Unfolded Protein Response Target Genes Research Articles

ATF6 protects against protein misfolding during cardiac hypertrophy

Cardiomyocytes activate the unfolded protein response (UPR) transcription factor ATF6 during pressure overload-induced hypertrophic growth. The UPR is thought to increase ER protein folding capacity and maintain proteostasis. ATF6 deficiency during pressure overload leads to heart failure, suggesting that ATF6 protects against myocardial dysfunction by preventing protein misfolding. However, conclusive evidence that ATF6 prevents toxic protein misfolding during cardiac hypertrophy is still pending. Here, we found that activation of the UPR, including ATF6, is a common response to pathological cardiac hypertrophy in mice. ATF6 KO mice failed to induce sufficient levels of UPR target genes in response to chronic isoproterenol infusion or transverse aortic constriction (TAC), resulting in impaired cardiac growth. To investigate the effects of ATF6 on protein folding, the accumulation of poly-ubiquitinated proteins as well as soluble amyloid oligomers were directly quantified in hypertrophied hearts of WT and ATF6 KO mice. Whereas only low levels of protein misfolding was observed in WT hearts after TAC, ATF6 KO mice accumulated increased quantities of misfolded protein, which was associated with impaired myocardial function. Collectively, the data suggest that ATF6 plays a critical adaptive role during cardiac hypertrophy by protecting against protein misfolding.

A Survey of Naturally Occurring Molecules as New Endoplasmic Reticulum Stress Activators with Selective Anticancer Activity

The last century has witnessed the establishment of neoplastic disease as the second cause of death in the world. Nonetheless, the road toward desirable success rates of cancer treatments is still long and paved with uncertainty. This work aims to select natural products that act via endoplasmic reticulum (ER) stress, a known vulnerability of malignant cells, and display selective toxicity against cancer cell lines. Among an in-house chemical library, nontoxic molecules towards noncancer cells were assessed for toxicity towards cancer cells, namely the human gastric adenocarcinoma cell line AGS and the lung adenocarcinoma cell line A549. Active molecules towards at least one of these cell lines were studied in a battery of ensuing assays to clarify the involvement of ER stress and unfolded protein response (UPR) in the cytotoxic effect. Several natural products are selectively cytotoxic against malignant cells, and the effect often relies on ER stress induction. Berberine was the most promising molecule, being active against both cell models by disrupting Ca2+ homeostasis, inducing UPR target gene expression and ER-resident caspase-4 activation. Our results indicate that berberine and emodin are potential leads for the development of more potent ER stressors to be used as selective anticancer agents.

Regulation of the unfolded protein response transducer IRE1α by SERPINH1 aggravates periodontitis with diabetes mellitus via prolonged ER stress

The hyperglycemic microenvironment induced by diabetes mellitus aggravates the inflammatory response, in which the IRE1α signal transduction pathway of the unfolded protein response (UPR) participates. However, the mechanism by which hyperglycemia regulates the IRE1α signaling pathway and affects endoplasmic reticulum (ER) homeostasis in human gingival epithelium in periodontitis with diabetes mellitus remains unknown. Our current data provide evidence that diabetes mellitus causes a hyperinflammatory response in the gingival epithelium, which accelerates periodontal inflammation. Next, we assessed UPR-IRE1α signaling in periodontitis with diabetes mellitus by examining human clinical gingival epithelium samples from healthy subjects, subjects with periodontitis and subjects with periodontitis with diabetes mellitus and by in vitro challenge of human epithelial cells with a hyperglycemic microenvironment. The results showed that a hyperglycemic microenvironment inhibited the IRE1α/XBP1 axis, decreased the expression of a UPR target gene (GRP78), and ultimately impaired the UPR, causing ER stress to be prolonged or more severe in human gingival epithelium. Subsequently, RNA sequencing (RNA-seq) data was analyzed to investigate the expression of ER-related genes in human gingival epithelium. Experiments verified that the mechanism by which periodontitis is aggravated in individuals with diabetes mellitus may involve decreased SERPINH1 expression. Furthermore, experiments in SERPINH1-knockdown and SERPINH1-overexpression models established in vitro indicated that SERPINH1 might act as an activator of IRE1α, maintaining human gingival epithelium homeostasis and reducing proinflammatory cytokine expression by preventing prolonged ER stress induced by high-glucose conditions. In conclusion, regulation of the UPR transducer IRE1α by SERPINH1 alleviates periodontitis with diabetes mellitus by mitigating prolonged ER stress. This finding provides evidence for the further study of periodontitis with diabetes mellitus.

