Unfolded Protein Response Response Research Articles

FicD regulates adaptation to the unfolded protein response in the murine liver

The unfolded protein response (UPR) is a cellular stress response that is activated when misfolded proteins accumulate in the endoplasmic reticulum (ER). Regulation of the UPR response must be adapted to the needs of the cell as prolonged UPR responses can result in disrupted cellular function and tissue damage. Previously, we discovered that the enzyme FicD (also known as Fic or HYPE) through its AMPylation and deAMPylation activity can modulate the UPR response via post-translational modification of BiP. FicD AMPylates BiP during homeostasis and deAMPylates BiP during stress. We hypothesized that FicD regulation of the UPR will play a role in mitigating the deleterious effects of UPR activation in tissues with frequent physiological stress. Here, we explore the role of FicD in the murine liver. As seen in our pancreatic studies, livers lacking FicD exhibit enhanced UPR signaling in response to short term physiologic fasting and feeding stress. However, in contrast to studies on the pancreas, livers, as a more regenerative tissue, remained remarkably resilient in the absence of FicD. The livers of FicD−/− did not show marked changes in UPR signaling or damage after either chronic high fat diet (HFD) feeding or acute pathological UPR induction. Intriguingly, FicD−/− mice showed changes in UPR induction and weight loss patterns following repeated pathological UPR induction. These findings indicate that FicD regulates UPR responses during mild physiological stress and in adaptation to repeated stresses, but there are tissue specific differences in the requirement for FicD regulation.

Elucidation of molecular mechanisms by which amyloid β1–42 fibrils exert cell toxicity

Abrupt aggregation of amyloid β1–42 (Aβ1–42) peptide in the frontal lobe is the expected underlying cause of Alzheimer's disease (AD). β-Sheet-rich oligomers and fibrils formed by Aβ1–42 exert high cell toxicity. A growing body of evidence indicates that lipids can uniquely alter the secondary structure and toxicity of Aβ1–42 aggregates. At the same time, underlying molecular mechanisms that determine this difference in toxicity of amyloid aggregates remain unclear. Using a set of molecular and biophysical assays to determine the molecular mechanism by which Aβ1–42 aggregates formed in the presence of cholesterol, cardiolipin, and phosphatidylcholine exert cell toxicity. Our findings demonstrate that rat neuronal cells exposed to Aβ1–42 fibrils formed in the presence of lipids with different chemical structure exert drastically different magnitude and dynamic of unfolded protein response (UPR) in the endoplasmic reticulum (ER) and mitochondria (MT). We found that the opposite dynamics of UPR in MT and ER in the cells exposed to Aβ1–42: cardiolipin fibrils and Aβ1–42 aggregates formed in a lipid-free environment. We also found that Aβ1–42: phosphatidylcholine fibrils upregulated ER UPR simultaneously downregulating the UPR response of MT, whereas Aβ1–42: cholesterol fibrils suppressed the UPR response of ER and upregulated UPR response of MT. We also observed progressively increasing ROS production that damages mitochondrial membranes and other cell organelles, ultimately leading to cell death.

Thapsigargin and Tunicamycin Block SARS-CoV-2 Entry into Host Cells via Differential Modulation of Unfolded Protein Response (UPR), AKT Signaling, and Apoptosis.

SARS-Co-V2 infection can induce ER stress-associated activation of unfolded protein response (UPR) in host cells, which may contribute to the pathogenesis of COVID-19. To understand the complex interplay between SARS-Co-V2 infection and UPR signaling, we examined the effects of acute pre-existing ER stress on SARS-Co-V2 infectivity. Huh-7 cells were treated with Tunicamycin (TUN) and Thapsigargin (THA) prior to SARS-CoV-2pp transduction (48 h p.i.) to induce ER stress. Pseudo-typed particles (SARS-CoV-2pp) entry into host cells was measured by Bright GloTM luciferase assay. Cell viability was assessed by cell titer Glo® luminescent assay. The mRNA and protein expression was evaluated by RT-qPCR and Western Blot. TUN (5 µg/mL) and THA (1 µM) efficiently inhibited the entry of SARS-CoV-2pp into host cells without any cytotoxic effect. TUN and THA's attenuation of virus entry was associated with differential modulation of ACE2 expression. Both TUN and THA significantly reduced the expression of stress-inducible ER chaperone GRP78/BiP in transduced cells. In contrast, the IRE1-XBP1s and PERK-eIF2α-ATF4-CHOP signaling pathways were downregulated with THA treatment, but not TUN in transduced cells. Insulin-mediated glucose uptake and phosphorylation of Ser307 IRS-1 and downstream p-AKT were enhanced with THA in transduced cells. Furthermore, TUN and THA differentially affected lipid metabolism and apoptotic signaling pathways. These findings suggest that short-term pre-existing ER stress prior to virus infection induces a specific UPR response in host cells capable of counteracting stress-inducible elements signaling, thereby depriving SARS-Co-V2 of essential components for entry and replication. Pharmacological manipulation of ER stress in host cells might provide new therapeutic strategies to alleviate SARS-CoV-2 infection.

