Endoplasmic Reticulum Homeostasis Research Articles

The endoplasmic reticulum kinase PERK interacts with the oxidoreductase ERO1 to metabolically adapt mitochondria

Endoplasmic reticulum (ER) homeostasis requires molecular regulators that tailor mitochondrial bioenergetics to the needs of protein folding. For instance, calnexin maintains mitochondria metabolism and mitochondria-ER contacts (MERCs) through reactive oxygen species (ROS) from NADPH oxidase 4 (NOX4). However, induction of ER stress requires a quick molecular rewiring of mitochondria to adapt to new energy needs. This machinery is not characterized. We now show that the oxidoreductase ERO1⍺ covalently interacts with protein kinase RNA-like ER kinase (PERK) upon treatment with tunicamycin. The PERK-ERO1⍺ interaction requires the C-terminal active site of ERO1⍺ and cysteine 216 of PERK. Moreover, we show that the PERK-ERO1⍺ complex promotes oxidization of MERC proteins and controls mitochondrial dynamics. Using proteinaceous probes, we determined that these functions improve ER-mitochondria Ca2+ flux to maintain bioenergetics in both organelles, while limiting oxidative stress. Therefore, the PERK-ERO1⍺ complex is a key molecular machinery that allows quick metabolic adaptation to ER stress.

New Frontiers on ER Stress Modulation: Are TRP Channels the Leading Actors?

The endoplasmic reticulum (ER) is a dynamic structure, playing multiple roles including calcium storage, protein synthesis and lipid metabolism. During cellular stress, variations in ER homeostasis and its functioning occur. This condition is referred as ER stress and generates a cascade of signaling events termed unfolded protein response (UPR), activated as adaptative response to mitigate the ER stress condition. In this regard, calcium levels play a pivotal role in ER homeostasis and therefore in cell fate regulation since calcium signaling is implicated in a plethora of physiological processes, but also in disease conditions such as neurodegeneration, cancer and metabolic disorders. A large body of emerging evidence highlighted the functional role of TRP channels and their ability to promote cell survival or death depending on endoplasmic reticulum stress resolution, making them an attractive target. Thus, in this review we focused on the TRP channels' correlation to UPR-mediated ER stress in disease pathogenesis, providing an overview of their implication in the activation of this cellular response.

Pseudorabies virus infection induces endoplasmic reticulum stress and unfolded protein response in suspension-cultured BHK-21 cells.

Viral infections cause endoplasmic reticulum (ER) stress and subsequently unfolded protein response (UPR) which restores ER homeostasis. In this study, levels of proteins or transcription of three UPR pathways were examined in suspension-cultured BHK-21 cells to investigate Pseudorabies virus (PRV) infection-induced ER stress, in which glucose-related proteins 78 kD and 94 kD (GRP78 and GRP94) were upregulated. The downstream double-stranded RNA-activated protein kinase-like ER kinase (PERK) pathway was activated with upregulation of ATF4, CHOP, and GADD34, and the inositol requiring kinase 1 (IRE1) pathway was triggered by the splicing of X box-binding protein 1 (XBP1) mRNA and the enhanced expression of p58IPK and EDEM. Furthermore, our results showed that the ER stress, induced by 0.005 µM thapsigargin, promoted PRV replication in suspension-cultured BHK-21 cells, and that PRV glycoprotein B (gB) overexpression triggered the PERK and IRE1 pathways.

Lipid desaturation regulates the balance between self-renewal and differentiation in mouse blastocyst-derived stem cells

