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PurposeThe unfolded protein response (UPR) is triggered when the protein folding capacity of the endoplasmic reticulum (ER) is overwhelmed and misfolded proteins accumulate in the ER, a condition referred to as ER stress. IRE1α is an ER-resident protein that plays major roles in orchestrating the UPR. Several lines of evidence implicate the UPR and its transducers in neurodegenerative diseases, including retinitis pigmentosa (RP), a group of inherited diseases that cause progressive dysfunction and loss of rod and cone photoreceptors. This study evaluated the contribution of IRE1α to photoreceptor development, homeostasis, and degeneration.MethodsWe used a conditional gene targeting strategy to selectively inactivate Ire1α in mouse rod photoreceptors. We used a combination of optical coherence tomography (OCT) imaging, histology, and electroretinography (ERG) to assess longitudinally the effect of IRE1α deficiency in retinal development and function. Furthermore, we evaluated the IRE1α-deficient retina responses to tunicamycin-induced ER stress and in the context of RP caused by the rhodopsin mutation RhoP23H.ResultsOCT imaging, histology, and ERG analyses did not reveal abnormalities in IRE1α-deficient retinas up to 3 months old. However, by 6 months of age, the Ire1α mutant animals showed reduced outer nuclear layer thickness and deficits in retinal function. Furthermore, conditional inactivation of Ire1α in rod photoreceptors accelerated retinal degeneration caused by the RhoP23H mutation.ConclusionsThese data suggest that IRE1α is dispensable for photoreceptor development but important for photoreceptor homeostasis in aging retinas and for protecting against ER stress-mediated photoreceptor degeneration.

The unfolded protein response (UPR) homeostatically matches endoplasmic reticulum (ER) protein-folding capacity to cellular secretory needs. However, under high or chronic ER stress, the UPR triggers apoptosis. This cell fate dichotomy is promoted by differential activation of the ER transmembrane kinase/endoribonuclease (RNase) IRE1α. We previously found that the RNase of IRE1α can be either fully activated or inactivated by ATP-competitive kinase inhibitors. Here we developed kinase inhibitors, partial antagonists of IRE1α RNase (PAIRs), that partially antagonize the IRE1α RNase at full occupancy. Biochemical and structural studies show that PAIRs promote partial RNase antagonism by intermediately displacing the helix αC in the IRE1α kinase domain. In insulin-producing β-cells, PAIRs permit adaptive splicing of Xbp1 mRNA while quelling destructive ER mRNA endonucleolytic decay and apoptosis. By preserving Xbp1 mRNA splicing, PAIRs allow B cells to differentiate into immunoglobulin-producing plasma cells. Thus, an intermediate RNase-inhibitory 'sweet spot', achieved by PAIR-bound IRE1α, captures a desirable conformation for drugging this master UPR sensor/effector.

Aims/introductionUnder irremediable endoplasmic reticulum (ER) stress, hyperactivated inositol-requiring enzyme 1α (IRE1α) triggers the terminal unfolded protein response (T-UPR), causing crucial cell dysfunction and apoptosis. We hypothesized that nicotinic acetylcholine receptor (nAChR) signaling regulates IRE1α activation to protect β-cells from the T-UPR under ER stress.Materials and methodsThe effects of nicotine on IRE1α activation and key T-UPR markers, thioredoxin-interacting protein and insulin/proinsulin, were analyzed by real-time polymerase chain reaction and western blotting in rat INS-1 and human EndoC-βH1 β-cell lines. Doxycycline-inducible IRE1α overexpression or ER stress agents were used to induce IRE1α activation. An α7 subunit-specific nAChR agonist (PNU-282987) and small interfering ribonucleic acid for α7 subunit-specific nAChR were used to modulate nAChR signaling.ResultsNicotine inhibits the increase in thioredoxin-interacting protein and the decrease in insulin 1/proinsulin expression levels induced by either forced IRE1α hyperactivation or ER stress agents. Nicotine attenuated X-box-binding protein-1 messenger ribonucleic acid site-specific splicing and IRE1α autophosphorylation induced by ER stress. Furthermore, PNU-282987 attenuated T-UPR induction by either forced IRE1α activation or ER stress agents. The effects of nicotine on attenuating thioredoxin-interacting protein and preserving insulin 1 expression levels were attenuated by pharmacological and genetic inhibition of α7 nAChR. Finally, nicotine suppressed apoptosis induced by either forced IRE1α activation or ER stress agents.ConclusionsOur findings suggest that nAChR signaling regulates IRE1α activation to protect β-cells from the T-UPR and apoptosis under ER stress partly through α7 nAChR. Targeting nAChR signaling to inhibit the T-UPR cascade may therefore hold therapeutic promise by thwarting β-cell death in diabetes.

