Endoplasmic Reticulum Oxidoreductase Research Articles

Proteomic associations with forced expiratory volume: a Mendelian randomisation study

BackgroundA decline in forced expiratory volume (FEV1) is a hallmark of respiratory diseases that are an important cause of morbidity among the elderly. While some data exist on biomarkers that are related to FEV1, we sought to do a systematic analysis of causal relations of biomarkers with FEV1.MethodsData from the population-based AGES-Reykjavik study were used. Serum proteomic measurements were done using 4782 DNA aptamers (SOMAmers). Data from 1479 participants with spirometric data were used to assess the association of SOMAmer measurements with FEV1 using linear regression. Bi-directional two-sample Mendelian randomisation (MR) analyses were done to assess causal relations of observationally associated SOMAmers with FEV1, using genotype and SOMAmer data from 5368 AGES-Reykjavik participants and genetic associations with FEV1 from a publicly available GWAS (n = 400,102).ResultsIn observational analyses, 530 SOMAmers were associated with FEV1 after multiple testing adjustment (FDR < 0.05). The most significant were Retinoic Acid Receptor Responder 2 (RARRES2), R-Spondin 4 (RSPO4) and Alkaline Phosphatase, Placental Like 2 (ALPPL2). Of the 257 SOMAmers with genetic instruments available, eight were associated with FEV1 in MR analyses. Three were directionally consistent with the observational estimate, Thrombospondin 2 (THBS2), Endoplasmic Reticulum Oxidoreductase 1 Beta (ERO1B) and Apolipoprotein M (APOM). THBS2 was further supported by a colocalization analysis. Analyses in the reverse direction, testing whether changes in SOMAmer levels were caused by changes in FEV1, were performed but no significant associations were found after multiple testing adjustments.ConclusionsIn summary, this large scale proteogenomic analyses of FEV1 reveals circulating protein markers of FEV1, as well as several proteins with potential causality to lung function.

Inhibition of ERO1a and IDO1 improves dendritic cell infiltration into pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal and treatment resistant cancers. Due to its desmoplastic and hypoxic nature along with an abundance of myeloid cell infiltration and scarce T cell infiltration, PDAC is considered a cold tumor. Here we sought to investigate myeloid cell infiltration and composition in PDAC spheroids by targeting the hypoxia-associated pathways endoplasmic reticulum oxidoreductase 1 alpha (ERO1a) and indoleamine 2,3-dioxygenase 1 (IDO1). Using MiaPaCa2 spheroids with hypoxic core, we assessed the roles of ERO1a and IDO1 inhibition in modulating monocyte infiltration and differentiation, followed by characterizing immunomodulatory factors secreted using LC-MS/MS. Inhibition of ERO1a and IDO1 significantly improved monocyte infiltration and differentiation into dendritic cells. LC-MS/MS analysis of the PDAC spheroid secretome identified downregulation of hypoxia and PDAC pathways, and upregulation of antigen presentation pathways upon inhibition of ERO1a and IDO1. Furthermore, immunomodulatory factors involved in immune infiltration and migration including interleukin-8, lymphocyte cytosolic protein 1, and transgelin-2, were upregulated upon inhibition of ERO1a and IDO1. Collectively, our results show that inhibition of ERO1a and IDO1 modulates the tumor microenvironment associated with improved monocyte infiltration and differentiation into dendritic cells to potentially influence therapeutic responses in patients with PDAC.

Apoptosis mechanisms induced by 15d-PMJ2 in HCT116 colon cancer cells: insights into CHOP10/TRB3/Akt signaling.