Abstract 3335: A novel and potent IRE1α RNase inhibitor, HM100168 as a promising therapeutic strategy in solid cancers

Abstract Protein folding homeostasis in the endoplasmic reticulum (ER) is regulated by a signaling network called the unfolded protein response (UPR). Inositol-requiring enzyme 1α (IRE1α) is an ER membrane-resident kinase/RNase that mediates signal transmission in the most evolutionarily conserved way of the UPR. The IRE1α-XBP1 pathway, one of the most essential branches of the UPR, is activated in various types of cancer. When IRE1α is activated, the spliced form of XBP1 (XBP1s) is generated to activate the transcription of many pro-survival genes to prevent cellular death and relieve cellular stress. IRE1α plays an instrumental pro-tumoral role in various cancers, such as glioblastoma, myeloma, lung adenocarcinoma, and breast cancers, as well as high IRE1α activity is associated with poor prognoses. Moreover, IRE1-XBP1 plays an essential role in the UPR and ER stress responses, which have been implicated in the development of drug resistance. Thereby, blockade of the IRE1α-XBP1 pathway is crucial as it is considered as a potential anti-cancer strategy. Here, we developed a potent and selective IRE1α RNase inhibitor, which have a hydroxy-aryl-aldehydes (HAA) moiety that binds to IRE1α within the RNase catalytic site. Our lead compound, HM100168, potently inhibited the RNase activity of human IRE1α at nanomolar concentrations without affecting its kinase activity. Experiments were performed on MCF7 breast cancer cells to confirm the inhibitory effect in the mRNA and protein levels of XBP1s, which was increased by tunicamycin (TM), a ER stress inducer. HM100168 strongly inhibited ​​XBP1s levels in a dose-dependent manner, displaying the potent IC50 values. We tested cell growth inhibition activity in 3D as well as 2D cell culture condition of various cancer cell lines. Interestingly, HM100168 effectively inhibited cell proliferation of diverse cancer cell lines in both 2D and 3D culture condition. In addition, we explored the promising anti-cancer effects of HM100168 and its synergistic potential when combined with anti-tumorigenic agents. As a result, HM100168 in combination with anti-tumorigenic agents significantly enhanced suppression of tumor growth in human cancer cell transplanted mice. In conclusion, we have identified a novel compound with unique properties that specifically blocked the endonuclease activity of IRE1α. HM100168 inhibited IRE1α endonuclease activity, without affecting its kinase activity after endoplasmic reticulum stress in vitro. The identification of this novel IRE1α inhibitor supports the hypothesis that the IRE1α-XBP1 axis is a promising target for anticancer therapy. Also, co-targeting of IRE1α-XBP1 in combination with anti-tumorigenic agents can be considered a novel approach to overcome cancer resistance. Further preclinical studies will be performed and reported soon after the establishment of a preclinical candidate. Citation Format: Jisook Kim, Seung Hyun Jung, Minjeong Kim, Wongi Park, Jooyun Byun, Soonki Park, Miyoung Lee, Yu-Yon Kim, Junghwa Park, Seokhyun Hong, Minhwa Kim, Young Gil Ahn. A novel and potent IRE1α RNase inhibitor, HM100168 as a promising therapeutic strategy in solid cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3335.