Sensor dimer disruption as a new mode of action to block the IRE1-mediated unfolded protein response

The unfolded protein response (UPR) is activated to cope with an accumulation of improperly folded proteins in the Endoplasmic reticulum (ER). The Inositol requiring enzyme 1α (IRE1α) is the most evolutionary conserved transducer of the UPR. Activated IRE1 forms ‘back-to-back’-dimers that enables the unconventional splicing of X-box Binding Protein 1 (XBP1) mRNA. The spliced XBP1 (XBP1s) mRNA is translated into a transcription factor controlling the expression of UPR target genes. Herein, we report a detailed in silico screening specifically targeting for the first time the dimer interface at the IRE1 RNase region. Using the database of FDA approved drugs, we identified four compounds (neomycin, pemetrexed, quercitrin and rutin) that were able to bind to and distort IRE1 RNase cavity. The activity of the compounds on IRE1 phosphorylation was evaluated in HEK293T cells and on IRE1 RNase activity using an in vitro fluorescence assay. These analyzes revealed sub-micromolar IC50 values. The current study reveals a new and unique mode of action to target and block the IRE1-mediated UPR signaling, whereby we may avoid problems associated with selectivity occurring when targeting the IRE1 kinase pocket as well as the inherent reactivity of covalent inhibitors targeting the RNase pocket.

Assays to Study IRE1 Activation and Signaling.

The endoplasmic reticulum (ER) stress sensor IRE1 is a a major player of the unfolded protein response (UPR), the main pathway driving adaptation processes to restore proteostasis.In addition, overactivation of IRE1 signaling contributes to a variety of pathologies including diabetes, neurodegenerative diseases, and cancer. Under ER stress, IRE1 auto-transphosphorylates and oligomerizes, triggering the activation of its endoribonuclease domain located in the cytosolic region. Active IRE1 catalyzes the splicing of the mRNA encoding for the XBP1 transcription factor, in addition to degrade several RNAs through a process known asregulated IRE1-dependent decay of mRNA (RIDD). Besides its role as an UPR transducer, several posttranslational modifications and protein-protein interactions can regulate IRE1 activity and modulate its signaling in the absence of stress.Thus, investigating the function of IRE1 in physiology and disease requires the use of complementary approaches. Here, we provide detailed protocols to perform four different assays to study IRE1 activation and signaling: (i) Phos-tag gels to evaluate the phosphorylation status of IRE1, (ii) microscopy using TREX-IRE1-GFP cells to measure IRE1 oligomerization, (iii) conventional RT-PCR to assess XBP1 mRNA processing, and (iv) quantitative PCR to determine the levels of canonical UPR target genes and the degradation of several mRNAs that are target of RIDD. We propose to use these experimental strategies as "gold standards" to study IRE1 signaling.

A Platform Technology for Monitoring the Unfolded Protein Response.

The unfolded protein response (UPR) is a complex signal transduction pathway that remodels gene expression in response to proteotoxic stress in the endoplasmic reticulum (ER) and is linked to the development of a range of diseases, including Alzheimer's disease, diabetes, and several types of cancer. UPR induction is typically monitored by measuring the expression level of UPR marker genes. Most tools for quantifying gene expression, including DNA microarrays and quantitative PCR with reverse transcription (RT-PCR), produce snapshots of the cell transcriptome, but are not ideal for measurements requiring temporal resolution of gene expression dynamics. Reporter assays for indirect detection of the UPR typically rely on extrachromosomal expression of reporters under the control of minimal or synthetic regulatory sequences that do not recapitulate the native chromosomal context of the UPR target genes. To address the need for tools to monitor chromosomal gene expression that recapitulate gene expression dynamics from the native chromosomal context and generate a readily detectable signal output, we developed a gene signal amplifier platform that links transcriptional and post-translational regulation of a fluorescent output to the expression of a chromosomal gene marker of the UPR. The platform is based on a genetic circuit that amplifies the output signal with high sensitivity and dynamic resolution and is implemented through chromosomal integration of the gene encoding the main control element of the genetic circuit to link its expression to that of the target gene, thereby generating a platform that can be easily adapted to monitor any UPR target through integration of the main control element at the appropriate chromosomal locus. By recapitulating the transcriptional and translational control mechanisms underlying the expression of UPR targets with high sensitivity, this platform provides a novel technology for monitoring the UPR with superior sensitivity and dynamic resolution.