Nuno Moura-Coelho,1– 4 João Paulo Cunha,4,5 Arnaldo Dias-Santos,2,6 Marco Dutra-Medeiros,2,6 Renato Papa-Vettorazzi,1 Felicidad Manero,1 José Güell1,3,7 1Cornea and Refractive Surgery Unit, Instituto Microcirugía Ocular (IMO) Barcelona, Barcelona, Spain; 2Ophthalmology, Faculdade de Ciências Médicas – Universidade Nova de Lisboa (NMS|FCM-UNL), Lisbon, Portugal; 3Anterior Segment, European School for Advanced Studies in Ophthalmology (ESASO), Lugano, Switzerland; 4Ophthalmology, Escola Superior de Tecnologias da Saúde de Lisboa (ESTeSL) – Instituto Politécnico de Lisboa, Lisbon, Portugal; 5Ophthalmology, Hospital CUF Cascais, Cascais, Portugal; 6Ophthalmology, Centro Hospitalar Universitário Lisboa Central (CHULC), Lisbon, Portugal; 7Ophthalmology, Universidad Autónoma de Barcelona (UAB), Barcelona, SpainCorrespondence: Nuno Moura-Coelho, Email nunomouracoelho.oft@gmail.comPurpose: To analyze the outcomes of Descemet’s membrane endothelial keratoplasty (DMEK) for corneal endothelial failure secondary to phakic intraocular lens implantation (PIOL) at a reference center for corneal transplantation in Spain.Design: Retrospective, single-surgeon case series.Methods: Single-center analysis of patients who underwent DMEK for PIOL-related corneal decompensation between July 2011 and July 2020 with at least 6 months of follow-up postoperatively. Primary outcome was final best-corrected visual acuity (BCVA, logMAR) compared to pre-DMEK BCVA. Secondary outcomes analyzed included post-DMEK refractive spherical equivalent, endothelial cell loss (%ECL), and graft failure.Results: Sixteen eyes (14 patients) underwent DMEK for PIOL-related corneal decompensation. Mean (SD) time to PIOL explantation was 9.3 (5.0) years, and median (P25-P75) time between PIOL explantation and DMEK surgery was 3 (2– 4) months. Median pre-DMEK BCVA was 0.80 (1.08– 0.60) logMAR. A statistically significant improvement in BCVA was observed 1 month after DMEK (p = 0.001), and median final BCVA was 0.15 (0.0– 0.35) logMAR (p = 0.002). Mean %ECL was 55.6 (18.7) % at 2-year follow-up and 61.7 (11.7) % in eyes with over 4 years of follow-up. Two eyes required re-bubbling (12.5%), one of which ended in primary graft failure (6.2%) and one eye had late endothelial graft failure (LEGF) at 4-year follow-up (1/15 grafts, 6.7%).Conclusion: In patients with PIOL-related corneal decompensation, DMEK leads to good and clinically significant refractive and visual outcomes in the medium-long term, with a good safety profile. Prospective studies are encouraged to ascertain whether these cases are at increased risk of accelerated endothelial cell loss and LEGF.Keywords: descemet membrane endothelial keratoplasty, endothelial keratoplasty, phakic intraocular lens, corneal endothelial failure