Abstract 17307: Pre-Existing Endoplasmic Reticulum (ER) Stress Inhibits Sars-Co-V2 Entry Into Host Cells Through Modulation of AKT Signaling and Unfolded Protein Response (UPR)

Introduction: It is well known that SARS-Co-V2 infection can induce ER stress-associated activation of unfolded protein response (UPR) in various host cells which may contribute to the pathogenesis of COVID-19. However, the interplay between SARS-Co-V2 infection and UPR signaling in pre-existing ER stress associated pathological conditions has not been well elucidated. Hypothesis: We hypothesized that modulation of a pre-existing ER stress in host cells could attenuate susceptibility to SARS-CoV-2 infection by activating a range of cellular defense. Methods: Increasing concentrations of Tunicamycin (Tm) and Thapsigargin (Tg) have been used to induce ER stress in Huh-7 cells. After 6h treatment, cells were infected with SARS-CoV-2 pseudotyped particles (SARS-CoV-2pp) for 48 p.i. SARS-Co-V2-2pp entry was measured using Bright Glo TM luciferase assay. Cell viability was measured by cell titer Glo ® luminescent cell viability assay. The mRNA and protein expression of UPR markers were evaluated using RT-qPCR and Western blot methods. Results: Tm (5 μg/ml) and Tg (1 μM) efficiently inhibited SARS-CoV-2pp entry into cells without any cytotoxic effect. Chemical ER stress-induced inhibition of SARS-CoV-2pp entry was associated with significant reduction of ACE2 expression in Tg-infected cells but not with Tm. Strikingly, Tm and Tg revealed differential effects in modulating the expression of ER stress genes in infected cells. Both Tm and Tg significantly reduced the expression of stress-inducible ER chaperone GRP78/BIP in infected cells. In contrast, the IRE1-XBP1s and PERK-eIF2α-ATF4-CHOP signaling pathways were downregulated in Tg-infected cells only. Additionally, insulin-mediated glucose uptake, phosphorylation of Ser 307 IRS-1 and downstream p-AKT were enhanced in Tg-infected cells without any change in ERK phosphorylation. Conclusions: These findings suggest that pre-existing ER stress could modulate a specific UPR response in infected cells capable of counteracting the stress-inducible elements signaling, thereby depriving SARS-Co-V2 of essential components for their entry and replication. Pharmacological manipulation of ER stress in host cells might provide new therapeutic strategies to alleviate SARS-CoV-2 infection.

Flavopiridol Suppresses Cell Proliferation and Migration and Induces Apoptotic Cell Death by Inhibiting Oncogenic FOXM1 Signaling in IDH Wild-Type and IDH-Mutant GBM Cells.

Glioblastoma multiforme (GBM) remains one of the most challenging solid cancers to treat due to its highly aggressive and drug-resistant nature. Flavopiridol is synthetic flavone that was recently approved by the FDA for the treatment of acute myeloid leukemia. Flavopiridol exhibits antiproliferative activity in several solid cancer cells and currently evaluated in clinical trials in several solid and hematological cancers. In this study, we investigated the molecular mechanisms underlying antiproliferative effects of flavopiridol in GBM cell lines with wild-type and mutant encoding isocitrate dehydrogenase 1 (IDH1). We found that flavopiridol inhibits proliferation, colony formation, and migration and induces apoptosis in IDH1 wild-type and IDH-mutant cells through inhibition of FOXM1 oncogenic signaling. Furthermore, flavopiridol treatment also inhibits of NF-KB, mediators unfolded protein response (UPR), including, GRP78, PERK and IRE1α, and DNA repair enzyme PARP, which have been shown to be potential therapeutic targets by downregulating FOXM1 in GBM cells. Our findings suggest for the first time that flavopiridol suppresses proliferation, survival, and migration and induces apoptosis in IDH1 wild-type and IDH1-mutant GBM cells by targeting FOXM1 oncogenic signaling which also regulates NF-KB, PARP, and UPR response in GBM cells. Flavopiridol may be a potential novel therapeutic strategy in the treatment of patients IDH1 wild-type and IDH1-mutant GBM.

Co-feeding strategy alleviates hypoxic stress in large-scale bioreactor for optimal production of secretory recombinant proteins in Pichia pastoris.

