Unfolded Protein Response Gene Expression Research Articles

Protein quality control systems in the endoplasmic reticulum and the cytosol coordinately prevent alachlor-induced proteotoxic stress in Saccharomyces cerevisiae

Alachlor, a widely used chloroacetanilide herbicide for controlling annual grasses in crops, has been reported to rapidly trigger protein denaturation and aggregation in the eukaryotic model organism Saccharomyces cerevisiae. Therefore, this study aimed to uncover cellular mechanisms involved in preventing alachlor-induced proteotoxicity. The findings reveal that the ubiquitin-proteasome system (UPS) played a crucial role in eliminating alachlor-denatured proteins by tagging them with polyubiquitin for subsequent proteasomal degradation. Exposure to alachlor rapidly induced an inhibition of proteasome activity by 90% within 30min. The molecular docking analysis suggests that this inhibition likely results from the binding of alachlor to β subunits within the catalytic core of the proteasome. Notably, our data suggest that nascent proteins in the endoplasmic reticulum (ER) are the primary targets of alachlor. Consequently, the unfolded protein response (UPR), responsible for coping with aberrant proteins in the ER, becomes activated within 1h of alachlor treatment, leading to the splicing of HAC1 mRNA into the active transcription activator Hac1p and the upregulation of UPR gene expression. These findings underscore the critical roles of the protein quality control systems UPS and UPR in mitigating alachlor-induced proteotoxicity by degrading alachlor-denatured proteins and enhancing the protein folding capacity of the ER. Environmental ImplicationsAlachlor is a chloroacetanilide herbicide globally used to control annual grasses in crops. Nevertheless, molecular mechanisms of its toxicity to other non-target eukaryotes remain unclear. Our results in the eukaryotic model Saccharomyces cerevisiae reveal that ER nascent proteins are the major target of alachlor. To cope with alachlor proteotoxicity, both protein quality control systems in the ER and the cytosol coordinately control intracellular protein homeostasis through their roles in clearing denatured proteins and enhancing protein folding capacity. These findings will provide a promising clue for further development of preventive healthcare and direct targeted therapies for herbicide poisoning.

PERK modulation, with GSK2606414, Sephin1 or salubrinal, failed to produce therapeutic benefits in the SOD1G93A mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) has been linked to overactivity of the protein kinase RNA-like ER kinase (PERK) branch of the unfolded protein response (UPR) pathway, both in ALS patients and mouse models. However, attempts to pharmacologically modulate PERK for therapeutic benefit have yielded inconsistent and often conflicting results. This study sought to address these discrepancies by comprehensively evaluating three commonly used, CNS-penetrant, PERK modulators (GSK2606414, salubrinal, and Sephin1) in the same experimental models, with the goal of assessing the viability of targeting the PERK pathway as a therapeutic strategy for ALS. To achieve this goal, a tunicamycin-challenge assay was developed using wild-type mice to monitor changes in liver UPR gene expression in response to PERK pathway modulation. Subsequently, multiple dosing regimens of each PERK modulator were tested in standardized, well-powered, gender-matched, and litter-matched survival efficacy studies using the SOD1G93A mouse model of ALS. The alpha-2-adrenergic receptor agonist clonidine was also tested to elucidate the results obtained from the Sephin1, and of the previously reported guanabenz studies, by comparing the effects of presence or absence of α-2 agonism. The results revealed that targeting PERK may not be an ideal approach for ALS treatment. Inhibiting PERK with GSK2606414 or activating it with salubrinal did not confer therapeutic benefits. While Sephin1 showed some promising therapeutic effects, it appears that these outcomes were mediated through PERK-independent mechanisms. Clonidine also produced some favorable therapeutic effects, which were unexpected and not linked to the UPR. In conclusion, this study highlights the challenges of pharmacologically targeting PERK for therapeutic purposes in the SOD1G93A mouse model and suggests that exploring other targets within, and outside, the UPR may be more promising avenues for ALS treatment.

