Eukaryotic Translation Initiation Factor 2α Research Articles

EIF4A3-Induced hsa_circ_0118578 Expression Enhances the Tumorigenesis of Papillary Thyroid Cancer.

Background: Circular RNA (circRNA) plays a regulatory role in the malignancy of papillary thyroid cancer (PTC). However, the role of a novel circRNA, hsa_circ_0118578, in PTC is not yet fully understood. This report focuses on unveiling hsa_circ_0118578's effect on PTC cell malignancy and reveals its mechanism in PTC progression. Methods: Levels of hsa_circ_0118578 in PTC were assessed by quantitative real-time polymerase chain reaction (qRT-PCR). The functional roles of hsa_circ_0118578 in PTC cell malignancy were evaluated through Transwell, 5-ethynyl-2'-deoxyuridine (EdU), and wound healing assays. A xenograft model in nude mice was used to examine the effects of hsa_circ_0118578's invivo. The interaction between eukaryotic translation initiation factor 4A3 (EIF4A3) and hsa_circ_0118578 was confirmed using RNA-binding protein immunoprecipitation, qRT-PCR, and western blotting. Results: The hsa_circ_0118578 with high expression in PTC tissues was associated with higher tumor node metastasis stage, lymph node metastasis, as well as poor differentiation. Cell functional assays demonstrated that silencing hsa_circ_0118578 inhibited PTC cell proliferation, invasion, and migration. In the xenograft assay, tumorigenicity of PTC cells invivo was reduced following hsa_circ_0118578 suppression. Additionally, EIF4A3, as an RNA-binding protein, was shown to interact with hsa_circ_0118578 to stabilize its expression in PTC cells. Conclusions: Upregulated hsa_circ_0118578 in PTC interacts with EIF4A3 to exert oncogenic effects by enhancing hsa_circ_0118578 stability, contributing to PTC development. These findings shed light on the oncogenic role of hsa_circ_0118578 in PTC and suggest it as a potential therapeutic target.

New insights into ER stress mediated by ATF6 and IRE1-XBP1 signals in yellow catfish under hypoxia

In aquaculture, fish are often exposed to the major stressor of hypoxia. However, the mechanism of hypoxia-induced stress remains unclear. The present study aimed to investigate the physiological and molecular responses of yellow catfish (Pelteobagrus fulvidraco) to hypoxia stress and to further elucidate its stress response mechanisms. Each fish (28.14 ± 0.75 g) in the hypoxia group was individually placed in a 1 L transparent plastic tank and transferred to a closed hypoxia environment, where the oxygen and carbon dioxide levels were adjusted to 2 % and 0.5 %, respectively. The results showed that hypoxia stress induced a stress response in yellow catfish, characterized by significant changes in lactate dehydrogenase (LD) and cortisol levels in plasma and liver, as well as increased apoptosis of brain cells and liver damage. Additionally, the HPA axis was activated, playing a crucial role in stress regulation during hypoxia exposure. And under hypoxia conditions, endoplasmic reticulum (ER) stress pathways were activated in yellow catfish liver, specifically showing significant upregulation of the activating transcription factor 6 (ATF6) and inositol-requiring enzyme 1 (IRE1)-X-box binding protein 1 (XBP1) signals, while the activated transcription factor 4 (ATF4) pathway remained inactive. The expression levels of atf6, ire1 and xbp1 genes, as well as ATF6, IRE1 and XBP1 proteins in the ATF6 and IRE1-XBP1 pathways, were significantly elevated. However, there was no significant differences in the expression levels of protein kinase R-like ER kinase (perk), eukaryotic translation initiation factor 2α (eif2α) and atf4 key genes, as well as PERK, EIF2α and ATF4 proteins in the PERK-ATF4 pathway. It is concluded for the first time that hypoxia significantly activates ER stress through the ATF6 and IRE1-XBP1 signaling pathways, but not the PERK-ATF4 pathway in yellow catfish. These findings not only provide new insights into the stress response mechanisms of fish but also offer the accurate molecular target of hypoxia-induced stress for aquaculture management.

Adjuvant alum regulates the eIF2a-GATA3 axis in CD4+ T cells to influence allergen immunotherapy.