Stem cells are defined by their ability to self-renew and differentiate, both shown in multiple studies to be regulated by metabolic processes. To decipher metabolic signatures of self-renewal in blastocyst-derived stem cells, we compared early differentiating embryonic stem cells (ESCs) and their extra-embryonic counterparts, trophoblast (T)SCs to their self-renewing counterparts. A metabolomics analysis pointed to the desaturation of fatty acyl chains as a metabolic signature of differentiating blastocyst-derived SCs via the upregulation of delta-6 desaturase (D6D; FADS2) and delta-5 desaturase (D5D; FADS1), key enzymes in the biosynthesis of polyunsaturated fatty acids (PUFAs). The inhibition of D6D or D5D by specific inhibitors or SiRNA retained stemness in ESCs and TSCs, and attenuated endoplasmic reticulum (ER) stress-related apoptosis. D6D inhibition in ESCs upregulated stearoyl-CoA desaturase-1 (Scd1), essential to maintain ER homeostasis. In TSCs, however, D6D inhibition downregulated Scd1. TSCs show higher Scd1 mRNA expression and high levels of monounsaturated fatty acyl chain products in comparison to ESCs. The addition of oleic acid, the product of Scd1 (essential for ESCs), to culture medium, was detrimental to TSCs. Interestingly, TSCs express a high molecular mass variant of Scd1 protein, hardly expressed by ESCs. Taken together, our data suggest that lipid desaturation is a metabolic regulator of the balance between differentiation and self-renewal of ESCs and TSCs. They point to lipid polydesaturation as a driver of differentiation in both cell types. Monounsaturated fatty acids (MUFAs), essential for ESCs are detrimental to TSCs.

Long noncoding RNA Gm2694 drives depressive-like behaviors in male mice by interacting with GRP78 to disrupt endoplasmic reticulum homeostasis.

Long noncoding RNAs (lncRNAs) are involved in various biological processes and implicated in the regulation of neuronal activity, but the potential role of lncRNAs in depression remains largely unknown. Here, we identified that lncRNA Gm2694 was increased in the medial prefrontal cortex (mPFC) of male mice subjected to chronic social defeat stress (CSDS). The down-regulation of Gm2694 in the mPFC alleviated CSDS-induced depressive-like behaviors through enhanced excitatory synaptic transmission. Furthermore, we found that Gm2694 preferentially interacted with the carboxyl-terminal domain of 78-kilodalton glucose-regulated protein (GRP78), which abrogated GRP78 function and disrupted endoplasmic reticulum homeostasis, resulting in a reduction of the surface expression of AMPA receptors (AMPARs). Overexpression of GRP78 in the mPFC promoted the surface expression of AMPARs and attenuated the CSDS-induced depressive-like behaviors of mice. Together, our results unraveled a previously unknown role of Gm2694 in regulating endoplasmic reticulum homeostasis and excitatory synaptic transmission in depression.

Current understanding of imeglimin action on pancreatic β-cells: Involvement of mitochondria and endoplasmic reticulum homeostasis.

Recent preclinical studies have provided insight on imeglimin's action on pancreatic β-cells and the mechanisms underlying its clinical benefits. Imeglimin may enhance glucose-induced insulin secretion (GIIS) and inhibit apoptosis of pancreatic ß-cells leading to preserved β-cell mass by maintaining or restoring the functional and structural integrity of the mitochondria and the endoplasmic reticulum homeostasis in pancreatic β-cells.

ER-associated RNA silencing promotes ER quality control

The endoplasmic reticulum (ER) coordinates mRNA translation and processing of secreted and endomembrane proteins. ER-associated degradation (ERAD) prevents the accumulation of misfolded proteins in the ER, but the physiological regulation of this process remains poorly characterized. Here, in a genetic screen using an ERAD model substrate in Caenorhabditis elegans, we identified an anti-viral RNA interference pathway, referred to as ER-associated RNA silencing (ERAS), which acts together with ERAD to preserve ER homeostasis and function. Induced by ER stress, ERAS is mediated by the Argonaute protein RDE-1/AGO2, is conserved in mammals and promotes ER-associated RNA turnover. ERAS and ERAD are complementary, as simultaneous inactivation of both quality-control pathways leads to increased ER stress, reduced protein quality control and impaired intestinal integrity. Collectively, our findings indicate that ER homeostasis and organismal health are protected by synergistic functions of ERAS and ERAD.

R(h)oad to antitumour therapy.

R(h)oad to antitumour therapy.

AA147 ameliorates post-cardiac arrest cerebral ischemia/reperfusion injury through the co-regulation of the ATF6 and Nrf2 signaling pathways.

