikromatografialla ja tutkia sen kiderakennetta pienen molekyylimodulaattorin kanssa, joka voisi mahdollisesti tehostaa sen toimintaa. Proteiini tuotettiin onnistuneesti ja puhdistettiin glutationi-S-transferaasi (GST)-merkinnän avulla ja eristetyn proteiinin aktiivisuus määritettiin. Rakenteellisia tutkimuksia ei suoritettu, koska kiteytymiseen tarvittavaa absoluuttista puhtautta ja saantoa ei saavutettu johtuen proteiinin menetyksestä geelisuodatuksen aikana ja saostumisen takia. Tulosten perusteella on todennäköistä, että proteiinin rakenne voitaisiin ratkaista ja biokemialliset ja rakenteelliset tutkimukset F10:llä ovat erittäin mahdollisia.The endoplasmic reticulum (ER) is an important organelle of the cell where a high number of proteins are synthesized and modified to obtain their final structure. Therefore, the ER stress, which is caused by accumulation of unfolded proteins in the ER, is not to be taken lightly since it could contribute to many diseases, such as cancer and neurodegenerative diseases. The response to the ER stress is the unfolded protein response (UPR), which is an adaptive system that helps in adjusting for increased folding needs within the ER. One of the main protein branches in the UPR is inositol requiring enzyme 1 (IRE1). IRE1 detects the status of protein folding inside the ER and initiates the UPR signaling pathway to achieve either normal folding status or cell death. The aim of this research was to express yeast IRE1 in E.coli and human IRE1 in insect cells, purify with affinity chromatography and study the IRE1’s crystal structure with a small molecule modulator that could possibly enhance its activity. The protein was expressed successfully and purified with glutathione S-transferase (GST) tag, and the activity of the pure protein was determined. The structural studies were not fully completed since the absolute purity and yield that was necessary for crystallization was not achieved due to loss of protein during gel filtration and precipitation. Based on the results it is likely that the structure of the protein could be solved and further biochemical and structural studies with F10 are possible

The regulation of the Hsp70 family molecular chaperone BiP via phosphorylation

As the sole member of the Hsp70 family found within the endoplasmic reticulum, BiP plays a critical role in the protein homeostasis of the ER in order to prevent build-up of unfolded proteins which would lead to ER stress. Due to this critical function and a complex allosteric cycle associated with it, the chaperone requires a complex multi-layered system of regulation. It is one aspect of this regulation, the limitedly characterised phosphorylation of BiP, which this study aimed to generate a greater understand of the mechanism of. In this regard, this study was able to identify and select two substrate binding domain located phosphorylation sites which were demonstrated to be only conserved within ER located Hsp70s (and non-conserved in Hsp70 located in other sub-cellular locations) based on conservational analysis of ER and cytoplasmic located Hsp70s. These sites were mutated to phosphomimetic mutations in order to simulate the effects of phosphorylation and characterise them. The study used a combination of methyl NMR to monitor changes in the conformational ensemble, biochemical assays to measure alterations to the rate of ATP hydrolysis and effects to the interactions with a known substrates IRE1 in the characterisation process. This approach allowed the characterisation of a regulation mechanism of BiP, in which phosphomimetic mutants and potentially phosphorylation caused a disruption of the domain undocked conformation whilst stabilising both the domain docked and intermediate transient conformation which results in the fine-tuning of BiP functions. These functions include increased ATPase activity as well as a reduced ability to deoligomerize IRE1. Additionally, the study was successful in the identification of the BiP binding site of IRE1-LD