Allergen-specific immunotherapy (AIT) is an aetiology-targeting therapy for allergic diseases. The therapeutic mechanism of AIT is not fully understood yet. Endoplasmic reticulum stress is associated with the pathogenesis of allergic disorders. This study aims to elucidate the effects of AIT on suppressing allergic response through regulating endoplasmic reticulum stress. In this study, patients with perennial allergic rhinitis were recruited. AIT was conducted for the patients. An allergic rhinitis (AR) mouse model was established with mite extracts as allergens. We found that AIT modulated the endoplasmic reticulum stress status in peripheral CD4+ T cells in patients with allergic rhinitis. The intensity of endoplasmic reticulum stress associated the PERK (protein kinase RNA-like endoplasmic reticulum kinase)-eIF2a (eukaryotic translation initiation factor 2a) axis in CD4+ T cells was upregulated by AIT, which was closely associated with the improvement in allergic rhinitis response after AIT. eIF2a interacted with GATA3 to downregulate the IL4 gene transcription in CD4+ T cells. High doses of aluminium hydroxide (alum) in AIT vaccines enhanced the activity of XBP1 to suppress eIF2a in CD4+ T cells. AIT containing a low dose of alum effectively mitigated the experimental allergic rhinitis, while the AIT without alum or a high dose of alum exacerbated the experimental allergic rhinitis. In conclusion, the alum adjuvant in allergen vaccines can regulate the activity of eIF2a to regulate the expression of Th2 cytokines in CD4+ T cells. Manipulating the alum dose in AIT vaccines has the potential to enhance the therapeutic effects of AIT.

Regulatory Role of eIF2αK4 in Amino Acid Transporter Expression in Mouse Brain Capillary Endothelial Cells.

Amino acid transporters are expressed in the brain capillary endothelial cells that form the blood-brain barrier (BBB), and their expression levels change during the neonatal period. This study aimed to investigate the molecular mechanisms regulating amino acid transporter levels in mouse brain capillary endothelial cells. Capillaries were isolated from the brains of neonatal and adult mice. Activation of eukaryotic translation initiation factor 2α kinase 4 (eIF2αK4) was analyzed in MBEC4 (mouse brain capillary endothelial) cells under amino acid-depleted conditions. Protein expression was determined using western blotting and proteomic analyses. Phosphorylation of eIF2α, a downstream target of eIF2αK4, was induced in the brain capillaries of neonates compared to adults. In vitro experiments using MBEC4 cells revealed that amino acid depletion induced eIF2α phosphorylation and expression of the amino acid transporter, solute carrier (Slc)-7a1. The eIF2αK4 inhibitor, GCN2iB, inhibited these inductions. Proteomic analysis revealed arginine depletion-dependent induction of amino acid transporters Slc1a4, Slc3a2, Slc7a1, Slc7a5, and Slc38a1. These effects were also inhibited by GCN2iB, suggesting the involvement of eIF2αK4 activation. In contrast, the expression of Slc2a1, Slc16a1, Abcb1b, Abcg2, transferrin receptor, insulin receptor, claudin-1, ZO-1, and Jam1 was not suppressed by the GCN2iB treatment. Overall, the eIF2αK4 pathway plays a regulatory role in amino acid transporter expression in brain capillary endothelial cells and facilitates the maintenance of amino acid homeostasis in the brain. This study provides new insights into the regulatory mechanisms underlying nutrient transport across the BBB.

The role of genetic and epigenetic modifications as potential biomarkers in the diagnosis and prognosis of thyroid cancer.