MUCOSAL SINGLE-CELL PROFILING OF CROHN’S-LIKE DISEASE OF THE POUCH REVEALS COMMON PATHOGENICS AND THERAPEUTIC TARGETS

Abstract BACKGROUND & AIMS After restorative proctocolectomy with ileal pouch-anal anastomosis (IPAA) for ulcerative colitis (UC), a subset of patients develop Crohn’s-like disease of the pouch (CDP), an inflammatory condition that affects both the ileoanal pouch and extra-pouch organs including pre-pouch ileum. The cellular and molecular identities of CDP are unknown, and its diagnosis and treatment remain challenging. To define the pathophysiology of CDP, we examined mucosal cells from patients with and without CDP using single cell analyses. METHODS Endoscopic samples from the pouch and pre-pouch ileum of 50 patients with an IPAA were collected for single-cell RNA sequencing (scRNA-seq) or mass cytometry (or CyTOF). We analyzed immune and non-immune cells from pouch and ileal tissues of patients with normal pouch/ileum and CDP (n = 5 or 6) using scRNA-seq. CyTOF was performed on mucosal immune cells from independent cohorts of patients with normal pouch/ileum, CDP, and pouchitis (n = 9 or 10). Mucosal samples from patients with familial adenomatous polyposis (FAP) after pouch formation were also analyzed by CyTOF. Some scRNA-seq and CyTOF findings were independently validated using immunohistochemistry (n = 6 or 8). RESULTS We revealed distinct immune and non-immune cell clusters in normal pouch and pre-pouch ileum. Colitogenic immune cells expanded in normal UC pouch compared to normal FAP pouch. Compared to normal pouch/pre-pouch ileum, CDP tissues exhibited expanded TCR clonotypes, elevated Th17 signaling, and diminished T cell exhaustion markers. Elevated frequencies of plasma cells, inflammatory fibroblasts and monocytes were noted in CDP pouch and ileum (Fig 1; only pouch data were shown). CDP also harbored elevated Th17-inducing cytokines such as IL23, IL1B, and IL6 produced by myeloid cells. ScRNA-seq and CyTOF identified increased CD14+TREM1+ pathogenic monocytes in both pouch and pre-pouch ileum of CDP. Ligand-receptor interactions further uncovered an essential role of proinflammatory myeloid cells in the pathogenesis of CDP. In addition, pouch and pre-pouch ileum of CDP showed prominent activation of the unfolded protein response (UPR) across all major cell compartments (Fig 2), which was not present in UC, CD or pouchitis based on integrated analysis of published databases. CONCLUSIONS CDP demonstrates altered immune and non-immune cell populations/states in the pouch and pre-pouch ileum. CDP represents a distinct entity of inflammatory bowel disease with extensive UPR activation, a unique feature that may serve as novel diagnostic markers and therapeutic targets using ER stress-alleviating agents including chemical chaperones. Fig 1 CDP reshapes immune and non-immune landscapes in pouch and pre-pouch ileum. (A) UMAP visualization of all cells from the pouch and ileum of CLDP and normal controls. (B) Diversity of epithelial cells, stromal cells, myeloid cells, T cells, innate lymphoid cells (ILCs), B cells and plasma cells in CLDP vs. normal controls in the pouch estimated by scRNA-seq (n = 5 or 6). *p<0.01. Fig 2 Extensive UPR activation in CDP tissues. (A) UPR gene expression in major cell compartments in CDP and normal controls, highlighted genes including CALR, XBP1, and SERP1 were upregulated in all 5 cell compartments. (B) ER stress/UPR pathways are enriched in CDP. (C) IHC of UPR proteins XBP1 and HSPA5/BiP.

Plant extracts modulate cellular stress to inhibit replication of mouse Coronavirus MHV-A59

The Covid-19 infection outbreak led to a global epidemic, and although several vaccines have been developed, the appearance of mutations has allowed the virus to evade the immune response. Added to this is the existing risk of the appearance of new emerging viruses. Therefore, it is necessary to explore novel antiviral therapies. Here, we investigate the potential in vitro of plant extracts to modulate cellular stress and inhibit murine hepatitis virus (MHV)-A59 replication. L929 cells were treated with P2Et (Caesalpinia spinosa) and Anamu SC (Petiveria alliacea) plant extracts during infection and virus production, ROS (reactive oxygen species), UPR (unfolded protein response), and autophagy were assessed. P2Et inhibited virus replication and attenuated both ROS production and UPR activation induced during infection. In contrast, the sustained presence of Anamu SC during viral adsorption and replication was required to inhibit viral infection, tending to induce pro-oxidant effects, and increasing UPR gene expression. Notably, the loss of the PERK protein resulted in a slight decrease in virus yield, suggesting a potential involvement of this UPR pathway during replication. Intriguingly, both extracts either maintained or increased the calreticulin surface exposure induced during infection. In conclusion, our findings highlight the development of antiviral natural plant extracts that differentially modulate cellular stress.