The loss of mixing efficiency inherent to bioreactor process operated at large-scale yields to the formation of concentration gradient and thus to heterogeneous culture conditions. For processes operated with methanol feeding, P. pastoris faces oscillatory culture conditions that significantly affect the cell ability to produce secretory recombinant proteins at high yield. Extended cell residence time in microenvironments of high methanol concentration and low oxygen availability that are typically found in the upper part of the bioreactor near the feeding point, triggers the unfolded protein response (UPR) and thus impairs proper protein secretion. Methanol co-feeding with sorbitol was shown herein to reduce the UPR response and to restore productivity of secreted protein.

Multiomics analysis of adaptation to repeated DNA damage in prostate cancer cells

ABSTRACT DNA damage is frequently utilized as the basis for cancer therapies; however, resistance to DNA damage remains one of the biggest challenges for successful treatment outcomes. Critically, the molecular drivers behind resistance are poorly understood. To address this question, we created an isogenic model of prostate cancer exhibiting more aggressive characteristics to better understand the molecular signatures associated with resistance and metastasis. 22Rv1 cells were repeatedly exposed to DNA damage daily for 6 weeks, similar to patient treatment regimes. Using Illumina Methylation EPIC arrays and RNA-seq, we compared DNA methylation and transcriptional profiles between the parental 22Rv1 cell line and the lineage exposed to prolonged DNA damage. Here we show that repeated DNA damage drives the molecular evolution of cancer cells to a more aggressive phenotype and identify molecular candidates behind this process. Total DNA methylation was increased while RNA-seq demonstrated these cells had dysregulated expression of genes involved in metabolism and the unfolded protein response (UPR) with Asparagine synthetase (ASNS) identified as central to this process. Despite the limited overlap between RNA-seq and DNA methylation, oxoglutarate dehydrogenase-like (OGDHL) was identified as altered in both data sets. Utilising a second approach we profiled the proteome in 22Rv1 cells following a single dose of radiotherapy. This analysis also highlighted the UPR in response to DNA damage. Together, these analyses identified dysregulation of metabolism and the UPR and identified ASNS and OGDHL as candidates for resistance to DNA damage. This work provides critical insight into molecular changes which underpin treatment resistance and metastasis.

Resistance Training Modulates Reticulum Endoplasmic Stress, Independent of Oxidative and Inflammatory Responses, in Elderly People.

Aging is related to changes in the redox status, low-grade inflammation, and decreased endoplasmic reticulum unfolded protein response (UPR). Exercise has been shown to regulate the inflammatory response, balance redox homeostasis, and ameliorate the UPR. This work aimed to investigate the effects of resistance training on changes in the UPR, oxidative status, and inflammatory responses in peripheral blood mononuclear cells of elderly subjects. Thirty elderly subjects volunteered to participate in an 8-week resistance training program, and 11 youth subjects were included for basal assessments. Klotho, heat shock protein 60 (HSP60), oxidative marker expression (catalase, glutathione, lipid peroxidation, nuclear factor erythroid 2-related factor 2, protein carbonyls, reactive oxygen species, and superoxide dismutase 1 and 2), the IRE1 arm of UPR, and TLR4/TRAF6/pIRAK1 pathway activation were evaluated before and following training. No changes in the HSP60 and Klotho protein content, oxidative status markers, and TLR4/TRAF6/pIRAK1 pathway activation were found with exercise. However, an attenuation of the reduced pIRE1/IRE1 ratio was observed following training. Systems biology analysis showed that a low number of proteins (RPS27A, SYVN1, HSPA5, and XBP1) are associated with IRE1, where XBP1 and RPS27A are essential nodes according to the centrality analysis. Additionally, a gene ontology analysis confirms that endoplasmic reticulum stress is a key mechanism modulated by IRE1. These findings might partially support the modulatory effect of resistance training on the endoplasmic reticulum in the elderly.

Inositol Requiring Enzyme 1, a metabolic sensor at the heart of dendritic cell biology