Ischemia/reperfusion caused by cardiac arrest (CA) disturbs endoplasmic reticulum (ER) homeostasis and redox balance in neurons. AA147, originally developed as a pharmacologic activator of the activating transcription factor 6 (ATF6), can protect multiple tissues from ischemia/reperfusion injury (IRI) by decreasing reactive oxygen species (ROS) and restoring ER function. However, it is unclear whether pharmacologic treatment of AA147 could ameliorate post-CA cerebral IRI and whether it is associated with proteostasis regulation and anti-oxidative stress mechanism. In the present study, mice were subjected to 9 min-CA surgery followed by cardiopulmonary resuscitation (CPR). AA147 or vehicle was administered 1 day before the operation and 15min after the return of spontaneous circulation. We found that AA147 restored neurological function and reduced dead neurons in mice suffering from CA. Moreover, AA147 inhibited CA/CPR-caused neuronal apoptosis and ER stress, indicated by reduced TUNEL-positive neurons, surged expression of Bcl-2/Bax, and down expression of cleaved caspase-3, caspase-12, C/EBP homologous protein (CHOP). The expression of ATF6 and its regulated gene glucose-regulated protein 78 (GRP78) increased significantly after the administration of AA147, suggesting the activation of the ATF6 pathway. In addition, AA147 also alleviated the upsurge of the ROS generation and MDA levels as well as increased SOD activity, accompanied by enhancement of the nuclear factor E2-related factor 2 (Nrf2) and its modulated heme-oxygenase-1 (HO-1) expressions. Cotreatment of AA147 with inhibitors of the ATF6 or Nrf2 significantly suppressed AA147-dependent reductions in ROS scavenging and neuronal death after CA/CPR. The results suggested that AA147 could confer neuroprotection against post-CA cerebral IRI through inhibition of oxidative stress along with ER stress-associated apoptosis, which is attributed to the coregulation of both ATF6 and Nrf2 signaling pathways activity. Our findings support the potential for AA147 as a therapeutic approach to improve post-CA brain injury.

ER Stress and the Unfolded Protein Response: Homeostatic Regulation Coordinate Plant Survival and Growth

The endoplasmic reticulum (ER), a eukaryotic organelle, is the major site of protein biosynthesis. The disturbance of ER function by biotic or abiotic stress triggers the accumulation of misfolded or unfolded proteins in the ER. The unfolded protein response (UPR) is the best-studied ER stress response. This transcriptional regulatory system senses ER stress, activates downstream genes that function to mitigate stress, and restores homeostasis. In addition to its conventional role in stress responses, recent reports indicate that the UPR is involved in plant growth and development. In this review, we summarize the current knowledge of ER stress sensing and the activation and downstream regulation of the UPR. We also describe how the UPR modulates both plant growth and stress tolerance by maintaining ER homeostasis. Lastly, we propose that the UPR is a major component of the machinery that balances the trade-off between plant growth and survival in a dynamic environment.

SHH/GLI2-TGF-β1 feedback loop between cancer cells and tumor-associated macrophages maintains epithelial-mesenchymal transition and endoplasmic reticulum homeostasis in cholangiocarcinoma