Thyroid cancer (TC) is the most common endocrine cancer, which contributes to more than 43,600 deaths and 586,000 cases worldwide every year. Among the TC types, PTC and FTC comprise 90% of all TCs. Genetic modifications in genes are responsible for encoding proteins of mitogen-associated protein kinase cascade, which is closely related with numerous cellular mechanisms, including controlling programmed cell death, differentiation, proliferation, gene expression, as well as in genes encoding the PI3K (phosphatidylinositol 3-kinase)/protein kinase B (AKT) cascade, which has contribution in controlling cell motility, adhesion, survival, and glucose metabolism, have been associated with the TC pathogenesis. Various genetic modifications including BRAF mutations, RAS mutations, RET mutations, paired-box gene 8/peroxisome proliferator-activated receptor-gamma fusion oncogene, RET/PTC rearrangements, telomerase reverse transcriptase mutations, neurotrophic tyrosine receptor kinase fusion genes, TP53 mutations, and eukaryotic translation initiation factor 1A X-linked mutations can effectively serve as potential biomarkers in both diagnosis and prognosis of TC. On the other hand, epigenetic modifications can lead to aberrant functions or suppression of a range of signalling cascades, which can ultimately result in cancer. Various studies have observed the link between epigenetic modification and multiple cancers including TC. It has been reported that several epigenetic alterations including histone modifications, aberrant DNA methylation, and epigenetic modulations of non-coding RNAs can play significant roles as potential biomarkers in the diagnosis and prognosis of TC. Therefore, a good understanding regarding the genetic and epigenetic modifications is not only essential for the diagnosis and prognosis of TC, but also for the development of novel therapeutics. In this review, most of the major TC-related genetic and epigenetic modifications and their potential as biomarkers for TC diagnosis and prognosis have been extensively discussed.

EIF4A3-mediated oncogenic circRNA hsa_circ_0001165 advances esophageal squamous cell carcinoma progression through the miR-381-3p/TNS3 pathway

Esophageal squamous cell carcinoma (ESCC) remains a major clinical challenge due to its poor prognosis and the scarcity effective therapeutic targets. Circular RNAs (circRNAs) are crucial in cancer progression. In this study, high-throughput sequencing was employed to profile ESCC tissues, revealing that hsa_circ_0001165 is notably elevated in both ESCC tumor samples and cell lines, with its expression is positively associated with patients' TNM staging. Knockdown of hsa_circ_0001165 resulted in reduced malignant biological behavior of ESCC cells in vitro and also inhibited tumor growth in vivo. Mechanism experimental analysis found that hsa_circ_0001165 expression is positively enhanced by eukaryotic translation initiation factor 4A3 (EIF4A3). Hsa_circ_0001165 acts as a miRNA sponge for miR-381-3p, increasing the expression of tensin-3 (TNS3) through a series of related mechanism assays include dual-luciferase reporter gene, RNA Immunoprecipitation and RNA-pulldown. The downregulation in miR-381-3p expression was observed in ESCC tissues, and the cell proliferation, invasion, and migration of ESCC were suppressed. The upregulated expression of hsa_circ_0001165 modulates the miR-381-3p/TNS3 axis and promotes aggressive phenotypes of ESCC. Hsa_circ_0001165 is regarded as a encouraging biomarker and potential therapeutic target for ESCC, presenting innovative options for both diagnostic and treatment approaches.

Modulation of the unfolded protein response by white spot syndrome virus via wsv406 targeting BiP to facilitate viral replication

Outbreaks of diseases are often linked to environmental stress, which can lead to endoplasmic reticulum (ER) stress and subsequently trigger the unfolded protein response (UPR). The replication of the white spot syndrome virus (WSSV), the most serious pathogen in shrimp aquaculture, has been shown to rely on the UPR signaling pathway, although the detailed mechanisms remain poorly understood. In this study, we discovered that WSSV enhances its replication by hijacking the UPR pathway via the viral protein wsv406. Our analysis revealed a significant upregulation of wsv406 in the hemocytes and gills of infected shrimp. Mass spectrometry analysis identified that wsv406 interacts specifically with the immunoglobulin heavy-chain-binding protein (BiP) in shrimp Litopenaeus vannamei. Further examination revealed that wsv406 binds to multiple domains of LvBiP, inhibiting its ATPase activity without disrupting its binding to UPR stress receptors. Silencing either wsv406 or LvBiP resulted in a reduction in WSSV replication and improved shrimp survival rates. Further, wsv406 activation of the PRKR-like ER kinase (PERK)-eukaryotic translation initiation factor 2α (eIF2α) and activating transcription factor 6 (ATF6) pathways was demonstrated by a decrease in the phosphorylation of eIF2α and the nuclear translocation of ATF6 when wsv406 was silenced during WSSV infection. This activation facilitated the transcription of WSSV genes, promoting viral replication. In summary, these findings reveal that wsv406 manipulates the host UPR by targeting LvBiP, thereby enhancing WSSV replication through the PERK-eIF2α and ATF6 pathways. These insights into the interaction between WSSV and host cellular machinery offer potential targets for developing therapeutic interventions to control WSSV outbreaks in shrimp aquaculture.