Three endoplasmic reticulum (ER) resident proteins, IRE1, PERK and ATF6 monitor the health status of the ER and initiate the unfolded protein response (UPR) to restore ER homeostasis during stress. IRE1, a conserved endonuclease, cleaves Xbp1 mRNA and generates the key transcription factor XBP1s. My lab studies the role of IRE1 in dendritic cells (DCs), antigen presenting cells that bridge innate and adaptive immune responses and are essential both for the generation of effective immunity and tolerance. We noticed that in vivo one subset of conventional DCs -cDC1s - displays constitutive IRE1 activity, in absence of a canonical UPR response. Despite strong activation of IRE1 and concomitant induction of XBP1 splicing in cDC1s, we never observed any typical XBP1-dependent gene expression and we undertook a RNA sequencing approach to retrieve a DC-specific gene signature for XBP1. To this end, we compared WT, XBP1KO and IRE1/XBP1DKO cDC1s, all isolated from the spleen. This comparison allowed us to dissect genes downregulated due to loss of XBP1 transcriptional activity versus genes downregulated due to the activation of Regulated IRE1 Dependent Decay (RIDD), a process driven by hyperactivation of IRE1 endonuclease activity in the absence of XBP1. Our analysis revealed that many genes in cDC1s appeared regulated in an IRE1 endonuclease-but not XBP1-dependent manner. Furthermore, the data point towards a novel physiological role for IRE1 in DC homeostasis and provide unique cues for IRE1 activation in DCs, which will be the main topic of this talk.

Cancer-induced Changes In Endoplasmic Stress And Autophagy Responses After Mechanical Overload In ApcMin/+ Mice

Cancer cachexia is characterized by the severe weight and muscle loss. The endoplasmic reticulum (ER) stress and autophagy perpetuate skeletal muscle homeostasis. Tumor cells elicit ER stress and unfolded protein response (UPR) in skeletal muscle. The inhibition of ER stress also activates protein degradation and autophagy, which magnifies muscle mass declination. While UPR moderates mechanical stress, the adaptations in UPR and autophagy in cancer cachexia are unknown. PURPOSE: To determine whether tumor burden alters UPR and autophagy responses to mechanical overload in mice. METHODS: Age-matched male wild-type (WT, n = 5) and ApcMin/+ (Min, n = 5) mice were utilized. Synergist ablation (SA) was performed on the left leg, while the right leg served as an internal control. Seven days after mechanical overload, the plantaris muscles were removed. After tissue homogenization, routine Western blotting was conducted using 80 ~ 120 μg of the total protein. Independent t-test (WT vs. Min) and two-way ANOVA (genotype x treatment) were used for statistical analysis. The coefficient of determination (r^2) was used to examine a significant correlation. RESULTS: Min mice showed a decrease of BW by 9.8% compared to their peak BW. Similarly, sham control plantaris weight in Min mice was smaller than that of WT mice (18.7 ± 0.6 mg vs. 15.1 ± 0.3 mg for WT and Min, respectively). In both mice, mechanical overload increased the plantaris weights, but the response was small in Min mice (43.3 ± 5.8% vs. 21.3 ± 4.6%, p ≤ 0.05). Western blot analysis resulted in Min mice having a reduced phospho-p70S6K after mechanical overload than WT mice (4.2-fold vs. 1.9-fold, p ≤ 0.05). Phosphorylated eukaryotic translation initiation factor 2α (p-eIF2α, 4.2-fold vs. 3.0-fold, p ≤ 0.05) and beclin (4.3-fold vs. 1.8-fold, p ≤ 0.05) levels were reduced in response to the stimulus in Min mice. A significant correlation was observed between p70S6K and eIF2α and between p70S6K and Beclin regardless of mouse genotype. The slopes of the regression curve in Min mice were smaller than WT mice (p ≤ 0.05). CONCLUSIONS: Reduced ER stress and autophagy responses to mechanical overload were associated with decreased mTORC1 signaling in cachectic mice. Supported by Louisiana BORSF (LEQSF(2017-20)-RD-A-22).

Potential role of endoplasmic reticulum stress in the pathophysiology of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is an endocrine disorder that affects million women worldwide, presenting a complex pathophysiology that has not been fully elucidated yet. Recently, it has been suggested that PCOS triggers the endoplasmic reticulum (ER) stress, thus being associated with unfolded protein response (UPR) activation. Indeed, the UPR response has been associated with several pathological conditions, including in the reproductive system. Several studies demonstrated that ovarian UPR markers are upregulated in PCOS, being associated with worst ovarian outcomes, and this was ameliorated by ER stress inhibition. In this review, we aim to summarize the main findings from previous studies covering this topic, in an attempt to clarify the potential role of ER stress and the UPR response in the pathophysiology of PCOS.