BackgroundTumor-associated macrophages (TAMs) play a dual role in tumors. However, the factors which drive the function of TAMs in cholangiocarcinoma remain largely undefined. MethodsSHH signaling pathway and endoplasmic reticulum stress (ERS) indicators were detected in clinical tissues and cholangiocarcinoma cell lines. TAMs were co-cultured with cholangiocarcinoma cells under conditions of hypoxia/normoxia. Polarized TAMs were counted by flow cytometry, and TGF-β1 levels in cell supernatants were detected by ELISA. The effects of glioma-associated oncogene GLI2 on TAMs themselves and cholangiocarcinoma cells were examined by conducting interference and overexpression assays. ResultsThe SHH signaling pathway and ERS were both activated in tumor tissues or tumor cell lines under conditions of hypoxia. In co-culture experiments, the presence of cholangiocarcinoma cells increased the proportion of M2-polarized TAMs and the secretion of TGF-β1 by TAMs, while knockdown of SHH expression reversed those increases. Overexpression of GLI2 in TAMS or stimulation of TAMS with Hh-Ag1.5 increased their levels of TGF-β1 expression. Furthermore, under co-culture conditions, interference with GLI2 expression in TAMs reduced the tumor cell migration, invasion, and ER homeostasis induced by Hh-Ag1.5-pretreated TAMs. Under conditions of hypoxia, the presence of cholangiocarcinoma cells promoted the expression of GLI2 and TGF-β1 in Tams, and in turn, TAMs inhibited the apoptosis and promoted the migration and invasion of cholangiocarcinoma cells. In vivo, an injection of cholangiocarcinoma cells plus TAMs contributed to the growth, EMT, and ER homeostasis of tumor tissue, while an injection of TAMs with GLI2 knockdown had the opposite effects. ConclusionCholangiocarcinoma cells regulated TAM polarization and TGF-β1 secretion via a paracrine SHH signaling pathway, and in turn, TAMs promoted the growth, EMT, and ER homeostasis of cholangiocarcinoma cells via TGF-β1.

The endoplasmic reticulum of trypanosomatids: An unrevealed road for chemotherapy.

The endoplasmic reticulum (ER) of higher eukaryotic cells forms an intricate membranous network that serves as the main processing facility for folding and assembling of secreted and membrane proteins. The ER is a highly dynamic organelle that interacts with other intracellular structures, as well as endosymbiotic pathogenic and non-pathogenic microorganisms. A strict ER quality control (ERQC) must work to ensure that proteins entering the ER are folded and processed correctly. Unfolded or misfolded proteins are usually identified, selected, and addressed to Endoplasmic Reticulum-Associated Degradation (ERAD) complex. Conversely, when there is a large demand for secreted proteins or ER imbalance, the accumulation of unfolded or misfolded proteins activates the Unfold Protein Response (UPR) to restore the ER homeostasis or, in the case of persistent ER stress, induces the cell death. Pathogenic trypanosomatids, such as Trypanosoma cruzi, Trypanosoma brucei and Leishmania spp are the etiological agents of important neglected diseases. These protozoans have a complex life cycle alternating between vertebrate and invertebrate hosts. The ER of trypanosomatids, like those found in higher eukaryotes, is also specialized for secretion, and depends on the ERAD and non-canonical UPR to deal with the ER stress. Here, we reviewed the basic aspects of ER biology, organization, and quality control in trypanosomatids. We also focused on the unusual way by which T. cruzi, T. brucei, and Leishmania spp. respond to ER stress, emphasizing how these parasites' ER-unrevealed roads might be an attractive target for chemotherapy.

Maprotiline restores ER homeostasis and rescues neurodegeneration via Histamine Receptor H1 inhibition in retinal ganglion cells

When the protein or calcium homeostasis of the endoplasmic reticulum (ER) is adversely altered, cells experience ER stress that leads to various diseases including neurodegeneration. Genetic deletion of an ER stress downstream effector, CHOP, significantly protects neuron somata and axons. Here we report that three tricyclic compounds identified through a small-scale high throughput screening using a CHOP promoter-driven luciferase cell-based assay, effectively inhibit ER stress by antagonizing their common target, histamine receptor H1 (HRH1). We further demonstrated that systemic administration of one of these compounds, maprotiline, or CRISPR-mediated retinal ganglion cell (RGC)-specific HRH1 inhibition, delivers considerable neuroprotection of both RGC somata and axons and preservation of visual function in two mouse optic neuropathy models. Finally, we determine that maprotiline restores ER homeostasis by inhibiting HRH1-mediated Ca2+ release from ER. In this work we establish maprotiline as a candidate neuroprotectant and HRH1 as a potential therapeutic target for glaucoma.