Agents that stimulate the endoplasmic reticulum (ER) stress pathway are being exploited pharmacologically to induce cancer cell death. Cytotoxic ER stress is typically regulated by the transcription factor, C/EBP homologous protein 10 (CHOP10). Products of CHOP10 transcription include the pro-apoptotic proteins: ER oxidoreductase 1α (ERO1α), death receptor-5 (DR5), and tribbles-related protein 3 (TRB3). Our previous findings showed cell death induced by 15-deoxy- Δ12,14 prostamide J2 (15d-PMJ2) occurred in an ER stress-dependent manner. However, the pathway by which 15d-PMJ2 regulates ER stress-mediated death downstream of CHOP10 has not been identified. Our results demonstrate 5µM 15d-PMJ2 increased CHOP10 expression and apoptosis in HCT116 colon cancer cells. In cells treated with pharmacological inhibitors of ER stress, 15d-PMJ2-induced apoptosis was reliant upon the ER stress pathway. To investigate the role of CHOP10 and its transcriptional products in apoptosis, genetic deletion of CHOP10 (CHOP10-KO) was performed using the CRISPR/Cas9 system. The apoptotic action of 15d-PMJ2 was blunted in cells lacking CHOP10 expression. The deletion of CHOP10 reduced the expression of DR5, ERO1α, and TRB3 although only the expression of TRB3 was significantly reduced. Therefore, we overexpressed TRB3 in CHOP10-KO cells and observed that the activation of Akt was inhibited and 15d-PMJ2-induced apoptosis was restored. Thus, a mechanism of apoptosis elicited by 15d-PMJ2 includes the stimulation of CHOP10/TRB3/Akt inhibition. Given the important role these signaling molecules play in cancer cell fate, 15d-PMJ2 may be an effective inducer of apoptosis in cancer cells.

Covalent inhibition of the endoplasmic reticulum oxido-reductase ERO1 for effective breast cancer therapy

Covalent inhibition of the endoplasmic reticulum oxido-reductase ERO1 for effective breast cancer therapy

Identification and functional analysis of endoplasmic reticulum oxidoreductase 1 (ERO1) from the green mud crab Scylla paramamosain: The first evidence of ERO1 involved in invertebrate immune response.

Endoplasmic reticulum oxidoreductase 1 (ERO1) is an important mediator in regulating disulfide bond formation and maintaining endoplasmic reticulum homeostasis. Its activity is transcriptionally regulated by the unfolded protein response (UPR) in the endoplasmic reticulum, which is known to be essential in immunity. However, whether ERO1 is involved in innate immunity in invertebrates remains unclear. In the present study, two subtypes of ERO1 from Scylla paramamosain were first identified and characterized. Sequence analysis revealed the conserved ERO1 domain and the oxidative capacity assay verified the oxidative capacity of SpERO1 recombinant protein. Moreover, SpERO1s were found to be ubiquitously expressed in all the tested tissues, with the highest expression observed in hemocytes. Two SpERO1s exhibited distinct expression patterns in response to Vibrio alginolyticus and White Spot Syndrome Virus (WSSV). Importantly, the downregulation of the expression of immune factors upon bacterial challenge in SpERO1-silenced crabs was observed. These results provided an initial foundation for further investigations into the role of ERO1 in the innate immunity of invertebrates.

Tumor necrosis factor-α reduces adiponectin production by decreasing transcriptional activity of peroxisome proliferator-activated receptor-γ in calf adipocytes