CoCl2-mimicked hypoxia induces the assembly of stress granules in trophoblast cells via eIF2α phosphorylation-dependent and -independent pathways.

Hypoxia has been implicated in preeclampsia (PE) pathophysiology. Stress granules (SGs) are present in the placenta of patients with PE. However, the pathways that contribute to SG aggregation in PE remain poorly understood. The objective of the current study is to investigate this issue. We first established an in vitro hypoxia model using human trophoblast cell line HTR-8/SVneo treated with cobalt chloride (CoCl2). CCK8 assay and wound healing assay were conducted to assess the viability and migration of HTR-8/SVneo cells after exposure to CoCl2-mimicked hypoxia. SG component expression in HTR-8/SVneo cells treated with CoCl2 alone, or in combination with indicated siRNAs was evaluated by reverse transcription quantitative PCR (RT-qPCR), western blot and immunofluorescence staining. Our results found CoCl2-mimicked hypoxia inhibits the proliferation and migration of HTR-8/SVneo cells. The treatment of CoCl2 can induce SG assembly in HTR-8/Svneo cells. Mechanistically, both heme-regulated inhibitors (HRI) mediated eukaryotic translation initiation factor (eIF)2α phosphorylation pathway and 4E binding protein 1 (4EBP1) pathway are involved in SG formation under the stress of CoCl2-mimicked hypoxia. Hypoxia-induced SGs in trophoblast cells might contribute to the etiology of PE.

DMRT3-mediated lncRNA OIP5-AS1 promotes the pyroptosis of bronchial epithelial cells by binding with EIF4A3 to enhance YAP mRNA stability.

Asthma is featured by persistent airway inflammation. Long noncoding RNAs (lncRNAs) are reported to play critical roles in asthma. However, the function of Opa interacting protein 5-antisense 1 (OIP5-AS1) in pyroptosis during the development of asthma remains unexplored. The blood samples of asthma patients (n = 32) as well as the baseline characteristics of asthma patients or healthy people were collected. An in vivo model of asthma was established using house dust mites (HDM). To mimic asthma in vitro, BEAS-2B cells were treated with HDM. Cell pyroptosis and apoptosis were examined by flow cytometry. The levels of interleukin-1 beta (IL-1β) and interleukin-18 (IL-18) were detected by enzyme-linked immunosorbent assay (ELISA). The binding among messenger RNAs (mRNAs) was assessed by chromatin immunoprecipitation (ChIP), dual luciferase report assay, RNA immunoprecipitation (RIP), co-immunoprecipitation (Co-IP), and RNA pull-down assay, respectively. The cellular localization was observed by fluorescence in situ hybridization (FISH) staining. The level of OIP5-AS1 was upregulated in asthma patients. HDM induced pyroptosis and increased the levels of IL-18, IL-1β, and lactate dehydrogenase (LDH) in BEAS-2B cells, which was obviously reversed by OIP5-AS1 knockdown. Consistently, the expressions of NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), c-caspase 1, and pyroptosis-related gasdermin D-1 (GSDMD-1) in BEAS-2B cells were upregulated by HDM treatment, while these phenomena were partially abolished by silencing of OIP5-AS1. Moreover, HDM promoted the progression of asthma in vivo, which was rescued by the downregulation of OIP5-AS1. OIP5-AS1 silencing decreased HDM-induced cell pyroptosis by inactivation of NLRP3. More importantly, OIP5-AS1 promoted the mRNA stability of yes-associated protein (YAP) via binding with eukaryotic translation initiation factor 4A3 (EIF4A3), and OIP5-AS1 was transcriptionally upregulated by doublesex and mab-3 related transcription factor 3 (DMRT3). DMRT3-mediated OIP5-AS1 aggravated the progression of asthma by mediation of the EIF4A3/YAP axis, which might provide a new therapeutic strategy against asthma.