Master regulators of the unfolded protein response (UPR), IRE1α and PERK, promote adaptation or apoptosis depending on the level of endoplasmic reticulum (ER) stress. Although the UPR is activated in many cancers, its effects on tumor growth remain unclear. Derived from endocrine cells, pancreatic neuroendocrine tumors (PanNET) universally hypersecrete one or more peptide hormones, likely sensitizing these cells to high ER protein-folding stress. To assess whether targeting the UPR is a viable therapeutic strategy, we analyzed human PanNET samples and found evidence of elevated ER stress and UPR activation. Genetic and pharmacologic modulation of IRE1α and PERK in cultured cells, xenograft, and spontaneous genetic (RIP-Tag2) mouse models of PanNETs revealed that UPR signaling was optimized for adaptation and that inhibiting either IRE1α or PERK led to hyperactivation and apoptotic signaling through the reciprocal arm, thereby halting tumor growth and survival. These results provide a strong rationale for therapeutically targeting the UPR in PanNETs and other cancers with elevated ER stress. SIGNIFICANCE: The UPR is upregulated in pancreatic neuroendocrine tumors and its inhibition significantly reduces tumor growth in preclinical models, providing strong rationale for targeting the UPR in these cancers.

BackgroundMyriad challenges to the proper folding and structural maturation of secretory pathway client proteins in the endoplasmic reticulum (ER) - a condition referred to as "ER stress" - activate intracellular signaling pathways termed the unfolded protein response (UPR).Scope of reviewThrough executing transcriptional and translational programs the UPR restores homeostasis in those cells experiencing manageable levels of ER stress. But the UPR also actively triggers cell degeneration and apoptosis in those cells that are encountering ER stress levels that exceed irremediable thresholds. Thus, UPR outputs are "double-edged". In pancreatic islet β-cells, numerous genetic mutations affecting the balance between these opposing UPR functions cause diabetes mellitus in both rodents and humans, amply demonstrating the principle that the UPR is critical for the proper functioning and survival of the cell.Major conclusionsSpecifically, we have found that the UPR master regulator IRE1α kinase/endoribonuclease (RNase) triggers apoptosis, β-cell degeneration, and diabetes, when ER stress reaches critical levels. Based on these mechanistic findings, we find that novel small molecule compounds that inhibit IRE1α during such "terminal" UPR signaling can spare ER stressed β-cells from death, perhaps affording future opportunities to test new drug candidates for disease modification in patients suffering from diabetes.

Diverse perturbations to endoplasmic reticulum (ER) functions compromise the proper folding and structural maturation of secretory proteins. To study secretory pathway physiology during such "ER stress," we employed an ER-targeted, redox-responsive, green fluorescent protein-eroGFP-that reports on ambient changes in oxidizing potential. Here we find that diverse ER stress regimes cause properly folded, ER-resident eroGFP (and other ER luminal proteins) to "reflux" back to the reducing environment of the cytosol as intact, folded proteins. By utilizing eroGFP in a comprehensive genetic screen in Saccharomyces cerevisiae, we show that ER protein reflux during ER stress requires specific chaperones and cochaperones residing in both the ER and the cytosol. Chaperone-mediated ER protein reflux does not require E3 ligase activity, and proceeds even more vigorously when these ER-associated degradation (ERAD) factors are crippled, suggesting that reflux may work in parallel with ERAD. In summary, chaperone-mediated ER protein reflux may be a conserved protein quality control process that evolved to maintain secretory pathway homeostasis during ER protein-folding stress.

Endoplasmic reticulum stress (ER stress) has been implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF), a disease of progressive fibrosis and respiratory failure. ER stress activates a signaling pathway called the unfolded protein response (UPR) that either restores homeostasis or promotes apoptosis. The bifunctional kinase/RNase IRE1α is a UPR sensor/effector that promotes apoptosis if ER stress remains high and irremediable (i.e., a "terminal" UPR). Using multiple small molecule inhibitors against IRE1α, we show that ER stress-induced apoptosis of murine alveolar epithelial cells can be mitigated in vitro. In vivo, we show that bleomycin exposure to murine lungs causes early ER stress to activate IRE1α and the terminal UPR prior to development of pulmonary fibrosis. Small-molecule IRE1α kinase-inhibiting RNase attenuators (KIRAs) that we developed were used to evaluate the contribution of IRE1α activation to bleomycin-induced pulmonary fibrosis. One such KIRA-KIRA7-provided systemically to mice at the time of bleomycin exposure decreases terminal UPR signaling and prevents lung fibrosis. Administration of KIRA7 14 days after bleomycin exposure even promoted the reversal of established fibrosis. Finally, we show that KIRA8, a nanomolar-potent, monoselective KIRA compound derived from a completely different scaffold than KIRA7, likewise promoted reversal of established fibrosis. These results demonstrate that IRE1α may be a promising target in pulmonary fibrosis and that kinase inhibitors of IRE1α may eventually be developed into efficacious anti-fibrotic drugs.