Characterization and modulation of endoplasmic reticulum stress response target genes in Kluyveromyces marxianus to improve secretory expressions of heterologous proteins

BackgroundKluyveromyces marxianus is a promising cell factory for producing bioethanol and that raised a demand for a high yield of heterologous proteins in this species. Expressions of heterologous proteins usually lead to the accumulation of misfolded or unfolded proteins in the lumen of the endoplasmic reticulum (ER) and then cause ER stress. To cope with this problem, a group of ER stress response target genes (ESRTs) are induced, mainly through a signaling network called unfolded protein response (UPR). Characterization and modulation of ESRTs direct the optimization of heterologous expressions. However, ESRTs in K. marxianus have not been identified so far.ResultsIn this study, we characterized the ER stress response in K. marxianus for the first time, by using two ER stress-inducing reagents, dithiothreitol (DTT) and tunicamycin (TM). Results showed that the Kar2–Ire1–Hac1 pathway of UPR is well conserved in K. marxianus. About 15% and 6% of genes were upregulated during treatment of DTT and TM, respectively. A total of 115 upregulated genes were characterized as ESRTs, among which 97 genes were identified as UPR target genes and 37 UPR target genes contained UPR elements in their promoters. Genes related to carbohydrate metabolic process and actin filament organization were identified as new types of UPR target genes. A total of 102 ESRTs were overexpressed separately in plasmids and their effects on productions of two different lignocellulolytic enzymes were systematically evaluated. Overexpressing genes involved in carbohydrate metabolism, including PDC1, PGK and VID28, overexpressing a chaperone gene CAJ1 or overexpressing a reductase gene MET13 substantially improved secretion expressions of heterologous proteins. Meanwhile, overexpressing a novel gene, KLMA_50479 (named ESR1), as well as overexpressing genes involved in ER-associated protein degradation (ERAD), including HRD3, USA1 andYET3, reduced the secretory expressions. ESR1 and the aforementioned ERAD genes were deleted from the genome. Resultant mutants, except the yet3Δ mutant, substantially improved secretions of three different heterologous proteins. During the fed-batch fermentation, extracellular activities of an endoxylanase and a glucanase in hrd3Δ cells improved by 43% and 28%, respectively, compared to those in wild-type cells.ConclusionsOur results unveil the transcriptional scope of the ER stress response in K. marxianus and suggest efficient ways to improve productions of heterologous proteins by manipulating expressions of ESRTs.

The Role of the UPR Pathway in the Pathophysiology and Treatment of Bipolar Disorder.

Bipolar disorder (BD) is a mood disorder that affects millions worldwide and is associated with severe mood swings between mania and depression. The mood stabilizers valproate (VPA) and lithium (Li) are among the main drugs that are used to treat BD patients. However, these drugs are not effective for all patients and cause serious side effects. Therefore, better drugs are needed to treat BD patients. The main barrier to developing new drugs is the lack of knowledge about the therapeutic mechanism of currently available drugs. Several hypotheses have been proposed for the mechanism of action of mood stabilizers. However, it is still not known how they act to alleviate both mania and depression. The pathology of BD is characterized by mitochondrial dysfunction, oxidative stress, and abnormalities in calcium signaling. A deficiency in the unfolded protein response (UPR) pathway may be a shared mechanism that leads to these cellular dysfunctions. This is supported by reported abnormalities in the UPR pathway in lymphoblasts from BD patients. Additionally, studies have demonstrated that mood stabilizers alter the expression of several UPR target genes in mouse and human neuronal cells. In this review, we outline a new perspective wherein mood stabilizers exert their therapeutic mechanism by activating the UPR. Furthermore, we discuss UPR abnormalities in BD patients and suggest future research directions to resolve discrepancies in the literature.