COMPREHENSIVE MOLECULAR PROFILING OF UVEAL MELANOMA EVALUATED WITH GENE EXPRESSION PROFILING, PREFERENTIALLY EXPRESSED ANTIGEN IN MELANOMA EXPRESSION, AND NEXT-GENERATION SEQUENCING.

To determine the association between gene-expression profiling (GEP), next-generation sequencing (NGS), preferentially expressed antigen in melanoma (PRAME) features, and metastatic risk in patients with uveal melanoma (UM). A retrospective analysis of patients with UM treated by brachytherapy or enucleation by a single ocular oncologist was conducted from November 2020 and July 2022. Clinicopathologic features, patient outcomes, GEP classification, NGS, and PRAME results were recorded. Comprehensive GEP, PRAME, and NGS testing was performed on 135 UMs. The presence of eukaryotic translation initiation factor 1A, X-chromosomal and splicing factor 3B subunit 1 mutations was significantly associated with GEP class 1A and GEP class 1B, respectively. The presence of BRCA- associated protein-1 mutation was significantly associated with GEP class 2. The average largest basal diameter for tumors with eukaryotic translation initiation factor 1A, X-chromosomal mutations was significantly smaller than those with splicing factor 3B subunit 1 mutations and BRCA1-associated protein-1 mutations. Class 2 tumors metastasized sooner than GEP class 1 tumors. Tumors with splicing factor 3B subunit 1 and/or BRCA1-associated protein-1 mutations metastasized sooner compared with tumors that had either no driver mutation or no mutations at all. Tumors with splicing factor 3B subunit 1 did not have a significantly different time to metastasis compared with tumors with BRCA1-associated protein-1 (P value = 0.97). Forty tumors (30%) were PRAME positive, and the remaining 95 tumors (70%) were PRAME negative. Tumors with PRAME-positive status did not have a significantly different time to metastasis compared with tumors without PRAME-positive status (P value = 0.11). GEP, NGS, and PRAME expression analysis help determine different levels of metastatic risk in UM. Although other prognostic tests exist, the following study reports on the use of NGS for metastatic prognostication in UM. However, limitations of NGS exist, especially with small lesions that are technically difficult to biopsy.

Lactobacillus delbrueckii alleviates lipopolysaccharide-induced muscle inflammation and atrophy in weaned piglets associated with inhibition of endoplasmic reticulum stress and protein degradation.

Pro-inflammatory cytokines in muscle play a pivotal role in physiological responses and in the pathophysiology of inflammatory disease and muscle atrophy. Lactobacillus delbrueckii (LD), as a kind of probiotics, has inhibitory effects on pro-inflammatory cytokines associated with various inflammatory diseases. This study was conducted to explore the effect of dietary LD on the lipopolysaccharide (LPS)-induced muscle inflammation and atrophy in piglets and to elucidate the underlying mechanism. A total of 36 weaned piglets (Duroc × Landrace × Large Yorkshire) were allotted into three groups with six replicates (pens) of two piglets: (1) Nonchallenged control; (2) LPS-challenged (LPS); (3) 0.2% LD diet and LPS-challenged (LD+LPS). On d 29, the piglets were injected intraperitoneally with LPS or sterilized saline, respectively. All piglets were slaughtered at 4 h after LPS or saline injection, the blood and muscle samples were collected for further analysis. Our results showed that dietary supplementation of LD significantly attenuated LPS-induced production of pro-inflammatory cytokines IL-6 and TNF-α in both serum and muscle of the piglets. Concomitantly, pretreating the piglets with LD also clearly inhibited LPS-induced nuclear translocation of NF-κB p65 subunits in the muscle, which correlated with the anti-inflammatory effects of LD on the muscle of piglets. Meanwhile, LPS-induced muscle atrophy, indicated by a higher expression of muscle atrophy F-box, muscle RING finger protein (MuRF1), forkhead box O 1, and autophagy-related protein 5 (ATG5) at the transcriptional level, whereas pretreatment with LD led to inhibition of these upregulations, particularly genes for MuRF1 and ATG5. Moreover, LPS-induced mRNA expression of endoplasmic reticulum stress markers, such as eukaryotic translational initiation factor 2α (eIF-2α) was suppressed by pretreatment with LD, which was accompanied by a decrease in the protein expression levels of IRE1α and GRP78. Additionally, LD significantly prevented muscle cell apoptotic death induced by LPS. Taken together, our data indicate that the anti-inflammatory effect of LD supply on muscle atrophy of piglets could be likely regulated by inhibiting the secretion of pro-inflammatory cytokines through the inactivation of the ER stress/NF-κB singling pathway, along with the reduction in protein degradation.