(Cell Metabolism 25, 883–897; April 4, 2017) In the originally published version of this article, the immunoblot image of the HDAC1 nuclear extract protein control in Figure 3I was incorrectly cropped such that it included one extraneous lane. The corrected and original versions of Figure 3I are shown here. Furthermore, in the Discussion, after the sentence “Such compensatory, dysregulated UPR effects may be general as Perk deletion, likewise, hyperactivates IRE1α in β cells, which suffer early apoptosis, leading to postnatal diabetes,” we incorrectly cited Harding, H.P., and Ron, D. (2002). Endoplasmic reticulum stress and the development of diabetes: a review. Diabetes 51, S455–S461. The correct citation is: Harding, H.P., Zeng, H., Zhang, Y., Jungries, R., Chung, P., Plesken, H., Sabatini, D.D., and Ron, D. (2001). Diabetes mellitus and exocrine pancreatic dysfunction in perk−/− mice reveals a role for translational control in secretory cell survival. Molecular Cell 7, 1153–1163. The authors apologize for any confusion these errors may have caused. [Figure presented]

The accumulation of unfolded proteins under endoplasmic reticulum (ER) stress leads to the activation of the multidomain protein sensor IRE1α as part of the unfolded protein response (UPR). Clustering of IRE1α lumenal domains in the presence of unfolded proteins promotes kinase trans-autophosphorylation in the cytosol and subsequent RNase domain activation. Interestingly, there is an allosteric relationship between the kinase and RNase domains of IRE1α, which allows ATP-competitive inhibitors to modulate the activity of the RNase domain. Here, we use kinase inhibitors to study how ATP-binding site conformation affects the activity of the RNase domain of IRE1α. We find that diverse ATP-competitive inhibitors of IRE1α promote dimerization and activation of RNase activity despite blocking kinase autophosphorylation. In contrast, a subset of ATP-competitive ligands, which we call KIRAs, allosterically inactivate the RNase domain through the kinase domain by stabilizing monomeric IRE1α. Further insight into how ATP-competitive inhibitors are able to divergently modulate the RNase domain through the kinase domain was gained by obtaining the first structure of apo human IRE1α in the RNase active back-to-back dimer conformation. Comparison of this structure with other existing structures of IRE1α and integration of our extensive structure activity relationship (SAR) data has led us to formulate a model to rationalize how ATP-binding site ligands are able to control the IRE1α oligomeric state and subsequent RNase domain activity.

Unbiased genetic studies have uncovered surprising molecular mechanisms in human cellular immunity and autoimmunity. We performed whole-exome sequencing and targeted sequencing in five families with an apparent mendelian syndrome of autoimmunity characterized by high-titer autoantibodies, inflammatory arthritis and interstitial lung disease. We identified four unique deleterious variants in the COPA gene (encoding coatomer subunit α) affecting the same functional domain. Hypothesizing that mutant COPA leads to defective intracellular transport via coat protein complex I (COPI), we show that COPA variants impair binding to proteins targeted for retrograde Golgi-to-ER transport. Additionally, expression of mutant COPA results in ER stress and the upregulation of cytokines priming for a T helper type 17 (TH17) response. Patient-derived CD4(+) T cells also demonstrate significant skewing toward a TH17 phenotype that is implicated in autoimmunity. Our findings uncover an unexpected molecular link between a vesicular transport protein and a syndrome of autoimmunity manifested by lung and joint disease.

Numerous genetic and environmental insults impede the ability of cells to properly fold and posttranslationally modify secretory and transmembrane proteins in the endoplasmic reticulum (ER), leading to a buildup of misfolded proteins in this organelle--a condition called ER stress. ER-stressed cells must rapidly restore protein-folding capacity to match protein-folding demand if they are to survive. In the presence of high levels of misfolded proteins in the ER, an intracellular signaling pathway called the unfolded protein response (UPR) induces a set of transcriptional and translational events that restore ER homeostasis. However, if ER stress persists chronically at high levels, a terminal UPR program ensures that cells commit to self-destruction. Chronic ER stress and defects in UPR signaling are emerging as key contributors to a growing list of human diseases, including diabetes, neurodegeneration, and cancer. Hence, there is much interest in targeting components of the UPR as a therapeutic strategy to combat these ER stress-associated pathologies.