SMALLER TRICHOMES WITH VARIABLE BRANCHES (SVB) and its homolog SVBL act downstream of transcription factor NAC089 and function redundantly in Arabidopsis unfolded protein response.

The unfolded protein response (UPR) is a cellular mechanism that alleviates endoplasmic reticulum stress to maintain protein homeostasis. Although SMALLER TRICHOMES WITH VARIABLE BRANCHES (SVB) is characterized as an emerging UPR factor downstream of the IRE-bZIP60 pathway, whether its homologs participate in the plant UPR remains unknown. Here, we showed that an SVB homolog, SVB-like (SVBL), functions redundantly with SVB in endoplasmic reticulum stress tolerance. The svb-1 svbl-1 double mutant showed a hypersensitivity phenotype and had higher UPR gene expression under endoplasmic reticulum stress than single mutants and the wild type. SVB responded to endoplasmic reticulum stress by accumulating in the root epidermis and phloem cells, but SVBL did not. Ectopic expression of the UPR factor NAC089 up-regulated both SVB and SVBL genes, suggesting that SVB and SVBL work downstream of NAC089. Thus, SVB and SVBL play distinct roles that are modulated by the common upstream regulator NAC089 to cope with endoplasmic reticulum stress in Arabidopsis.

Triggering of Endoplasmic Reticulum Stress by Tannic Acid Inhibits the Proliferation and Migration of Colorectal Cancer Cells.

Due to the pivotal role of endoplasmic reticulum (ER) stress in cancers, interfering with its function can cause the accumulation of unfolded proteins, which ultimately leads to the activation of the unfolded protein response (UPR) signaling pathway and apoptosis. Therefore, the use of plant compounds such as tannic acid with UPR-inducing properties can be proposed as a possible treatment method for cancer. In this study, we investigated the effect of tannic acid on cell migration, colony formation, growth, and UPR-induced apoptosis in the SW48 colorectal cancer cell line. The MTT assay was performed to investigate the cytotoxic effect of tannic acid. We performed the qPCR method to elucidate the effect of tannic acid on the expression of Bim, MMP-9, Bcl-xL, cyclin D1, CHOP, and ATF4 genes. We also used the colony formation and migration experiments to investigate the effect of this compound on the colony formation and migration ability of tumor cells. Finally, we used Hoechst staining to measure cell apoptosis. Tannic acid inhibited the cell survival, clonogenic, and migration of colon cancer cells. This compound increased the expression of ER stress-mediated UPR genes, ATF4 and CHOP. Moreover; tannic acid increased the expression of pro-apoptotic proteins like Bim, while at the same time causing a sharp decline in the expression of anti-apoptotic protein Bcl-xL. A decline in MMP-9 expression confirmed the anti-metastatic role of this compound. Taken together, tannic acid can induce apoptosis via ER stress-mediated UPR pathway, and has a suppressive effect on cell viability, growth, migration, colony formation, and metastasis, suggesting it may be a potential drug in colorectal cancer treatment.

Bridging Energy Need and Feeding Behavior: The Impact of eIF2α Phosphorylation in AgRP Neurons.

This study examines the impact of eukaryotic translation initiation factor 2α (eIF2α) phosphorylation, triggered by an energy deficit, on hypothalamic AgRP neurons and its subsequent influence on whole-body energy homeostasis. Impaired eIF2α phosphorylation diminishes the unfolded protein response and autophagy, both of which are crucial for energy deficit-induced activation of AgRP neurons. This study highlights the significance of eIF2α phosphorylation as a cellular marker indicating the availability of energy in AgRP neurons and as a molecular switch that regulates homeostatic feeding behavior.