Functional elucidation of the EIF4A3–circR-4225–miR-507–TNFSF11 regulatory axis in LUAD and its role in tumor progression

Lung adenocarcinoma (LUAD) is the most common subtypes of NSCLC. However, the therapeutic effects for LUAD are unsatisfactory at current stage, so it is important to find new molecular targets and therapeutic strategies. circRNAs can regulate the expression of target genes by binding to microRNAs (miRNAs) to form competitive endogenous RNAs (ceRNAs). Therefore, we investigated the functions of circR-4225 in the tumor progression of LUAD and its molecular mechanism in this paper. circR-4225 is up-regulated in LUAD tissues. EIF4A3, a member of the eukaryotic translation initiation factor 4A (EIF4A) family, promotes the expression of circR-4225. circR-4225 acts as a molecular sponge to down-regulate miR-507, which promotes the up-regulation of the expression of its target gene–tumor necrosis factor superfamily member 11 (TNFSF11). Knockdown of circR-4225 in the LUAD cell lines can inhibit cell proliferation and viability, and promote apoptosis of the LUAD cell lines, which can be reverted by inhibiting miR-507 or overexpressing TNFSF11. To sum it up, this study demonstrated that circR-4225 was significantly up-regulated in LUAD tissues, and circR-4225 promoted LUAD progression by sponging miR-507 and up-regulating TNFSF11. This study can provide new molecular targets for early diagnosis and treatment of LUAD.

EIF4A3-induced hsa_circ_0078136 inhibits the tumorigenesis of retinoblastoma via IL-17 signaling pathway.

Retinoblastoma (RB) is one of the most common intraocular cancers, with the highest prevalence among infants and young children under the age five. Numerous findings across the literature illustrate the involvement and significance of circular RNAs (circRNAs) in human malignancies, including RB. The current investigation attempted to decipher the exact roles and underlying mechanisms of a novel circRNA, hsa_circ_0078136, in RB progression. The hsa_circ_0078136 expression was evaluated in RB tumors and cell lines via qRT-PCR. The significance of hsa_circ_0078136 in RB was examined by performing CCK8 assay, transwell assays, western blotting of apoptotic and IL-17 signaling ligand molecules, and a subcutaneous xenograft tumor model. In addition, the interaction of circRNA and eukaryotic translation initiation factor 4A3 (EIF4A3) was determined with bioinformatics, western blot, and RIP assay. The hsa_circ_0078136 expression was reduced in RB tumor samples and cells. Additionally, its overexpression restricted the oncogenic properties of RB cells in vitro. Moreover, hsa_circ_0078136 overexpression lowered the protein levels of cytokine ligand molecules of IL-17 signaling pathway in RB cell lines. In vivo, hsa_circ_0078136 overexpression in subcutaneous tumor xenografts reduced tumor growth. We also observed that EIF4A3 binds to the downstream flanking sequence of hsa_circ_0078136 in the SHRPH pre-mRNA transcript, and EIF4A3 overexpression reduced hsa_circ_0078136 expression, suggesting that EIF4A3 inhibited hsa_circ_0078136 formation. Our results demonstrate that hsa_circ_0078136 is regulated by EIF4A3 and functions as a tumor suppressor via the IL-17 signaling pathway in RB.

EIF4A3-Induced Upregulation of hsa_circ_0049396 Attenuates the Tumorigenesis of Nasopharyngeal Carcinoma by Regulating the Hippo-YAP Pathway.