Fic-mediated AMPylation tempers the unfolded protein response during physiological stress

The proper balance of synthesis, folding, modification, and degradation of proteins, also known as protein homeostasis, is vital to cellular health and function. The unfolded protein response (UPR) is activated when the mechanisms maintaining protein homeostasis in the endoplasmic reticulum become overwhelmed. However, prolonged or strong UPR responses can result in elevated inflammation and cellular damage. Previously, we discovered that the enzyme filamentation induced by cyclic-AMP (Fic)can modulate the UPR response via posttranslational modification of binding immunoglobulin protein (BiP) by AMPylation during homeostasis and deAMPylation during stress. Loss of fic in Drosophila leads to vision defects and altered UPR activation in the fly eye. To investigate the importance of Fic-mediated AMPylation in a mammalian system, we generated a conditional null allele of Fic in mice and characterized the effect of Fic loss on the exocrine pancreas. Compared to controls, Fic-/- mice exhibit elevated serum markers for pancreatic dysfunction and display enhanced UPR signaling in the exocrine pancreas in response to physiological and pharmacological stress. In addition, both fic-/- flies and Fic-/- mice show reduced capacity to recover from damage by stress that triggers the UPR. These findings show that Fic-mediated AMPylation acts as a molecular rheostat that is required to temper the UPR response in the mammalian pancreas during physiological stress. Based on these findings, we propose that repeated physiological stress in differentiated tissues requires this rheostat for tissue resilience and continued function over the lifetime of an animal.

Cyclosporine A but not tacrolimus induces pro‐apoptotic endoplasmic reticulum stress in renal tubular cells

Calcineurin inhibitors (CNI), cyclosporine A (CsA) or tacrolimus (Tac), belong to the first‐line immunosuppressive strategies in organ‐transplanted patients. Both CNI may exert renal side effects, which are typically stronger in patients receiving CsA. Continued clinical demand for CsA requires improved understanding of its nephrotoxicity. Calcineurin inhibition by CsA occurs via complexes with cyclophilins, whereas Tac recruits FKBP12 instead. We hypothesized that impaired chaperone function of cyclophilins may aggravate CsA cytotoxicity due to induction of endoplasmic reticulum (ER) stress and pro‐apoptotic unfolded protein response (UPR). To address this hypothesis, effects of CsA vs. Tac (10 µM for 6 h) on the UPR signaling were evaluated in cultured human embryonic kidney cells (HEK293), human renal proximal tubular (PT) epithelial cells (HRPTEpC), and isolated rat PTs using quantitative PCR, immunoblotting, and immunofluorescence staining. An established ER stress inducer, thapsigargin (Tg) served as a positive control. CsA and Tg, but not Tac, induced ER stress and pro‐apoptotic UPR in HEK293 cells, which was reflected by increased levels of key UPR products (BiP, CHOP, spliced XBP1, and phosphorylated IRE1a) and cleaved caspase 3 (cCas3). Similar effects were observed in HRPTEpC cells and isolated rat PTs. Knockdown of cyclophilin A or B isoforms in HEK293 cells using siRNA augmented CHOP and cCas3 to an extent comparable with CsA treatment suggesting relevance of cyclophilin activity for intact proteostasis. Application of chemical chaperones, TUDCA or 4‐PBA, alleviated the CsA‐induced UPR suggesting that boosting the protein folding may have protective effects against CsA cytotoxicity. Along the same line, genetic suppression of UPR in HEK293 cells by CRISPR/Cas9‐mediated deletion of the key ER stress sensors, PERK or ATF6, blunted the pro‐apoptotic UPR in response to CsA.In summary, these results suggest that nephrotoxic effects of CsA are partially mediated by suppression of cyclophilins, ER stress, and pro‐apoptotic UPR. Pharmacological modulation of UPR bears the potential to alleviate CsA nephrotoxicity.

Regulation of the unfolded protein response during dehydration stress in African clawed frogs, Xenopus laevis.