The inability of cells to properly fold, modify and assemble secretory and transmembrane proteins leads to accumulation of misfolded proteins in the endoplasmic reticulum (ER). Under these conditions of 'ER stress', cell survival depends on homeostatic benefits from an intracellular signaling pathway called the unfolded protein response (UPR). When activated, the UPR induces transcriptional and translational programs that restore ER homeostasis. However, under high-level or chronic ER stress, these adaptive changes ultimately become overshadowed by alternative 'terminal UPR' signals that actively commit cells to degeneration, culminating in programmed cell death. Chronic ER stress and maladaptive UPR signaling are implicated in the etiology and pathogenesis of myriad human diseases. Naturally, this has generated widespread interest in targeting key nodal components of the UPR as therapeutic strategies. Here we summarize the state of this field with emphasis placed on two of the master UPR regulators, PERK and IRE1, which are both capable of being drugged with small molecules.

Depending on endoplasmic reticulum (ER) stress levels, the ER transmembrane multidomain protein IRE1α promotes either adaptation or apoptosis. Unfolded ER proteins cause IRE1α lumenal domain homo-oligomerization, inducing trans autophosphorylation that further drives homo-oligomerization of its cytosolic kinase/endoribonuclease (RNase) domains to activate mRNA splicing of adaptive XBP1 transcription factor. However, under high/chronic ER stress, IRE1α surpasses an oligomerization threshold that expands RNase substrate repertoire to many ER-localized mRNAs, leading to apoptosis. To modulate these effects, we developed ATP-competitive IRE1α Kinase-Inhibiting RNase Attenuators-KIRAs-that allosterically inhibit IRE1α's RNase by breaking oligomers. One optimized KIRA, KIRA6, inhibits IRE1α in vivo and promotes cell survival under ER stress. Intravitreally, KIRA6 preserves photoreceptor functional viability in rat models of ER stress-induced retinal degeneration. Systemically, KIRA6 preserves pancreatic β cells, increases insulin, and reduces hyperglycemia in Akita diabetic mice. Thus, IRE1α powerfully controls cell fate but can itself be controlled with small molecules to reduce cell degeneration.

BCL-2 family members are known to be implicated in survival in numerous biological settings. Here, we provide evidence that in injury and repair processes in lungs, BCL-2 mainly acts to attenuate endoplasmic reticulum (ER) stress and limit extracellular matrix accumulation. Days after an intratracheal bleomycin challenge, mice lose a fraction of their alveolar type II epithelium from terminal ER stress driven by activation of the critical ER sensor and stress effector IRE1α. This fraction is dramatically increased by BCL-2 inhibition, because IRE1α activation is dependent on its physical association with the BCL-2–proapoptotic family member BAX, and we found BCL-2 to disrupt this association in vitro. In vivo, navitoclax (a BCL-2/BCL-xL inhibitor) given 15–21 days after bleomycin challenge evoked strong activation of IRE-1α in mesenchymal cells and markers of ER stress, but not apoptosis. Remarkably, after BCL-2 inhibition, bleomycin-exposed mice demonstrated persistent collagen accumulation at Day 42, compared with resolution in controls. Enhanced fibrosis proved to be due to the RNAase activity of IRE1α downregulating MRC2 mRNA and protein, a mediator of collagen turnover. The critical role of MRC2 was confirmed in precision-cut lung slice cultures of Day-42 lungs from bleomycin-exposed wild-type and MRC2 null mice. Soluble and tissue collagen accumulated in precision-cut lung slice cultures from navitoclax-treated, bleomycin-challenged mice compared with controls, in a manner nearly identical to that of challenged but untreated MRC2 null mice. Thus, apart from mitochondrial-based antiapoptosis, BCL-2 functions to attenuate ER stress responses, fostering tissue homeostasis and injury repair. [ABSTRACT FROM AUTHOR]