Adiponectin (encoded by ADIPOQ) is an adipokine that orchestrates energy homeostasis by modulating glucose and fatty acid metabolism in peripheral tissues. During the periparturient period, dairy cows often develop adipose tissue inflammation and decreased plasma adiponectin levels. Proinflammatory cytokine tumor necrosis factor-α (TNF-α) plays a pivotal role in regulating the endocrine functions of adipocytes, but whether it affects adiponectin production in calf adipocytes remains obscure. Thus, the present study aimed to determine whether TNF-α could affect adiponectin production in calf adipocytes and to identify the underlying mechanism. Adipocytes isolated from Holstein calves were differentiated and used for (1) BODIPY493/503 staining; (2) treatment with 0.1 ng/mL TNF-α for different times (0, 8, 16, 24, or 48 h); (3) transfection with peroxisome proliferator-activated receptor-γ (PPARG) small interfering RNA for 48 h followed by treatment with or without 0.1 ng/mL TNF-α for 24 h; and (4) overexpression of PPARG for 48 h followed by treatment with or without 0.1 ng/mL TNF-α for 24 h. After differentiation, obvious lipid droplets and secretion of adiponectin were observed in adipocytes. Treatment with TNF-α did not alter mRNA abundance of ADIPOQ but reduced the total and high molecular weight (HMW) adiponectin content in the supernatant of adipocytes. Quantification of mRNA abundance of endoplasmic reticulum (ER)/Golgi resident chaperones involved in adiponectin assembly revealed that ER protein 44 (ERP44), ER oxidoreductase 1α (ERO1A), and disulfide bond-forming oxidoreductase A-like protein (GSTK1) were downregulated in TNF-α-treated adipocytes, while 78-kDa glucose-regulated protein and Golgi-localizing γ-adaptin ear homology domain ARF binding protein-1 were unaltered. Moreover, TNF-α diminished nuclear translocation of PPARγ and downregulated mRNA abundance of PPARG and its downstream target gene fatty acid synthase, suggesting that TNF-α suppressed the transcriptional activity of PPARγ. In the absence of TNF-α, overexpression of PPARG enhanced the total and HMW adiponectin content in supernatant and upregulated the mRNA abundance of ADIPOQ, ERP44, ERO1A, and GSTK1 in adipocytes. However, knockdown of PPARG reduced the total and HMW adiponectin content in supernatant and downregulated the mRNA abundance of ADIPOQ, ERP44, ERO1A, and GSTK1 in adipocytes. In the presence of TNF-α, overexpression of PPARG decreased, while knockdown of PPARG further exacerbated TNF-α-induced reductions in total and HMW adiponectin secretion and gene expression of ERP44, ERO1A, and GSTK1. Overall, TNF-α reduces adiponectin assembly in the calf adipocyte, which may be partly mediated by attenuation of PPARγ transcriptional activity. Thus, locally elevated levels of TNF-α in adipose tissue may be one reason for the decrease in circulating adiponectin in periparturient dairy cows.

Meet the First Authors

Meet the First Authors

A Critical Role for ERO1α in Arterial Thrombosis and Ischemic Stroke.

Platelet adhesion and aggregation play a crucial role in arterial thrombosis and ischemic stroke. Here, we identify platelet ERO1α (endoplasmic reticulum oxidoreductase 1α) as a novel regulator of Ca2+ signaling and a potential pharmacological target for treating thrombotic diseases. Intravital microscopy, animal disease models, and a wide range of cell biological studies were utilized to demonstrate the pathophysiological role of ERO1α in arteriolar and arterial thrombosis and to prove the importance of platelet ERO1α in platelet activation and aggregation. Mass spectrometry, electron microscopy, and biochemical studies were used to investigate the molecular mechanism. We used novel blocking antibodies and small-molecule inhibitors to study whether ERO1α can be targeted to attenuate thrombotic conditions. Megakaryocyte-specific or global deletion of Ero1α in mice similarly reduced platelet thrombus formation in arteriolar and arterial thrombosis without affecting tail bleeding times and blood loss following vascular injury. We observed that platelet ERO1α localized exclusively in the dense tubular system and promoted Ca2+ mobilization, platelet activation, and aggregation. Platelet ERO1α directly interacted with STIM1 (stromal interaction molecule 1) and SERCA2 (sarco/endoplasmic reticulum Ca2+-ATPase 2) and regulated their functions. Such interactions were impaired in mutant STIM1-Cys49/56Ser and mutant SERCA2-Cys875/887Ser. We found that ERO1α modified an allosteric Cys49-Cys56 disulfide bond in STIM1 and a Cys875-Cys887 disulfide bond in SERCA2, contributing to Ca2+ store content and increasing cytosolic Ca2+ levels during platelet activation. Inhibition of Ero1α with small-molecule inhibitors but not blocking antibodies attenuated arteriolar and arterial thrombosis and reduced infarct volume following focal brain ischemia in mice. Our results suggest that ERO1α acts as a thiol oxidase for Ca2+ signaling molecules, STIM1 and SERCA2, and enhances cytosolic Ca2+ levels, promoting platelet activation and aggregation. Our study provides evidence that ERO1α may be a potential target to reduce thrombotic events.