The unfolded protein response (UPR) is a wide-ranging cellular response to accumulation of malfolded proteins in the endoplasmic reticulum (ER) and acts as a quality control mechanism to halt protein processing and repair/destroy malfolded proteins under stress conditions of many kinds. Among vertebrate species, amphibians experience the greatest challenges in maintaining water and osmotic balance, the high permeability of their skin making them very susceptible to dehydration and challenging their ability to maintain cellular homeostasis. The present study evaluates the involvement of the UPR in dealing with dehydration-mediated disruption of protein processing in the tissues of African clawed frogs, Xenopus laevis. This primarily aquatic frog must deal with seasonal drought conditions in its native southern Africa environment. Key markers of cellular stress that impact protein processing were identified in six tissues of frogs that had lost 28% of total body water, as compared with fully hydrated controls. This included upregulation of glucose-regulated proteins (GRPs) that are resident chaperones in the ER, particularly 2-ninefold increases in GRP58, GRP75, and/or GRP94 in the lung and skin. Activating transcription factors (ATF3, ATF4, ATF6) that mediate UPR responses also responded to dehydration stress, particularly in skeletal muscle where both ATF3 and ATF4 rose strongly in the nucleus. Other protein markers of the UPR including GADD34, GADD153, EDEM, and XBP-1 also showed selective upregulation in frog tissues in response to dehydration and nuclear levels of the transcription factors XBP-1 and P-CREB rose indicating up-regulation of genes under their control.

Comparison of the Unfolded Protein Response in Cellobiose Utilization of Recombinant Angel- and W303-1A-Derived Yeast Expressing β-Glucosidase.

The unfolded protein response (UPR) is one of the most important protein quality control mechanisms in cells. At least, three factors are predicted to activate the UPR in yeast cells during fermentation. Using UPRE-lacZ as a reporter, we constructed two indicator strains, KZ and WZ, based on Angel-derived K-a and W303-1A strains, respectively, and investigated their UPR response to tunicamycin, ethanol, and acetic acid. Then, four strains carrying plasmids BG-cwp2 and BG were obtained to realize the displaying and secretion of β-glucosidase, respectively. The results of cellobiose utilization assays indicated interactions between the UPR and the metabolic burden between the strain source, anchoring moiety, oxygen supply, and cellobiose concentration. Meanwhile, as expected, growth (OD600), β-glucosidase, and β-galactosidase activities were shown to have a positive inter-relationship, in which the values of the KZ-derived strains were far lower than those of the WZ-derived strains. Additionally, extra metabolic burden by displaying over secreting was also much more serious in strain KZ than in strain WZ. The maximum ethanol titer of the four strains (KZ (BG-cwp2), KZ (BG), WZ (BG-cwp2), and WZ (BG)) in oxygen-limited 10% cellobiose fermentation was 3.173, 5.307, 5.495, and 5.486% (v/v), respectively, and the acetic acid titer ranged from 0.038 to 0.060% (v/v). The corresponding maximum values of the ratio of β-galactosidase activity to that of the control were 3.30, 5.29, 6.45, and 8.72, respectively. Under aerobic conditions with 2% cellobiose, those values were 3.79, 4.97, 6.99, and 7.67, respectively. A comparison of the results implied that β-glucosidase expression durably induced the UPR, and the effect of ethanol and acetic acid depended on the titer produced. Further study is necessary to identify ethanol- or acid-specific target gene expression. Taken together, our results indicated that the host strain W303-1A is a better secretory protein producer, and the first step to modify strain K-a for cellulosic ethanol fermentation would be to relieve the bottleneck of UPR capacity. The results of the present study will help to identify candidate host strains and optimize expression and fermentation by quantifying UPR induction.

Activation of the UPR sensor ATF6α is regulated by its redox-dependent dimerization and ER retention by ERp18

SignificanceMembrane and secretory proteins are synthesized in the endoplasmic reticulum (ER). Perturbations to ER function disrupts protein folding, causing misfolded proteins to accumulate, a condition known as ER stress. Cells adapt to stress by activating the unfolded protein response (UPR), which ultimately restores proteostasis. A key player in the UPR response is ATF6α, which requires release from ER retention and modulation of its redox status during activation. Here, we report that ER stress promotes formation of a specific ATF6α dimer, which is preferentially trafficked to the Golgi for processing. We show that ERp18 regulates ATF6α by mitigating its dimerization and trafficking to the Golgi and identify redox-dependent oligomerization of ATF6α as a key mechanism regulating its function during the UPR.