Circular RNAs (circRNAs) and eukaryotic translation initiation factor 4A3 (EIF4A3) have been reported to participate in the pathogenesis of nasopharyngeal carcinoma (NPC), but their mechanism has not been fully understood. This research aimed to confirm the role and regulatory mechanism of hsa_circ_0049396 interacting with EIF4A3 in NPC tumorigenesis. Quantitative real time polymerase chain reaction (qRT-PCR) was executed to detect the levels of hsa_circ_0049396 and EIF4A3. Cell function experiments and nude mice xenograft assay were used to confirm the role of hsa_circ_0049396 in NPC. The regulatory effect of EIA4A3 on hsa_circ_0049396 was determined by circInteractome prediction, RNA binding protein immunoprecipitation (RIP) assay, and qRT-PCR. In addition, the Hippo-YAP pathway-related proteins and EIF4A3 protein were detected by western blotting. hsa_circ_0049396 was proved to be downregulated in NPC samples, and its low expression indicated the poor prognosis of NPC. After upregulating hsa_circ_0049396 in NPC cells, the proliferation, migration, invasion, and tumor growth invivo were suppressed by inhibiting the Hippo-YAP pathway. Moreover, EIF4A3 bound to the flanking regions of the hsa_circ_0049396 to enhance hsa_circ_0049396 expression in NPC cells. hsa_circ_0049396 mediated by EIF4A3 in NPC can attenuate NPC tumorigenesis by inhibiting the Hippo-YAP pathway. This finding may provide a potential early diagnostic biomarker or drug target to improve the precision medicine approaches of NPC.

Amidino-rocaglates (ADRs), a class of synthetic rocaglates, are potent inhibitors of SARS-CoV-2 replication through inhibition of viral protein synthesis

Coronaviruses are highly transmissible respiratory viruses that cause symptoms ranging from mild congestion to severe respiratory distress. The recent outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has underscored the need for new antivirals with broad-acting mechanisms to combat increasing emergence of new variants. Currently, there are only a few antivirals approved for treatment of SARS-CoV-2. Previously, the rocaglate natural product silvestrol and synthetic rocaglates such as CR-1-31b were shown to have antiviral effects by inhibiting eukaryotic translation initiation factor 4A1 (eIF4A) function and virus protein synthesis. In this study, we evaluated amidino-rocaglates (ADRs), a class of synthetic rocaglates with the most potent eIF4A-inhibitory activity to-date, for inhibition of SARS-CoV-2 infection. This class of compounds showed low nanomolar potency against multiple SARS-CoV-2 variants and in multiple cell types, including human lung-derived cells, with strong inhibition of virus over host protein synthesis and low cytotoxicity. The most potent ADRs were also shown to be active against two highly pathogenic and distantly related coronaviruses, SARS-CoV and MERS-CoV. Mechanistically, cells with mutations of eIF4A1, which are known to reduce rocaglate interaction displayed reduced ADR-associated loss of cellular function, consistent with targeting of protein synthesis. Overall, ADRs and derivatives may offer new potential treatments for SARS-CoV-2 with the goal of developing a broad-acting anti-coronavirus agent.

DHPS-Mediated Hypusination Regulates METTL3 Self-m6A-Methylation Modification to Promote Melanoma Proliferation and the Development of Novel Inhibitors.

Discovering new treatments for melanoma will benefit human health. The mechanism by which deoxyhypusine synthase (DHPS) promotes melanoma development remains elucidated. Multi-omics studies have revealed that DHPS regulates m6A modification and maintains mRNA stability in melanoma cells. Mechanistically, DHPS activates the hypusination of eukaryotic translation initiation factor 5A (eIF5A) to assist METTL3 localizing on its mRNA for m6A modification, then promoting METTL3 expression. Structure-based design, synthesis, and activity screening yielded the hit compound GL-1 as a DHPS inhibitor. Notably, GL-1 directly inhibits DHPS binding to eIF5A, whereas GC-7 cannot. Based on the clarification of the mode of action of GL-1 on DHPS, it is found that GL-1 can promote the accumulation of intracellular Cu2+ to induce apoptosis, and antibody microarray analysis shows that GL-1 inhibits the expression of several cytokines. GL-1 shows promising antitumor activity with good bioavailability in a xenograft tumor model. These findings clarify the molecular mechanisms by which DHPS regulates melanoma proliferation and demonstrate the potential of GL-1 for clinical melanoma therapy.