Deficiency of an endoplasmic reticulum oxidoreductase and activation of protein translation synergistically impair breast tumor resilience

Deficiency of an endoplasmic reticulum oxidoreductase and activation of protein translation synergistically impair breast tumor resilience

Suppression of Microglial ERO1a Alleviates Inflammation and Enhances the Efficacy of Rehabilitative Training After Ischemic Stroke.

Microglia mediated inflammation plays a crucial role in cellular events and functional recovery post ischemic stroke. In the current study, we profiled the proteome changes of microglia treated with oxygen and glucose deprivation (OGD). Bioinformatics analysis identified that differentially expressed proteins (DEPs) were enriched in pathways associated with oxidate phosphorylation and mitochondrial respiratory chain at both 6h and 24h post OGD. We next focused on one validated target named endoplasmic reticulum oxidoreductase 1 alpha (ERO1a) to study its role in stroke pathophysiology. We showed that over-expression of microglial ERO1a exacerbated inflammation, cell apoptosis and behavioral outcomes post middle cerebral artery occlusion (MCAO). In contrast, suppression of microglial ERO1a significantly reduced activation of both microglia and astrocyte, along with cell apoptosis. Furthermore, knocking down microglial ERO1a improved the efficacy of rehabilitative training and enhanced the mTOR activity in spared corticospinal neurons. Our study provided novel insights into the identification of therapeutic targets and the design of rehabilitative protocols to treat ischemic stroke and other traumatic CNS injuries.

Knockdown of ERO1L attenuates tumor growth, migration and invasion in lung adenocarcinoma through Wnt/β‑catenin pathway

ABSTRACT Recent studies confirm the critical roles of endoplasmic reticulum oxidoreductase 1 alpha (ERO1L) in malignant behavior of various cancers. Nevertheless, what function ERO1L plays in lung adenocarcinoma (LUAD) remains uncovered. The expressions and clinical significance of ERO1L in LUAD were investigated using the TCGA dataset. The ERO1L levels were examined by RT-qPCR. The LUAD cell proliferation was valued using colony formation as well as CCK-8 assays. The invasion and migration abilities of LUAD cells were detected through Transwell in addition to wound healing assays. The effects of ERO1L on LUAD cell apoptosis were determined by flow cytometric analysis. Moreover, we also established mouse xenograft models of LUAD cells to confirm the functions of ERO1L in vivo. The ERO1L levels in tumors were identified by immunohistochemistry. Western blot was used for the detection of the levels of Wnt/βcatenin signaling-related proteins. The TCGA database revealed that ERO1L expressions were higher in LUAD tissues than those in non-cancerous tissues. ERO1L overexpression was related to poorer overall survival of LUAD patients. In addition, ERO1L silence suppresses LUAD cell clone formation, proliferation, migration as well as invasion but induces apoptosis. Moreover, we also verified that ERO1L silence could promote LUAD growth in vivo. Based on the mechanism analysis, ERO1L was confirmed to regulate LUAD development via Wnt/βcatenin cascade signal. ERO1L, the expression of which was increased in LUAD tissues, functioned as an oncogene. ERO1L silence significantly attenuated LUAD tumorigenesis, likely via inhibition of Wnt/βcatenin signaling, indicating that ERO1L could be exploited as a promising biomarker in LUAD treatment.