Arabidopsis TBP-ASSOCIATED FACTOR 12 ortholog NOBIRO6 controls root elongation with unfolded protein response cofactor activity

Plant root growth is indeterminate but continuously responds to environmental changes. We previously reported on the severe root growth defect of a double mutant in bZIP17 and bZIP28 (bz1728) modulating the unfolded protein response (UPR). To elucidate the mechanism by which bz1728 seedlings develop a short root, we obtained a series of bz1728 suppressor mutants, called nobiro, for rescued root growth. We focused here on nobiro6, which is defective in the general transcription factor component TBP-ASSOCIATED FACTOR 12b (TAF12b). The expression of hundreds of genes, including the bZIP60-UPR regulon, was induced in the bz1728 mutant, but these inductions were markedly attenuated in the bz1728nobiro6 mutant. In view of this, we assigned transcriptional cofactor activity via physical interaction with bZIP60 to NOBIRO6/TAF12b. The single nobiro6/taf12b mutant also showed an altered sensitivity to endoplasmic reticulum stress for both UPR and root growth responses, demonstrating that NOBIRO6/TAF12b contributes to environment-responsive root growth control through UPR.

Impact of ER Stress and ER-Mitochondrial Crosstalk in Huntington's Disease.

Accumulation of misfolded proteins is a common phenomenon of several neurodegenerative diseases. The misfolding of proteins due to abnormal polyglutamine (PolyQ) expansions are linked to the development of PolyQ diseases including Huntington’s disease (HD). Though the genetic basis of PolyQ repeats in HD remains prominent, the primary molecular basis mediated by PolyQ toxicity remains elusive. Accumulation of misfolded proteins in the ER or disruption of ER homeostasis causes ER stress and activates an evolutionarily conserved pathway called Unfolded protein response (UPR). Protein homeostasis disruption at organelle level involving UPR or ER stress response pathways are found to be linked to HD. Due to dynamic intricate connections between ER and mitochondria, proteins at ER-mitochondria contact sites (mitochondria associated ER membranes or MAMs) play a significant role in HD development. The current review aims at highlighting the most updated information about different UPR pathways and their involvement in HD disease progression. Moreover, the role of MAMs in HD progression has also been discussed. In the end, the review has focused on the therapeutic interventions responsible for ameliorating diseased states via modulating either ER stress response proteins or modulating the expression of ER-mitochondrial contact proteins.

Endothelial PERK-ATF4-JAG1 axis activated by T-ALL remodels bone marrow vascular niche.

The endoplasmic reticulum unfolded protein response (UPR) is a conserved adaptive signaling in ER homeostasis and has emerged as critical in highly proliferating cells and potential treatment target for acute T-cell lymphoblastic leukemia (T-ALL).Methods: in this study, we assessed the transcriptomic and phenotypic alterations in UPR response of the bone marrow endothelial cells (ECs) in mice engrafted with T-ALL and in bone marrow specimens from patients who have T-ALL. We used PERK inhibitor and generated endothelial specific PERK knockout mice to study the impact of PERK on leukemia progression and hematopoiesis. We performed chromatin immunoprecipitation (ChIP) to study the mechanistic regulation of JAG1 by ATF4. We characterized small extracellular vesicles (SEV) from leukemia-developing mice and studied the effect of SEVs on EC function.Results: we found that T-ALL development induced a robust activation of protein kinase RNA-like endoplasmic reticulum kinase (PERK)-dominant UPR in the bone marrow endothelial vascular niche. The activation of PERK-eIF2a-ATF4 axis remodels the vascular niche, upregulates angiogenic factors including VEGFα and ATF4-regulated JAG1, and suppresses the expression of SCF and CXCL12, which are important to HSC maintenance and regeneration. Further, targeting endothelial PERK significantly improved T-ALL outcome. EC-specific deletion of PERK abolished the aberrant JAG1 up-regulation, improved HSC maintenance, promoted leukemia apoptosis, and improved overall survival. Finally, we showed that small extracellular vesicles are critical mediators of endothelial PERK-eIF2a-ATF4 activation and JAG1 up-regulation in leukemia. Corroborating animal model studies, activation of PERK-ATF4-JAG1 is prominent in human T-ALL bone marrow and T-ALL xenografts.Conclusion: our studies thus revealed for the first time that the leukemia-initiated PERK-ATF4-JAG1 axis plays a critical role in the remodeling of the bone marrow vascular niche and that targeting vascular niche UPR is a potential therapeutic opportunity in T-ALL.