The plasma membrane-associated transcription factor NAC091 regulates unfolded protein response in Arabidopsis thaliana

Adverse environmental stresses may cause the accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER), and the unfolded protein response (UPR) pathway is initiated to mitigate the ER stress. Previous studies demonstrate that NAC062, a plasma membrane-associated transcription factor, plays important roles in promoting cell survival under ER stress conditions in Arabidopsis thaliana. In this study, we identified another plasma membrane-associated transcription factor, NAC091 (also known as ANAC091/TIP), as an important UPR mediator. ER stress induces the expression of NAC091, which is mainly dependent on the ER stress regulators bZIP60 and bZIP28. In addition, NAC091 has transcriptional activation activity, and the truncated form of NAC091 devoid of the C-terminal transmembrane domain (TMD) forms a homodimer in the nucleus. Under ER stress conditions, NAC091 relocates from the plasma membrane to the nucleus and regulates the expression of canonical UPR genes involved in cell survival. Further, the loss-of-function mutant of NAC091 confers impaired ER stress tolerance. Together, these results reveal the important role of NAC091 in ER stress response in Arabidopsis, and demonstrate that NAC091 relays the ER stress signal from the plasma membrane to the nucleus to alleviate ER stress and promote cell survival in plants.

Regulation of plasmacytoid dendritic cells byER stress-mediated metabolic responses

Abstract Plasmacytoid dendritic cells (pDCs) chronically produce type I interferon (IFN-I) in autoimmune diseases, including systemic sclerosis (SSc) and systemic lupus erythematosus (SLE). We report that the IRE1α-XBP1 branch of the unfolded protein response (UPR) inhibits IFN-α production by TLR7- or TLR9-activated pDCs. In SSc patients, UPR gene expression was reduced in pDCs, which inversely correlated with IFN-I–stimulated gene expression. CXCL4, a chemokine highly secreted in SSc patients, downregulated IRE1α-XBP1–controlled genes and promoted IFN-α production by pDCs. Mechanistically, IRE1α-XBP1 activation rewired glycolysis to serine biosynthesis by inducing phosphoglycerate dehydrogenase (PHGDH) expression. This process reduced pyruvate access to the tricarboxylic acid (TCA) cycle and blunted mitochondrial ATP generation, which are essential for pDC IFN-I responses. Notably, PHGDH expression was reduced in pDCs from patients with SSc and SLE, and pharmacological blockade of TCA cycle reactions inhibited IFN-I responses in pDCs from these patients. Hence, modulating the IRE1α-XBP1–PHGDH axis may represent a hitherto unexplored strategy for alleviating chronic pDC activation in autoimmune disorders.

Effect of Common ER Stress-Inducing Drugs on the Growth and Lipid Phenotypes of Chlamydomonas and Arabidopsis.

Endoplasmic reticulum (ER) stress is caused by the stress-induced accumulation of unfolded proteins in the ER. Several compounds are used to induce the unfolded protein response (UPR) in animals, with different modes of action, but which ER stress-inducing drugs induce ER stress in microalgae or land plants is unclear. In this study, we examined the effects of seven chemicals that were reported to induce ER stress in animals on the growth, UPR gene expression and fatty acid profiles of Chlamydomonas reinhardtii (Chlamydomonas) and Arabidopsis thaliana (Arabidopsis): 2-deoxyglucose, dithiothreitol (DTT), tunicamycin (TM), thapsigargin, brefeldin A (BFA), monensin (MON) and eeyarestatin I. In both model photosynthetic organisms, DTT, TM, BFA and MON treatment induced ER stress, as indicated by the induction of spliced bZIP1 and bZIP60, respectively. In Chlamydomonas, DTT, TM and BFA treatment induced the production of transcripts related to lipid biosynthesis, but MON treatment did not. In Arabidopsis, DTT, TM, BFA and MON inhibited seed germination and seedling growth with the activation of bZIP60. These findings lay the foundation for using four types of ER stress-inducing drugs in photosynthetic organisms, and they help uncover the mode of action of each compound.