Abstract 4466: Inhibition of ERO1a and IDO1 improves dendritic cell infiltration into pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most hypoxic, lethal, and treatment resistant cancers. Due to its desmoplasias along with the hypoxic state and scarce infiltration of T cells, PDAC is considered a cold tumor. Using a proteomic approach, we previously identified that the expression of endoplasmic reticulum oxidoreductase 1 alpha (ERO1α) is induced by hypoxia and correlates with reduced survival in PDAC. Furthermore, we observed an increased kynurenine formation under hypoxia, suggesting a role of indoleamine 2,3-dioxygenases (IDO1) in PDAC. Since ERO1α and IDO are involved in driving immune suppression through influencing the generation of immature myeloid cells, we sought to investigate whether inhibition of these pathways modulates the myeloid cell composition in PDAC. Using MiaPaCa2 as a PDAC spheroid model with hypoxic core grown in serum-free defined media and immune cell infiltration, we assessed the functional role of ERO1a with IDO1 in modulating monocyte infiltration and differentiation into pro- and anti-inflammatory phenotypes, followed by characterizing the immunomodulatory factors secreted using tandem mass spectrometry. Inhibition of ERO1a and IDO1 significantly improved U937 monocyte infiltration (p-value = ****) and differentiation into dendritic cells (p-value = ****). Secretome analysis of proteins identified with high false discovery rate (q-value&amp;lt;0.01) revealed a downregulation of hypoxia and pancreatic cancer pathways in PDAC spheroids. In the presence of monocytes, upregulation of immune-related pathways for antigen presentation (15-fold) and myeloid cell maintenance signaling (2-fold) were observed. Moreover, specific immune-modulatory factors involved in immune infiltration and migration including interleukin 8, lymphocyte cytosolic protein 1 and transgelin-2 were upregulated at 27-, 20-, and 14-fold respectively. Taken together, these results indicate that inhibition of ERO1a and IDO1 drives an inflamed tumor microenvironment associated with improved monocyte infiltration and differentiation into dendritic cells to potentially enable T cell infiltration and killing of PDAC tumors. Citation Format: Apple Tay, Andreas Lundqvist, Siu Kwan Sze. Inhibition of ERO1a and IDO1 improves dendritic cell infiltration into pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4466.

Oxytocin action on components of endoplasmic reticulum in hippocampal neuronal cells

Despite the known importance of the endoplasmic reticulum (ER) in protein synthesis and vesicular transport, it is not clear whether neuropeptide and neuromodulator oxytocin can directly affect components of the ER in neuronal cells. Therefore, in the present study, we hypothesize that incubation of hippocampal neuronal cells in a presence of oxytocin 1) plays a role in the regulation of the expression of selected ER chaperone components and molecules involved in unfolded protein response pathway 2) affects distribution of the intracellular fluorescence signal highly selective for the ER. We found that oxytocin (1 μM) after 60 min significantly decreased the gene expression of oxidoreductase Ero1β, chaperone glucose-regulated proteins (Grp) 78 and Grp94. A significant decrease in GRP78 protein levels in response to oxytocin treatment occurred after 30, 60 and 120 min. We also observed a time-dependent increase in calreticulin protein levels with a statistically significant increase observed after 360 min. We found that the dynamics of the ER network changes significantly within 2 h of incubation under the influence of oxytocin. In conclusion we have shown that ER chaperones, oxidoreductases and trafficking molecules in neuronal cells are changing in response to oxytocin treatment in a short-term scenario potentially relevant for growth of dendrites and axons.

Platelet ERO1α Is Crucial for Ca2+ Mobilization and Platelet Activation in Atherothrombosis