IRE1 signaling regulates chondrocyte apoptosis and death fate in the osteoarthritis.

IRE1 is an important central regulator of unfolded protein response (UPR) in the endoplasmic reticulum (ER) because of its ability to regulate cell fate as a function of stress sensing. When misfolded proteins accumulated in chondrocytes ER, IRE1 disintegrates with BIP/GRP78 and undergoes dimer/oligomerization and transautophosphorylation. These two processes are mediated through an enzyme activity of IRE1 to activate endoribonuclease and generates XBP1 by unconventional splicing of XBP1 messenger RNA. Thereby promoting the transcription of UPR target genes and apoptosis. The deficiency of inositol‐requiring enzyme 1α (IRE1α) in chondrocytes downregulates prosurvival factors XBP1S and Bcl‐2, which enhances the apoptosis of chondrocytes through increasing proapoptotic factors caspase‐3, p‐JNK, and CHOP. Meanwhile, the activation of IRE1α increases chondrocyte viability and reduces cell apoptosis. However, the understanding of IRE1 responses and cell death fate remains controversial. This review provides updated data about the role IRE1 plays in chondrocytes and new insights about the potential efficacy of IRE1 regulation in cartilage repair and osteoarthritis treatment.

Roles of XBP1s in Transcriptional Regulation of Target Genes.

The spliced form of X-box binding protein 1 (XBP1s) is an active transcription factor that plays a vital role in the unfolded protein response (UPR). Under endoplasmic reticulum (ER) stress, unspliced Xbp1 mRNA is cleaved by the activated stress sensor IRE1α and converted to the mature form encoding spliced XBP1 (XBP1s). Translated XBP1s migrates to the nucleus and regulates the transcriptional programs of UPR target genes encoding ER molecular chaperones, folding enzymes, and ER-associated protein degradation (ERAD) components to decrease ER stress. Moreover, studies have shown that XBP1s regulates the transcription of diverse genes that are involved in lipid and glucose metabolism and immune responses. Therefore, XBP1s has been considered an important therapeutic target in studying various diseases, including cancer, diabetes, and autoimmune and inflammatory diseases. XBP1s is involved in several unique mechanisms to regulate the transcription of different target genes by interacting with other proteins to modulate their activity. Although recent studies discovered numerous target genes of XBP1s via genome-wide analyses, how XBP1s regulates their transcription remains unclear. This review discusses the roles of XBP1s in target genes transcriptional regulation. More in-depth knowledge of XBP1s target genes and transcriptional regulatory mechanisms in the future will help develop new therapeutic targets for each disease.

Abstract 2689: Investigating the molecular mechanisms linking disrupted growth homeostasis to tumourigenesis in Li-Fraumeni Syndrome

Abstract Li-Fraumeni Syndrome (LFS) is a highly penetrant cancer predisposition syndrome caused by germline TP53 mutations. Patients face an increased lifetime risk to develop a broad spectrum of cancers. Implementation of comprehensive cancer screening protocols has led to improvements in survival for LFS patients. However, these interventions pose a significant burden to both patient quality of life and health resource utilization. While these interventions will always be important, recent evidence has suggested an additional approach which may improve patient outcomes: pharmacological prevention of cancer onset. mTORC1 inhibition selectively targets tumor initiation. While rapamycin, an mTORC1 inhibitor, has not shown efficacy in treatment of established cancers, it is reported to prevent tumor onset in mice that are genetically predisposed to cancer, including mouse models of LFS. mTORC1 is associated with protein synthesis and cellular growth. Hyperactivated cellular growth is associated with gain-of-function (GOF) TP53 mutations that underlie many cases of LFS. Wild-type p53 (wtp53) inhibits mTORC1 and, as a consequence, biosynthesis. We have shown that mutant p53 with oncogenic GOF (mutp53) may promote the opposite effect. Under conditions of inappropriately elevated protein synthesis, as is seen in cancer, stress responses, such as the unfolded protein response (UPR), are activated. UPR activation contributes to the secretion of pro-tumorigenic cytokines. We hypothesized that under conditions of elevated protein synthesis, mutp53 engages in a feedforward activation of biosynthesis which hyperactivates UPR-mediated secretion of pro-tumorigenic cytokines. In order to harness the cancer risk reduction potential of mTORC1 inhibition, we undertook investigation of the stress responses which may underlie tumorigenesis in LFS. We sought to establish whether mTORC1 ablation in the tumor microenvironment could influence growth of tumor xenografts formed from the co-injection of LFS patient-derived mutp53 fibroblasts and RH4 rhabdomyosarcoma cells, a representative LFS spectrum cancer cell line. While both wtp53 fibroblasts and mutp53 fibroblasts supported tumor growth, only the tumor growth reinforced by mutp53 fibroblasts was suppressed by rapamycin. Additionally, we utilized protein arrays to profile conditioned media collected from mutp53 and wtp53 fibroblast cultures. Mutp53 fibroblasts secreted higher levels of various pro-tumorigenic cytokines compared to wtp53 fibroblasts, which was partially negated by rapamycin. UPR target gene expression is elevated in fibroblasts expressing mutp53, and this effect is negated by rapamycin. Altogether, our work supports a role for mutp53-driven, mTORC1-mediated hyperactivated protein biosynthesis and subsequent engagement of the UPR, resulting in production of a pro-tumorigenic secretory milieu. Citation Format: Camilla Giovino, Frank Telfer, Nish Patel, Sangeetha Paramathas, Ran Kafri, David Malkin. Investigating the molecular mechanisms linking disrupted growth homeostasis to tumourigenesis in Li-Fraumeni Syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2689.