Atg8 and Ire1 in combination regulate the autophagy-related endoplasmic reticulum stress response in Candida albicans

The unfolded protein response (UPR) is an important pathway to prevent endoplasmic reticulum (ER) stress in eukaryotic cells. In Saccharomyces cerevisiae, Ire1 is a key regulatory factor required for HAC1 gene splicing for further production of functional Hac1 and activation of UPR gene expression. Autophagy is another mechanism involved in the attenuation of ER stress by ER-phagy, and Atg8 is a core protein in autophagy. Both autophagy and UPR are critical for ER stress response, but whether they act individually or in combination in Candida albicans is unknown. In this study, we explored the interaction between Ire1 and the autophagy protein Atg8 for the ER stress response by constructing the atg8Δ/Δire1Δ/Δ double mutant in the pathogenic fungus C. albicans. Compared to the single mutants atg8Δ/Δ or ire1Δ/Δ, atg8Δ/Δire1Δ/Δ exhibited much higher sensitivity to various ER stress-inducing agents and more severe attenuation of UPR gene expression under ER stress. Further investigations showed that the double mutant had a defect in ER-phagy, which was associated with attenuated vacuolar fusion under ER stress. This study revealed that Ire1 and Atg8 in combination function in the activation of the UPR and ER-phagy to maintain ER homeostasis under ER stress in C. albicans.

Chronic activation of pDCs in autoimmunity is linked to dysregulated ER stress and metabolic responses.

Plasmacytoid dendritic cells (pDCs) chronically produce type I interferon (IFN-I) in autoimmune diseases, including systemic sclerosis (SSc) and systemic lupus erythematosus (SLE). We report that the IRE1α-XBP1 branch of the unfolded protein response (UPR) inhibits IFN-α production by TLR7- or TLR9-activated pDCs. In SSc patients, UPR gene expression was reduced in pDCs, which inversely correlated with IFN-I-stimulated gene expression. CXCL4, a chemokine highly secreted in SSc patients, downregulated IRE1α-XBP1-controlled genes and promoted IFN-α production by pDCs. Mechanistically, IRE1α-XBP1 activation rewired glycolysis to serine biosynthesis by inducing phosphoglycerate dehydrogenase (PHGDH) expression. This process reduced pyruvate access to the tricarboxylic acid (TCA) cycle and blunted mitochondrial ATP generation, which are essential for pDC IFN-I responses. Notably, PHGDH expression was reduced in pDCs from patients with SSc and SLE, and pharmacological blockade of TCA cycle reactions inhibited IFN-I responses in pDCs from these patients. Hence, modulating the IRE1α-XBP1-PHGDH axis may represent a hitherto unexplored strategy for alleviating chronic pDC activation in autoimmune disorders.

Palmitate and thapsigargin have contrasting effects on ER membrane lipid composition and ER proteostasis in neuronal cells

The endoplasmic reticulum (ER) is an organelle that performs several key functions such as protein synthesis and folding, lipid metabolism and calcium homeostasis. When these functions are disrupted, such as upon protein misfolding, ER stress occurs. ER stress can trigger adaptive responses to restore proper functioning such as activation of the unfolded protein response (UPR). In certain cells, the free fatty acid palmitate has been shown to induce the UPR. Here, we examined the effects of palmitate on UPR gene expression in a human neuronal cell line and compared it with thapsigargin, a known depletor of ER calcium and trigger of the UPR. We used a Gaussia luciferase-based reporter to assess how palmitate treatment affects ER proteostasis and calcium homeostasis in the cells. We also investigated how ER calcium depletion by thapsigargin affects lipid membrane composition by performing mass spectrometry on subcellular fractions and compared this to palmitate. Surprisingly, palmitate treatment did not activate UPR despite prominent changes to membrane phospholipids. Conversely, thapsigargin induced a strong UPR, but did not significantly change the membrane lipid composition in subcellular fractions. In summary, our data demonstrate that changes in membrane lipid composition and disturbances in ER calcium homeostasis have a minimal influence on each other in neuronal cells. These data provide new insight into the adaptive interplay of lipid homeostasis and proteostasis in the cell.