Endoplasmic reticulum oxidoreductase 1α (ERO1α) is a key oxidase of protein disulfide isomerase (PDI) during protein folding in the ER. Platelet ERO1α was reported to interact with PDI and αIIbβ3 integrin and regulate platelet function. However, the regulatory mechanism and pathophysiological role remain unknown. In the present study, we demonstrate that platelet ERO1α orchestrates intracellular Ca2+ signaling during platelet activation and contributes to thrombogenesis. Intravital microscopy using megakaryocyte-specific Ero1α conditional knockout (CKO) and KO mice reveals that platelet ERO1α is crucial for platelet thrombus formation in mouse models of laser-induced cremaster arteriolar thrombosis and FeCl3-induced carotid arterial thrombosis. Deletion of Ero1α does not affect bleeding times and blood loss at the site of tail amputation, implying that ERO1α is not required for hemostasis. Studies using Ero1α-null platelets, novel function-blocking antibodies, and small-molecule inhibitors reveal that intracellular, but not extracellular, ERO1α promotes P-selectin exposure, αIIbβ3 integrin activation, and platelet aggregation induced by multiple agonists, such as thrombin and collagen-related peptide. Importantly, the regulatory function of ERO1α is independent of PDI activity. Mass spectrometry and immunoprecipitation assays identify stromal interaction molecule 1 (STIM1) and sarco/endoplasmic reticulum Ca2+-ATPase 2 (SERCA2) as ERO1α binding partners. We have found that ERO1α-STIM1 binding is enhanced during platelet activation, whereas ERO1α-SERCA2 binding is reduced. Intriguingly, platelet ERO1α interacts with STIM1 through Cys49 and Cys56 residues and promotes its function by oxidizing the Cys49-Cys56 disulfide bond, contributing to Ca2+ store content and enhancing Ca2+ mobilization during platelet activation. Using a high throughput screen, we identify a small-molecule ERO1α inhibitor, B20, which inhibits ERO1α activity with an IC50 value of 10.0 μM but does not affect the activities of H2O2 and PDI. Treatment of platelets with B20 recapitulates the functional defects as seen in Ero1α-null platelets. Intravenous injection of B20 and eptifibatide, an αIIbβ3 integrin antagonist, into WT mice significantly inhibits platelet thrombus formation following laser- or FeCl3-induced vascular injury. While treatment of mice with eptifibatide markedly prolongs tail bleeding times and causes blood loss, B20 does not impair hemostatic function. Furthermore, treatment with B20 reduces the infarct volume in ischemic stroke. These results indicate that platelet ERO1α is a novel regulator of Ca2+ signaling and promotes platelet activation and aggregation in a PDI-independent manner, contributing to the pathogenesis of arterial thrombosis and thromboinflammation. Targeting ERO1α may be an attractive therapeutic strategy for treating patients with thrombotic disease.

Porcine circovirus type 2 induces CHOP-ERO1α-ROS-mediated apoptosis in PK-15 cells

Porcine circovirus type 2 (PCV2) infection induces endoplasmic reticulum (ER) stress and oxidative stress. These cellular responses could be connected with apoptosis. However, the mechanisms that link ER stress and oxidative stress in PCV2-induced apoptosis are poorly characterized. Here, we demonstrate that PCV2 infection increased expression of proapoptotic protein C/EBP homologous protein (CHOP) and ER oxidoreductase 1 alpha (ERO1α). Inhibition of CHOP by RNA silencing or inhibition of ERO1α by short hairpin RNA or EN460 repressed PCV2-induced reactive oxygen species (ROS) generation, cytosolic calcium level, and apoptotic rate in PK-15 cells. Overexpression of ERO1α enhanced PCV2-induced oxidative stress, caspase-3 cleavage, and apoptosis rate. Treatment of PCV2-infected cells with ROS scavenger N-acetyl-L-cysteine downregulated PCV2-induced ROS production, cytosolic calcium level, and apoptosis rate, but intriguingly decreased expression of CHOP and ERO1α. Thus, we propose that PCV2 induces apoptosis through ER Stress via CHOP-ERO1α-ROS signaling in host cells.