Abstract 2026: KDM1A inhibition augment ER stress inducers efficacy to reduce glioblastoma stemness

Abstract Background: Glioblastoma (GBM) is the most common malignant brain tumor with a dismal prognosis and median survival of 20 months. Standard therapy consists of surgical resection, external beam radiation therapy, adjuvant chemotherapy with temozolomide, and tumor-treating fields. Despite these therapies, GBM patients inevitably experience tumor progression and eventually succumb to their disease. Glioma stem cells (GSCs) play a central role in GBM development and contribute to treatment resistance. Recently we showed that lysine-specific histone demethylase 1A (KDM1A/LSD1) is essential for GSCs stemness, and inhibition of KDM1A induces unfolded protein response (UPR) in GSCs. In this study, we tested the hypothesis that inhibition of KDM1A sensitizes GSCs to ER stress inducers by inducing UPR. Methods: KDM1A knockdown (KDM1A-KD) cells were generated using KDM1A specific shRNA. We studied the effect of KDM1A-KD or pharmacological KDM1A inhibitors (NCD38 and NCL-1) in combination with ER stress inducers (thapsigargin and brefeldin A) on GSCs viability using CellTiter-Glo assay. Stemness was determined using extreme limiting dilution analysis (ELDA) and sphere formation assays. Mechanistic studies were conducted using RNA-seq, ChIP, RT-qPCR, and Western blotting analysis. Furthermore, the in vivo efficacy of KDM1A inhibitor and ER stress inducer was studied using orthotopic models of GBM. Results: Cell viability assays demonstrated that knockdown or inhibition of KDM1A sensitized GSCs to ER stress inducers thapsigargin and brefeldin A. Furthermore, KDM1A-KD, NCD38, or NCL-1, in combination with ER stress inducers, significantly decreased the stemness and sphere-forming ability of GSCs. RNA-seq analysis revealed that UPR was activated after knockdown or inhibition of KDM1A in GSCs. Western blot and RT-qPCR analysis showed that a combination of KDM1A inhibitors and ER stress inducers increased UPR signaling in GSCs. ChIP analysis indicated that KDM1A inhibition enriched the active histone methylation mark (H3K4me2) at the promoter of UPR target genes. In vivo studies showed that KDM1A inhibition activates UPR in tumors and improved overall survival. Conclusions: Our results support that KDM1A knockdown or inhibition sensitizes GSCs to ER stress inducers and that the use of KDM1A inhibitors in conjunction with ER stress inducers is a potential novel therapy for GBM patients. Citation Format: Yi He, Prabhakar Pitta Venkata, Salvador Alejo, Yihong Chen, Bridgitte Palacios, Gabrielle Gray, Uday P. Pratap, Suryavathi Viswanadhapalli, Siyuan Zheng, Rajeshwar R. Tekmal, Andrew J. Brenner, Gangadhara R. Sareddy. KDM1A inhibition augment ER stress inducers efficacy to reduce glioblastoma stemness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2026.