BackgroundStress of the endoplasmic reticulum (ER) leading to activation of the unfolded protein response (UPR) and alveolar epithelial cell (AEC) apoptosis may play a role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Our objectives were to determine whether circulating caspase-cleaved cytokeratin-18 (cCK-18) is a marker of AEC apoptosis in IPF, define the relationship of cCK-18 with activation of the UPR, and assess its utility as a diagnostic biomarker.MethodsIPF and normal lung tissues were stained with the antibody (M30) that specifically binds cCK-18. The relationship between markers of the UPR and cCK-18 was determined in AECs exposed in vitro to thapsigargin to induce ER stress. cCK-18 was measured in serum from subjects with IPF, hypersensitivity pneumonitis (HP), nonspecific interstitial pneumonia (NSIP), and control subjects.ResultscCK-18 immunoreactivity was present in AECs of IPF lung, but not in control subjects. Markers of the UPR (phosphorylated IRE-1α and spliced XBP-1) were more highly expressed in IPF type II AECs than in normal type II AECs. Phosphorylated IRE-1α and cCK-18 increased following thapsigargin-induced ER stress. Serum cCK-18 level distinguished IPF from diseased and control subjects. Serum cCK-18 was not associated with disease severity or outcome.ConclusionscCK-18 may be a marker of AEC apoptosis and UPR activation in patients with IPF. Circulating levels of cCK-18 are increased in patients with IPF and cCK-18 may be a useful diagnostic biomarker.

The endoplasmic reticulum (ER) is the primary organelle for folding and maturation of secretory and transmembrane proteins. Inability to meet protein-folding demand leads to "ER stress," and activates IRE1α, an ER transmembrane kinase-endoribonuclease (RNase). IRE1α promotes adaptation through splicing Xbp1 mRNA or apoptosis through incompletely understood mechanisms. Here, we found that sustained IRE1α RNase activation caused rapid decay of select microRNAs (miRs -17, -34a, -96, and -125b) that normally repress translation of Caspase-2 mRNA, and thus sharply elevates protein levels of this initiator protease of the mitochondrial apoptotic pathway. In cell-free systems, recombinant IRE1α endonucleolytically cleaved microRNA precursors at sites distinct from DICER. Thus, IRE1α regulates translation of a proapoptotic protein through terminating microRNA biogenesis, and noncoding RNAs are part of the ER stress response.

Overwhelming of protein folding in the endoplasmic reticulum (ER)--referred to as "ER stress"--activates a set of intracellular signaling pathways termed the unfolded protein response (UPR). Beneficial outputs of the UPR promote adaptation in cells experiencing manageably low levels of ER stress. However, if ER stress reaches critically high levels, the UPR uses destructive outputs to trigger programmed cell death. Genetic mutations in various UPR components cause inherited syndromes of diabetes mellitus in both rodents and humans, implicating the UPR in the proper functioning and survival of pancreatic islet β cells. Markers of chronically elevated ER stress, terminal UPR signaling, and apoptosis are evident in β cells in these rare disorders; these markers are similarly present in islets of human patients with common forms of diabetes. These findings promise to enhance our molecular understanding of human diabetes significantly and may lead to new and effective therapies.

When unfolded proteins accumulate to irremediably high levels within the endoplasmic reticulum (ER), intracellular signaling pathways called the unfolded protein response (UPR) become hyperactivated to cause programmed cell death. We discovered that thioredoxin-interacting protein (TXNIP) is a critical node in this "terminal UPR." TXNIP becomes rapidly induced by IRE1α, an ER bifunctional kinase/endoribonuclease (RNase). Hyperactivated IRE1α increases TXNIP mRNA stability by reducing levels of a TXNIP destabilizing microRNA, miR-17. In turn, elevated TXNIP protein activates the NLRP3 inflammasome, causing procaspase-1 cleavage and interleukin 1β (IL-1β) secretion. Txnip gene deletion reduces pancreatic β cell death during ER stress and suppresses diabetes caused by proinsulin misfolding in the Akita mouse. Finally, small molecule IRE1α RNase inhibitors suppress TXNIP production to block IL-1β secretion. In summary, the IRE1α-TXNIP pathway is used in the terminal UPR to promote sterile inflammation and programmed cell death and may be targeted to develop effective treatments for cell degenerative diseases.