Combined Toxicological Effects of Di (2-Ethylhexyl) Phthalate and UV-B Irradiation through Endoplasmic Reticulum Stress—Tight Junction Disruption in Human HaCaT Keratinocytes

Di (2-ethylhexyl) phthalate (DEHP) is widely used as a plasticizer, and human exposure to DEHP is widespread and frequent. However, information about the combined effect of DEHP and ultraviolet (UV)-B on the skin are still limited. We investigated the cytotoxic effects of DEHP and UV-B on HaCaT keratinocytes and evaluated the related underlying mechanisms involving endoplasmic reticulum (ER) stress signals and the disruption of junction complexes as an effective target for skin inflammation. Our results revealed that co-treatment with DEHP and UV-B irradiation alleviated the cell cytotoxicity and markedly decreased X-box binding protein 1 (XBP1), endoplasmic reticulum oxidoreductase 1 alpha (Ero1α), and C/EBP homologous protein (CHOP) whereas a single dose of 40 mJ/cm2 UV-B generated mild ER stress to slightly less or similar levels as that seen with DEHP. DEHP was also shown to inhibit tight junctions (TJs) after UV-B irradiation, increased apoptosis by altering apoptotic gene Bax and stress kinases, JNK, and p38 MAPK. Furthermore, exposure of HaCaT cells to DEHP and UV-B irradiation resulted in the marked suppression of the nuclear factor kappa B (NF-κB)/p65 signaling pathway. Taken together, our data suggest that nontoxic DEHP and UV-B irradiation regulated ER stress and epidermal TJ disruption with the induction of apoptosis activation and the secretion of proinflammatory cytokines such as interleukin 1 beta (IL-1β) and IL-6 in human keratinocytes. Further investigation is needed to confirm the mechanisms implicated in its toxicity and determine the effects of exposure to DEHP and UV-B irradiation on markers involved in this study.

SARS-CoV-2 ORF8 reshapes the ER through forming mixed disulfides with ER oxidoreductases

The replication machinery of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is closely associated with the endoplasmic reticulum (ER) in host cells. Activation of the unfolded protein response (UPR) is a strategy hijacked by coronavirus to facilitate its replication and suppress host innate immunity. Here, we have found that SARS-CoV-2 ORF8 protein accumulates in the ER and escapes the degradation system by forming mixed disulfide complexes with ER oxidoreductases. ORF8 induces the activation of three UPR pathways through targeting key UPR components, remodels ER morphology and accelerates protein trafficking. Moreover, small molecule reducing agents release ORF8 from the mixed disulfide complexes and facilitate its degradation, therefore mitigate ER stress. Our study reveals a unique mechanism by which SARS-CoV-2 ORF8 escapes degradation by host cells and regulates ER reshaping. Targeting ORF8-involved mixed disulfide complexes could be a new strategy to alleviate SARS-CoV-2 induced ER stress and related diseases.

Homocysteine facilitates endoplasmic reticulum stress and apoptosis of hepatocytes by suppressing ERO1α expression via cooperation between DNMT1 and G9a.

Endoplasmic reticulum (ER) stress and apoptosis play a critical role in liver injury. Endoplasmic reticulum oxidoreductase 1α (ERO1α) is an oxidase that exists in the luminal side of the ER membrane, participating in protein folding and secretion and inhibiting apoptosis, but the underlying mechanism on liver injury induced by homocysteine (Hcy) remains obscure. In this study, hyperhomocysteinemia (HHcy) mice model was established in cbs +/− mice by feeding a high‐methionine diet for 12 weeks; and cbs +/− mice fed with high‐methionine diet exhibited more severe liver injury compared to cbs +/+ mice. Mechanistically, we found that Hcy promoted ER stress and apoptosis of hepatocytes and thereby aggravated liver injury through inhibiting ERO1α expression; accordingly, overexpression of ERO1α remarkably alleviated ER stress and apoptosis of hepatocytes induced by Hcy. Epigenetic modification analysis revealed that Hcy significantly increased levels of DNA methylation and H3 lysine 9 dimethylation (H3K9me2) on ERO1α promoter, which attributed to upregulated DNA methyltransferase 1 (DNMT1) and G9a, respectively. Further study showed that DNMT1 and G9a cooperatively regulated ERO1α expression in hepatocytes exposed to Hcy. Taken together, our work demonstrates that Hcy activates ER stress and apoptosis of hepatocytes by downregulating ERO1α expression via cooperation between DNMT1 and G9a, which provides new insight into the mechanism of Hcy‐induced ER stress and apoptosis of hepatocytes in liver injury.