Expression of unfolded protein response genes in post-transplantation liver biopsies

BackgroundCholestatic liver diseases are a major source of morbidity and mortality that can progress to end-stage liver disease and hyperbilirubinemia is a hallmark of cholestasis. There are few effective medical therapies for primary biliary cholangitis, primary sclerosing cholangitis and other cholestatic liver diseases, in part, due to our incomplete understanding of the pathogenesis of cholestatic liver injury. The hepatic unfolded protein response (UPR) is an adaptive cellular response to endoplasmic reticulum stress that is important in the pathogenesis of many liver diseases and recent animal studies have demonstrated the importance of the UPR in the pathogenesis of cholestatic liver injury. However, the role of the UPR in human cholestatic liver diseases is largely unknown.MethodsRNA was extracted from liver biopsies from patients after liver transplantation. RNA-seq was performed to determine the transcriptional profile and hepatic UPR gene expression that is associated with liver injury and cholestasis.ResultsTranscriptome analysis revealed that patients with hyperbilirubinemia had enhanced expression of hepatic UPR pathways. Alternatively, liver biopsy samples from patients with acute rejection had enhanced gene expression of LAG3 and CDK1. Pearson correlation analysis of serum alanine aminotransferase, aspartate aminotransferase and total bilirubin levels demonstrated significant correlations with the hepatic expression of several UPR genes, as well as genes involved in hepatic bile acid metabolism and inflammation. In contrast, serum alkaline phosphatase levels were correlated with the level of hepatic bile acid metabolism gene expression but not liver UPR gene expression.ConclusionsOverall, these data indicate that hepatic UPR pathways are increased in cholestatic human liver biopsy samples and supports an important role of the UPR in the mechanism of human cholestatic liver injury.

Abstract P3063: Downregulation Of The T Cell Unfolded Protein Response Precedes T Cell Cardiac Infiltration In Experimental Heart Failure With Preserved Ejection Fraction

Introduction: Diastolic dysfunction in Heart Failure with Preserved Ejection Fraction (HFpEF) is associated with T cell systemic inflammation and downregulation of myocardial unfolded protein response (UPR) genes. The T cell UPR can modulate T cell effector function and immune responses, yet this is largely unexplored in HFpEF. Hypothesis: We hypothesized T cells contribute to cardiometabolic HFpEF and that metabolic and nitrosative stress-induced alterations of the T cell UPR modulate T cell activation and diastolic function. Methods: Male C57/BL6 (wild-type, WT) or T cell receptor alpha-deficient ( Tcra-/-) were fed a high-fat diet (HFD) and L-NAME for 1, 3, and 5 weeks, or standard chow (STD). We assessed diastolic function by invasive hemodynamic analyses. Immune cells were characterized in the heart and spleen by flow cytometry, and cardiac pathology was assessed by histology. The UPR gene expression was evaluated in splenic CD4 + T cells over time by qPCR. Results: Unlike WT mice, Tcra-/- mice fed HFD/L-NAME for 5 weeks did not develop diastolic dysfunction or cardiomyocyte hypertrophy, demonstrating a critical role for T cells in experimental cardiometabolic HFpEF. Indeed, cardiac CD4 + T cells were increased in WT mice fed HFD/L-NAME for 5 weeks compared to STD, concordant with an expansion of splenic CD44 hi CD62l lo interferon-gamma (IFNγ) + effector cells. Strikingly, splenic T cells also experienced downregulation of the UPR proteins activating transcription factor 4 ( Atf4 ), Atf6 , and X box-binding protein 1 ( Xbp1s/u ). We found that the Atf4 arm of the T cell UPR is the earliest responder to HFD/L-NAME, with observed downregulation of Atf4 and its downstream effector C/EBP homologous protein ( Chop ) as early as 3 weeks, prior to onset of diastolic dysfunction, followed by the subsequent downregulation of Atf6 and Xbp1s/u . Conclusions: T cells contribute to diastolic dysfunction and cardiomyocyte hypertrophy in experimental cardiometabolic HFpEF. Early modulation of T cell Atf4 precedes diastolic dysfunction, enhanced IFNγ + effector T cell activation, and global T cell UPR downregulation. Ongoing studies are focused on understanding the functional consequences of impaired T cell UPR in cardiometabolic HFpEF.