OTUB1 facilitates bladder cancer progression by stabilizing ATF6 in response to endoplasmic reticulum stress.

The unfolded protein response (UPR) plays an important role in carcinogenesis, but the functional role and mechanism of UPR‐associated bladder carcinogenesis remain to be characterized. Upon UPR activation, ATF6α is activated to upregulate the transcription of UPR target genes. Although the mechanism of ATF6 activation has been studied extensively, the negative regulation of ATF6 stabilization is not well understood. Here, we report that the deubiquitinase otubain 1 (OTUB1) facilitates bladder cancer progression by stabilizing ATF6 in response to endoplasmic reticulum stress. OTUB1 expression is raised in bladder cancer patients. Genetic ablation of OTUB1 markedly inhibited bladder cancer cell proliferation, viability, and migration both in vitro and in vivo. Mechanistically, luciferase pathway screening showed that ATF6 signaling was clearly activated compared with other pathways. OTUB1 was found to activate ATF6 signaling by inhibiting its ubiquitylation, thereby remodeling the stressed cells through transcriptional regulation. Our results show that high OTUB1 expression promotes bladder cancer progression by stabilizing ATF6 and that OTUB1 is a potential therapeutic target in bladder cancer.

Enforced dimerization between XBP1s and ATF6f enhances the protective effects of the UPR in models of neurodegeneration

Alteration to endoplasmic reticulum (ER) proteostasis is observed in a variety of neurodegenerative diseases associated with abnormal protein aggregation. Activation of the unfolded protein response (UPR) enables an adaptive reaction to recover ER proteostasis and cell function. The UPR is initiated by specialized stress sensors that engage gene expression programs through the concerted action of the transcription factors ATF4, ATF6f, and XBP1s. Although UPR signaling is generally studied as unique linear signaling branches, correlative evidence suggests that ATF6f and XBP1s may physically interact to regulate a subset of UPR target genes. In this study, we designed an ATF6f/XBP1s fusion protein termed UPRplus that behaves as a heterodimer in terms of its selective transcriptional activity. Cell-based studies demonstrated that UPRplus has a stronger effect in reducing the abnormal aggregation of mutant huntingtin and α-synuclein when compared to XBP1s or ATF6 alone. We developed a gene transfer approach to deliver UPRplus into the brain using adeno-associated viruses (AAVs) and demonstrated potent neuroprotection invivo in preclinical models of Parkinson's disease and Huntington's disease. These results support the concept in which directing UPR-mediated gene expression toward specific adaptive programs may serve as a possible strategy to optimize the beneficial effects of the pathway in different disease conditions.

A Human IRE1 Inhibitor Blocks the Unfolded Protein Response in the Pathogenic Fungus Aspergillus fumigatus and Suggests Noncanonical Functions within the Pathway.

The unfolded protein response (UPR) is a signaling network that maintains homeostasis of the endoplasmic reticulum (ER). In the human-pathogenic fungus Aspergillus fumigatus, the UPR is initiated by activation of an endoribonuclease (RNase) domain in the ER transmembrane stress sensor IreA, which splices the downstream mRNA hacAu into its active form, hacAi, encoding the master transcriptional regulator of the pathway. Small-molecule inhibitors against IRE1, the human ortholog of IreA, have been developed for anticancer therapy, but their effects on the fungal UPR are unexplored. Here, we demonstrate that the IRE1 RNase inhibitor 4μ8C prevented A. fumigatus from increasing the levels of hacAi mRNA, thereby blocking induction of downstream UPR target gene expression. Treatment with 4μ8C had minimal effects on growth in minimal medium but severely impaired growth on a collagen substrate that requires high levels of hydrolytic enzyme secretion, mirroring the phenotype of other fungal UPR mutants. 4μ8C also increased sensitivity to carvacrol, a natural compound that disrupts ER integrity in fungi, and hygromycin B, which correlated with reduced expression of glycosylation-related genes. Interestingly, treatment with 4μ8C was unable to induce all of the phenotypes attributed to the loss of the canonical UPR in a ΔhacA mutant but showed remarkable similarity to the phenotype of an RNase-deficient IreA mutant that is also unable to generate the hacAi mRNA. These results establish proof of principle that pharmacological inhibition of the canonical UPR pathway is feasible in A. fumigatus and support a noncanonical role for the hacAu mRNA in ER stress response.IMPORTANCE The unfolded protein response (UPR) is a signaling pathway that maintains endoplasmic reticulum (ER) homeostasis, with functions that overlap virulence mechanisms in the human-pathogenic mold Aspergillus fumigatus The canonical pathway centers on HacA, its master transcriptional regulator. Translation of this protein requires the removal of an unconventional intron from the cytoplasmic mRNA of the hacA gene, which is achieved by an RNase domain located in the ER-transmembrane stress sensor IreA. Here, we show that targeting this RNase activity with a small-molecule inhibitor effectively blocked UPR activation, resulting in effects that mirror the consequences of genetic deletion of the RNase domain. However, these phenotypes were surprisingly narrow in scope relative to those associated with a complete deletion of the hacA gene. These findings expand the understanding of UPR signaling in this species by supporting the existence of noncanonical functions for the unspliced hacA mRNA in ER stress response.