Abstract 14972: Sestrin2 Prevents Excessive Cardiac Ischemia/Reperfusion Injury by Regulating Unfolded Protein Response

Introduction: Cardiac ischemia/reperfusion (I/R) can alter redox and calcium homeostasis, causing proteins misfolded or unfolded in endoplasmic reticulum (ER) and activating unfolded protein response (UPR). Appropriate UPR is an adaptive response of the cell, however excessive and prolonged UPR can further deteriorate calcium overload and reactive oxygen species (ROS) overproduction. We have found that Sestrin2 (Sesn2), an endogenous inhibitor of the mammalian target of rapamycin complex 1 (mTORC1) is upregulated after cardiac I/R and lack of Sesn2 in hearts may cause more severe I/R injury. Hypothesis: Sesn2 maintains the ER homeostasis as an adaptive response to attenuate calcium overload and reduce ROS overproduction during myocardial I/R. Methods: Ligation/release of the left anterior descending coronary arteries (LAD) were performed in the C57BL/6J and cardiac specific Sesn2 knock out (Sens2 cKO ) mice to induce myocardial I/R injury. Results: We found that absence of Sesn2 in the hearts increased myocardial infarction by 49.4%, and decreased the systolic function of the heart by 22.9%, compared to wild type (WT) mice. I/R-induced UPR is a time-dependent process, and it reached its peak at 6 hours post reperfusion, but returned to normal after 24 hours of reperfusion in WT mice, however, Sesn2 deficiency resulted in higher and prolonged activation curve of UPR, as well as more myocardial apoptosis, compared to WT mice. We also found that Sesn2 deficiency impaired calcium transient and increased ROS after I/R. Furthermore, the activation of mTORC1 pathway after I/R was also significantly increased and prolonged in Sesn2 cKO hearts. Inhibiting the mTORC1 by rapamycin after I/R reduced myocardial apoptosis in both WT and Sesn2 cKO mice. Conclusions: During I/R, the regulation of the mTORC1 signaling pathway by Sesn2 may be a key factor in maintaining ER homeostasis to reduce cardiac I/R injury.

Cancer cell-intrinsic XBP1 drives immunosuppressive reprogramming of intratumoral myeloid cells by promoting cholesterol production

A hostile microenvironment in tumor tissues disrupts endoplasmic reticulum homeostasis and induces the unfolded protein response (UPR). A chronic UPR in both cancer cells and tumor-infiltrating leukocytes could facilitate the evasion of immune surveillance. However, how the UPR in cancer cells cripples the anti-tumor immune response is unclear. Here, we demonstrate that, in cancer cells, the UPR component X-box binding protein 1 (XBP1) favors the synthesis and secretion of cholesterol, which activates myeloid-derived suppressor cells (MDSCs) and causes immunosuppression. Cholesterol is delivered in the form of small extracellular vesicles and internalized by MDSCs through macropinocytosis. Genetic or pharmacological depletion of XBP1 or reducing the tumor cholesterol content remarkably decreases MDSC abundance and triggers robust anti-tumor responses. Thus, our data unravel the cell-non-autonomous role of XBP1/cholesterol signaling in the regulation of tumor growth and suggest its inhibition as a useful strategy for improving the efficacy of cancer immunotherapy.

Role of ER Stress in Xenobiotic-Induced Liver Diseases and Hepatotoxicity.

The liver is a highly metabolic organ and plays a crucial role in the transportation, storage, and/or detoxication of xenobiotics. Liver damage induced by xenobiotics (e.g., heavy metal, endocrine disrupting chemicals, Chinese herbal medicine, or nanoparticles) has become a pivotal reason for liver diseases, leading to great clinical challenge and much attention for the past decades. Given that endoplasmic reticulum (ER) is the prominent organelle involved in hepatic metabolism, ER dysfunction, namely, ER stress, is clearly observed in various liver diseases. In response to ER stress, a conserved adaptive signaling pathway known as unfolded protein response (UPR) is activated to restore ER homeostasis. However, the prolonged ER stress with UPR eventually leads to the death of hepatocytes, which is a pathogenic event in many hepatic diseases. Therefore, analyzing the perturbation in the activation or inhibition of ER stress and the UPR signaling pathway is likely an effective marker for investigating the molecular mechanisms behind the toxic effects of xenobiotics on the liver. We review the role of ER stress in hepatic diseases and xenobiotic-induced hepatotoxicity, which not only provides a theoretical basis for further understanding the pathogenesis of liver diseases and the mechanisms of hepatotoxicity induced by xenobiotics but also presents a potential target for the prevention and treatment of xenobiotic-related liver diseases.