Clinical and biological relevance of CREB3L1 in Philadelphia chromosome-negative myeloproliferative neoplasms

Cyclic AMP-response element-binding protein 3-like 1 (CREB3L1) is a gene involved in the unfolded protein response (UPR). Recently, we demonstrated that CREB3L1 is specifically overexpressed in the platelets of patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). In this study, we aimed to show the clinical and biological relevance of CREB3L1 in these hematological diseases. Overexpression of CREB3L1 was specific to platelets in MPNs and associated with a higher risk of thrombosis and fibrotic transformation in essential thrombocythemia (ET) and polycythemia vera (PV) cases, respectively. Furthermore, we found that UPR genes were downregulated in platelets of patients with ET and PV, which were more pronounced in patients harboring the JAK2 V617F mutation. However, CREB3L1 overexpression does not alter UPR gene expression or cell proliferation in UT-7/TPO/CALRm cells exogenously expressing mutated calreticulin and HEL cells harboring endogenous JAK2 V617F. Furthermore, CREB3L1 overexpression did not modulate sensitivity to endoplasmic reticulum stress in these cell lines. Taken together, our data show 1) a potential role of CREB3L1 expression in platelets as a new marker of high-risk MPNs and 2) an association between CREB3L1 overexpression and UPR gene downregulation in these patients’ platelets, with CREB3L1 not altering UPR in our in vitro models and possibly further in vivo mechanisms being involved.

Proinflammatory T Cells with Downregulated Unfolded Protein Response Genes Contribute to Experimental Heart Failure with Preserved Ejection Fraction (HFpEF)

IntroductionHeart Failure with Preserved Ejection Fraction (HFpEF) is an uncurable widespread syndrome characterized by impaired cardiac relaxation (diastolic function), preserved cardiac contractility (systolic function), and the concordance of several comorbidities, such as obesity and hypertension. While T cell inflammation is known to contribute to HF with reduced cardiac contractility (HF with reduced EF), whether T cell inflammation occurs in HFpEF remains elusive. Recently, downregulation of the unfolded protein response (UPR) in cardiomyocytes was identified as a cardiometabolic HFpEF‐unique molecular signature. Interestingly, downregulation of the UPR has been reported to improve T cell anti‐tumor inflammatory responses. We hypothesized T cells contribute to diastolic dysfunction in HFpEF following T cell UPR downregulation in response to metabolic and nitrosative stress.MethodsWe modeled cardiometabolic HFpEF in male C57/Bl6 (WT), Nur77‐GFP mice, reporter mice for T cell antigen engagement, and T cell receptor‐a (Tcra−/−) mice using high‐fat diet (HFD) and L‐NAME‐supplemented drinking water to mimic obesity and hypertension, respectively. Mice fed standard chow (STD) were used as controls. Invasive hemodynamic analyses were used to measure cardiac function. Immune populations in the heart, mediastinal lymph nodes (MdLN), and spleen were characterized using flow cytometry. CD4+ T cells from MdLN and spleen were isolated using magnetic‐assisted cell sorting, and UPR gene expression was assessed using qPCR.ResultsWe observed significant increases in cardiac CD4+ T cells in WT mice fed HFD/L‐NAME, concordant with diastolic dysfunction and preserved EF, compared to STD mice. We found increased effector CD4+CD62LloCD44hiIFNγ+ T cells in spleens and MdLN isolated from WT HFD/L‐NAME mice compared to STD controls. Nur77‐GFP mice revealed no antigen recognition by CD4+ T cells in the heart following HFD/L‐NAME. Tcra−/−mice fed HFD/L‐NAME did not develop diastolic dysfunction or cardiomyocyte hypertrophy. Strikingly, qPCR analysis of splenic T cells of WT HFD/L‐NAME mice revealed significantly decreased expression of spliced X box‐binding protein 1 (XBP1s), compared to controls, with no significant changes in unspliced XBP1 or activating transcription factor 4 (ATF4). ATF6 and C/EBP homologous protein (CHOP) gene expression was also reduced compared to T cells from control mice.ConclusionOur data demonstrate that diastolic dysfunction and cardiomyocyte hypertrophy are T cell dependent in preclinical cardiometabolic HFpEF and reveal downregulation of two branches of the T cell UPR as a potential novel mechanism that contributes to T cell inflammation in HFpEF.

Transcriptional competition shapes proteotoxic ER stress resolution.