Inhibition of the PI 3-kinase pathway disrupts the unfolded protein response and reduces sensitivity to ER stress-dependent apoptosis.

Class Ia phosphoinositide 3-kinases (PI3K) are critical mediators of insulin and growth factor action. We have demonstrated that the p85α regulatory subunit of PI3K modulates the unfolded protein response (UPR) by interacting with and regulating the nuclear translocation of XBP-1s, a transcription factor essential for the UPR. We now show that PI3K activity is required for full activation of the UPR. Pharmacological inhibition of PI3K in cells blunts the ER stress-dependent phosphorylation of IRE1α and PERK, decreases induction of ATF4, CHOP, and XBP-1 and upregulates UPR target genes. Cells expressing a human p85α mutant (R649W) previously shown to inhibit PI3K, exhibit decreased activation of IRE1α and PERK and reduced induction of CHOP and ATF4. Pharmacological inhibition of PI3K, overexpression of a mutant of p85α that lacks the ability to interact with the p110α catalytic subunit (∆p85α) or expression of mutant p85α (R649W) in vivo, decreased UPR-dependent induction of ER stress response genes. Acute tunicamycin treatment of R649W+/- mice revealed reduced induction of UPR target genes in adipose tissue, whereas chronic tunicamycin exposure caused sustained increases in UPR target genes in adipose tissue. Finally, R649W+/- cells exhibited a dramatic resistance to ER stress-dependent apoptosis. These data suggest that PI3K pathway dysfunction causes ER stress that may drive the pathogenesis of several diseases including Type 2 diabetes and various cancers.

Testing Inhibitory Drugs of the Human UPR Sensor IRE1 on Aspergillus fumigatus

Aspergillus fumigatus is a fungus that is associated with life‐threatening pulmonary infections. Unfortunately, current antifungal drugs to treat this infection are limited, making the search for novel molecular targets a high priority. A. fumigatus has an intracellular stress response pathway known as the unfolded protein response (UPR), which is key to its ability to cause infection. Inhibitors of the human UPR pathway have been developed, but are untested on A. fumigatus. Here, the ability of STF 083010 and toyocamycin, human UPR inhibitors, to inhibit the corresponding fungal UPR pathway and reduce the fitness of A. fumigatus was tested. Culturing of A. fumigatus in the presence or absence of the compound under conditions that require UPR intervention demonstrated that toyocamycin did not affect the fungus, whereas STF 083010 inhibits colony growth and is fungistatic. Additionally, STF 083010 increases the toxicity of ER stress agents and cell wall damaging agents, and it also prevents the fungus from utilizing polymeric substrates. Finally, the ability of STF 083010 to inhibit the induction of UPR target genes by a strong stimulus was examined by qPCR. The results demonstrate that STF 083010 blocks the ability of DTT to induce the expression of hacAi and bipA, indicating suppression of the fungal UPR. These results provide the first evidence that pharmacologic inhibition of the UPR pathway is feasible in A. fumigatus, and that the loss of fitness by STF 083010 could be a novel approach to antifungal therapy.