MicroRNA-218-5p affects lung adenocarcinoma progression through targeting endoplasmic reticulum oxidoreductase 1 alpha

ABSTRACT Lung adenocarcinoma (LUAD) severely threatens the health of people owing to its lethality. Nonetheless, the underlying mechanisms on LUAD development remain unclear to a great extent. This work aimed to probe the functions of miR-218-5p in LUAD. MiR-218-5p and endoplasmic reticulum oxidoreductase 1 alpha (ERO1A) were screened as differently downregulated and upregulated RNAs in LUAD, respectively, by bioinformatics analyses. The results of cell functional assays stated that enforced expression of miR-218-5p notably restrained cell viability, invasion, and migration in LUAD. MiR-218-5p may interact with 3’-untranslated region of ERO1A mRNA as analyzed by bioinformatics. Afterward, western blot and dual-luciferase reporter gene analyses were introduced to identify their interaction. ERO1A overexpression reversed the suppressive impacts of miR-218-5p on LUAD cell progression, indicating the implication of miR-218-5p/ERO1A axis in suppressing cancer development. We also observed that this regulatory axis suppressed angiogenesis in LUAD. Taken together, miR-218-5p/ERO1A axis exerted an imperative role in LUAD cell progression, which provides a valuable clue for the development of LUAD therapeutic regimen.

Ero1α-Dependent ERp44 Dissociation From RyR2 Contributes to Cardiac Arrhythmia.

Oxidative stress in cardiac disease promotes proarrhythmic disturbances in Ca2+ homeostasis, impairing luminal Ca2+ regulation of the sarcoplasmic reticulum (SR) Ca2+ release channel, the RyR2 (ryanodine receptor), and increasing channel activity. However, exact mechanisms underlying redox-mediated increase of RyR2 function in cardiac disease remain elusive. We tested whether the oxidoreductase family of proteins that dynamically regulate the oxidative environment within the SR are involved in this process. A rat model of hypertrophy induced by thoracic aortic banding (TAB) was used for ex vivo whole heart optical mapping and for Ca2+ and reactive oxygen species imaging in isolated ventricular myocytes (VMs). The SR-targeted reactive oxygen species biosensor ERroGFP showed increased intra-SR oxidation in TAB VMs that was associated with increased expression of Ero1α (endoplasmic reticulum oxidoreductase 1 alpha). Pharmacological (EN460) or genetic Ero1α inhibition normalized SR redox state, increased Ca2+ transient amplitude and SR Ca2+ content, and reduced proarrhythmic spontaneous Ca2+ waves in TAB VMs under β-adrenergic stimulation (isoproterenol). Ero1α overexpression in Sham VMs had opposite effects. Ero1α inhibition attenuated Ca2+-dependent ventricular tachyarrhythmias in TAB hearts challenged with isoproterenol. Experiments in TAB VMs and human embryonic kidney 293 cells expressing human RyR2 revealed that an Ero1α-mediated increase in SR Ca2+-channel activity involves dissociation of intraluminal protein ERp44 (endoplasmic reticulum protein 44) from the RyR2 complex. Site-directed mutagenesis and molecular dynamics simulations demonstrated a novel redox-sensitive association of ERp44 with RyR2 mediated by intraluminal cysteine 4806. ERp44-RyR2 association in TAB VMs was restored by Ero1α inhibition, but not by reducing agent dithiothreitol, as hypo-oxidation precludes formation of covalent bond between RyR2 and ERp44. A novel axis of intraluminal interaction between RyR2, ERp44, and Ero1α has been identified. Ero1α inhibition exhibits promising therapeutic potential by stabilizing RyR2-ERp44 complex, thereby reducing spontaneous Ca2+ release and Ca2+-dependent tachyarrhythmias in hypertrophic hearts, without causing hypo-oxidative stress in the SR.