Through dynamic activities of conserved master transcription factors (mTFs), the unfolded protein response (UPR) relieves proteostasis imbalance of the endoplasmic reticulum (ER), a condition known as ER stress1,2. Because dysregulated UPR is lethal, the competence for fate changes of the UPR mTFs must be tightly controlled3,4. However, the molecular mechanisms underlying regulatory dynamics of mTFs remain largely elusive. Here, we identified the abscisic acid-related regulator G-class bZIP TF2 (GBF2) and the cis-regulatory element G-box as regulatory components of the plant UPR led by the mTFs, bZIP28 and bZIP60. We demonstrate that, by competing with the mTFs at G-box, GBF2 represses UPR gene expression. Conversely, a gbf2 null mutation enhances UPR gene expression and suppresses the lethality of a bzip28 bzip60 mutant in unresolved ER stress. By demonstrating that GBF2 functions as a transcriptional repressor of the UPR, we address the long-standing challenge of identifying shared signalling components for a better understanding of the dynamic nature and complexity of stress biology. Furthermore, our results identify a new layer of UPR gene regulation hinged upon an antagonistic mTFs-GFB2 competition for proteostasis and cell fate determination.

Caffeine and MDMA (Ecstasy) Exacerbate ER Stress Triggered by Hyperthermia.

Drugs of abuse can cause local and systemic hyperthermia, a known trigger of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). Another trigger of ER stress and UPR is ER calcium depletion, which causes ER exodosis, the secretion of ER-resident proteins. In rodent models, club drugs such as 3,4-methylenedioxymethamphetamine (MDMA, ‘ecstasy’) can create hyperthermic conditions in the brain and cause toxicity that is affected by the environmental temperature and the presence of other drugs, such as caffeine. In human studies, MDMA stimulated an acute, dose-dependent increase in core body temperature, but an examination of caffeine and MDMA in combination remains a topic for clinical research. Here we examine the secretion of ER-resident proteins and activation of the UPR under combined exposure to MDMA and caffeine in a cellular model of hyperthermia. We show that hyperthermia triggers the secretion of normally ER-resident proteins, and that this aberrant protein secretion is potentiated by the presence of MDMA, caffeine, or a combination of the two drugs. Hyperthermia activates the UPR but the addition of MDMA or caffeine does not alter the canonical UPR gene expression despite the drug effects on ER exodosis of UPR-related proteins. One exception was increased BiP/GRP78 mRNA levels in MDMA-treated cells exposed to hyperthermia. These findings suggest that club drug use under hyperthermic conditions exacerbates disruption of ER proteostasis, contributing to cellular toxicity.

CRISPR/Cas9 and Transgene Verification of Gene Involvement in Unfolded Protein Response and Recombinant Protein Production in Barley Grain.

The use of plants as heterologous hosts to produce recombinant proteins has some intriguing advantages. There is, however, the potential of overloading the endoplasmic reticulum (ER) capacity when producing recombinant proteins in the seeds. This leads to an ER-stress condition and accumulating of unfolded proteins. The unfolded protein response (UPR) is activated to alleviate the ER-stress. With the aim to increase the yield of human epidermal growth factor (EGF) and mouse leukemia inhibitory factor (mLIF) in barley, we selected genes reported to have increased expression during ER-induced stress. The selected genes were calreticulin (CRT), protein disulfide isomerase (PDI), isopentenyl diphosphate isomerase (IPI), glutathione-s-transferase (GST), HSP70, HSP26, and HSP16.9. These were knocked out using CRISPR/Cas9 or overexpressed by conventional transgenesis. The generated homozygous barley lines were crossed with barley plants expressing EGF or mLIF and the offspring plants analyzed for EGF and mLIF protein accumulation in the mature grain. All manipulated genes had an impact on the expression of UPR genes when plantlets were subjected to tunicamycin (TN). The PDI knockout plant showed decreased protein body formation, with protein evenly distributed in the cells of the endosperm. The two genes, GST and IPI, were found to have a positive effect on recombinant protein production. mLIF expression was increased in a F2 homozygous GST knockout mutant background as compared to a F2 GST wild-type offspring. The overexpression of IPI in a F1 cross showed a significant increase in EGF expression. We demonstrate that manipulation of UPR related genes can have a positive effect on recombinant protein accumulation.