Inhibition of autophagy sensitizes lignan-induced endoplasmic reticulum stress-mediated cell death

Relationship between autophagy and endoplasmic reticulum (ER) stress and their application to treat cancer have been actively studied these days. Recently, a lignan [(−)-(2R, 3R)-1,4-O-diferuloylsecoisolariciresinol, DFS] from Alnus japonica has been found to reduce the viability of colon cancer cells. In this study, we have observed DFS-induced autophagy in a variety of cancer cell lines. In addition, DFS led to ER stress, based on the activation of unfolded protein response (UPR) transducers and an elevated expression of UPR target genes in prostate and colon cancer cells. Further investigation has shown that DFS triggered the activation of AMP-activated protein kinase (AMPK) signaling and nuclear translocation of transcription factor EB (TFEB). Furthermore, the cytotoxicity of DFS was potentiated by the co-treatment of autophagy inhibitor in these cancer cells. This study has provided a noble implication that the combination of DFS and autophagy inhibition exerts a synergistic effect in cancer treatment.

Vitamin D Receptor Activation in Liver Macrophages Protects Against Hepatic Endoplasmic Reticulum Stress in Mice.

Hepatic endoplasmic reticulum (ER) stress, whether triggered by intrinsic or extrinsic factors, can be resolved by the unfolded protein response (UPR). Sustained UPR activation leads to cell death and inflammatory response and contributes to liver disease progression. Hepatic tissue macrophages are key players in orchestrating liver inflammation, and ER stress can enhance macrophage activation. However, it is not well defined how the interplay between ER stress and inflammation is regulated during hepatic stress response. Here we demonstrate that vitamin D receptor (VDR) activation mitigates hepatic ER stress response, whereas VDR knockout mice undergo persistent UPR activation and apoptosis in response to chemical ER stress inducer. Moreover, VDR deficiency promotes hepatic macrophage infiltration and increases gene expression and systematic levels of proinflammatory cytokines, including interleukin (IL)-1β, IL-6, and tumor necrosis factor α. VDR expression is induced in hepatic macrophages by ER stress, and VDR plays a dual regulatory role in macrophages by protecting against ER stress and promoting anti-inflammatory polarization. Co-culture with VDR-activated bone marrow-derived macrophages suppresses UPR target genes in primary hepatocytes treated with ER stress inducers. Thus, the immunomodulatory functions of VDR in macrophages are critical in hepatic ER stress resolution in mice. VDR signaling in macrophages regulates a shift between proinflammatory and anti-inflammatory activation during ER stress-induced inflammation to promote hepatic ER stress resolution.

No evidence for cell-to-cell transmission of the unfolded protein response in cell culture.

The unfolded protein response (UPR) is one of the major cell‐autonomous proteostatic stress responses. The UPR has been implicated in the pathogenesis of neurodegenerative diseases and is therefore actively investigated as therapeutic target. In this respect, cell non‐autonomous effects of the UPR including the reported cell‐to‐cell transmission of UPR activity may be highly important. A pharmaca‐based UPR induction was employed to generate conditioned media (CM) from CM‐donating neuronal (‘donor’) cells (SK‐N‐SH and primary mouse neurons). As previously reported, upon subsequent transfer of CM to naive neuronal ‘acceptor’ cells, we confirmed UPR target mRNA and protein expression by qPCR and automated microscopy. However, UPR target gene expression was also induced in the absence of donor cells, indicating carry‐over of pharmaca. Genetic induction of single pathways of the UPR in donor cells did not result in UPR transmission to acceptor cells. Moreover, no transmission was detected upon full UPR activation by nutrient deprivation or inducible expression of the heavy chain of immunoglobulin M in donor HeLa cells. In addition, in direct co‐culture of donor cells expressing the immunoglobulin M heavy chain and fluorescent UPR reporter acceptor HeLa cells, UPR transmission was not observed. In conclusion, carry‐over of pharmaca is a major confounding factor in pharmaca‐based UPR transmission protocols that are therefore unsuitable to study cell‐to‐cell UPR transmission. In addition, the absence of UPR transmission in non‐pharmaca‐based models of UPR activation indicates that cell‐to‐cell UPR transmission does not occur in cell culture.