Pharmacological Manipulation of UPR: Potential Antiviral Strategy Against Chikungunya Virus.

Viruses invade the host cells and maneuver the cellular translation machinery to translate the viral proteins in substantial amounts, which may disturb Endoplasmic Reticulum homeostasis leading to induction of Unfolded Protein Response (UPR), a host response pathway involved in viral pathogenesis. Here, we investigated the effect of UPR pathways on the pathogenesis of chikungunya virus infection. We observed thatchikungunya virus mediated themodulation of UPR. A positive modulation was observed in the activation of IRE1 and ATF6 branch while the PERK branch of UPR observed suppressed upon virus infection. We further investigated the effect of the inhibition of UPR pathways on chikungunya virus replication using inhibitors for each branch. Cells treated with 3-ethoxy-5,6-dibromosalicylaldehyde (IRE1 inhibitor) and AEBSF (ATF6 inhibitor) significantly inhibits the viral replication process. This study has provided a novel perspective in designing antivirals against chikungunya virus. The online version contains supplementary material available at 10.1007/s12088-022-01046-5.

An Endoplasmic Reticulum Targeting Type I Photosensitizer for Effective Photodynamic Therapy against Hypoxic Tumor Cells.

Organelle-targeted type I photodynamic therapy (PDT) shows great potential to overcome the hypoxic microenvironment in solid tumors. The endoplasmic reticulum (ER) is an indispensable organelle in cells with important biological functions. When the ER is damaged due to the production of reactive oxygen species (ROS), the accumulation of misfolded proteins will interfere with ER homeostasis, resulting in ER stress. Here, an ER-targeted benzophenothiazine-based photosensitizer NBS-ER was presented. ER targeting modification significantly reduced the dark toxicity and improved phototoxicity index (PI). NBS-ER could effectively produce O2 - ⋅ with near-infrared irradiation, making its phototoxicity under hypoxia close to that under normoxia. Meanwhile, the photoinduced ROS triggered ER stress and induced apoptosis. In addition, NBS-ER possessed excellent photodynamic therapeutic effect in 4T1-tumor-bearing mice.

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.

Nanocarrier-Assisted Delivery of Metformin Boosts Remodeling of Diabetic Periodontal Tissue via Cellular Exocytosis-Mediated Regulation of Endoplasmic Reticulum Homeostasis.

Endoplasmic reticulum (ER) dysfunction is a potential contributor to the impaired repair capacity of periodontal tissue in diabetes mellitus (DM) patients. Restoring ER homeostasis is thus critical for successful regenerative therapy of diabetic periodontal tissue. Recent studies have shown that metformin can modulate DM-induced ER dysfunction, yet its mechanism remains unclear. Herein, we show that high glucose elevates the intracellular miR-129-3p level due to exocytosis-mediated release failure and subsequently perturbs ER calcium homeostasis via downregulating transmembrane and coiled-coil domain 1 (TMCO1), an ER Ca2+ leak channel, in periodontal ligament stem cells (PDLSCs). This results in the degradation of RUNX2 via the ubiquitination-dependent pathway, in turn leading to impaired PDLSCs osteogenesis. Interestingly, metformin could upregulate P2X7R-mediated exosome release and decrease intracellular miR-129-3p accumulation, which restores ER homeostasis and thereby rescues the impaired PDLSCs. To further demonstrate the in vivo effect of metformin, a nanocarrier for sustained local delivery of metformin (Met@HALL) in periodontal tissue is developed. Our results demonstrate that compared to controls, Met@HALL with enhanced cytocompatibility and pro-osteogenic activity could boost the remodeling of diabetic periodontal tissue in rats. Collectively, our findings unravel a mechanism of metformin in restoring cellular ER homeostasis, enabling the development of a nanocarrier-mediated ER targeting strategy for remodeling diabetic periodontal tissue.