Analysis of the effect of hypusination in myeloid cells on colitis and colitis-associated cancer

Hypusine is an amino acid synthesized by the enzyme deoxyhypusine synthase (DHPS). It is critical for the activity of eukaryotic translation initiation factor 5A (EIF5A). We reported that hypusination i) in macrophages supports the innate response towards pathogenic bacteria and ii) in epithelial cells maintains intestinal homeostasis. Herein, we investigated the effect of myeloid hypusination on the outcome of colitis and colitis-associated cancer. We found that patients with Crohn's disease exhibit increased levels of DHPS and EIF5AHyp in cells infiltrating the colon lamina propria. However, the specific deletion of Dhps in myeloid cells had no impact on clinical, histological, or inflammatory parameters in mice treated with dextran sulfate sodium (DSS). Further, tumorigenesis and level of dysplasia were not affected by myeloid deletion of Dhps in the azoxymethane-DSS model. The composition of the fecal and the mucosa-associated microbiome was similar in animals lacking or not DHPS in myeloid cells. Thus, hypusination in myeloid cells does not regulate colitis associated with epithelial injury and colitis-associated cancer. Enhancement of the DHPS/hypusine pathway in patients with inflammatory bowel disease could have therapeutic impact through epithelial effects, but modulation of hypusination in myeloid cells will be unlikely to affect the disease.

Inhibition of the eukaryotic initiation factor-2α kinase PERK decreases risk of autoimmune diabetes in mice.

Preventing the onset of autoimmune type 1 diabetes (T1D) is feasible through pharmacological interventions that target molecular stress-responsive mechanisms. Cellular stresses, such as nutrient deficiency, viral infection, or unfolded proteins, trigger the integrated stress response (ISR), which curtails protein synthesis by phosphorylating eukaryotic translation initiation factor-2α (eIF2α). In T1D, maladaptive unfolded protein response (UPR) in insulin-producing β cells renders these cells susceptible to autoimmunity. We found that inhibition of the eIF2α kinase PKR-like ER kinase (PERK), a common component of the UPR and ISR, reversed the mRNA translation block in stressed human islets and delayed the onset of diabetes, reduced islet inflammation, and preserved β cell mass in T1D-susceptible mice. Single-cell RNA-Seq of islets from PERK-inhibited mice showed reductions in the UPR and PERK signaling pathways and alterations in antigen-processing and presentation pathways in β cells. Spatial proteomics of islets from these mice showed an increase in the immune checkpoint protein programmed death-ligand 1 (PD-L1) in β cells. Golgi membrane protein 1, whose levels increased following PERK inhibition in human islets and EndoC-βH1 human β cells, interacted with and stabilized PD-L1. Collectively, our studies show that PERK activity enhances β cell immunogenicity and that inhibition of PERK may offer a strategy for preventing or delaying the development of T1D.

Extracellular vesicle-delivered hsa_circ_0090081 regulated by EIF4A3 enhances gastric cancer tumorigenesis

BackgroundCircular RNA (circRNA) and extracellular vesicles (EVs) in tumors are crucial for the malignant phenotype of tumor cells. Nevertheless, the mechanisms and clinical effects of EV-delivered hsa_circ_0090081 in gastric cancer (GC) are unclear. This study aimed to reveal the effect of eukaryotic translation initiation factor 4A3 (EIF4A3)-mediated hsa_circ_0090081 expression and EV-delivered hsa_circ_0090081 on GC progression.MethodsqRT-PCR was conducted to clarify hsa_circ_0090081 and EIF4A3 levels in GC tissues. Transmission electronic microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blotting identified the EVs isolated from GC cells by ultracentrifugation. The roles of hsa_circ_0090081, EIF4A3, and EV-delivered hsa_circ_0090081 in GC cells were analyzed using Transwell, EdU, and CCK-8 assays. The regulatory role between EIF4A3 and hsa_circ_0090081 was investigated using RIP, qRT-PCR, and Pearson’s analysis.ResultsOur study showed that hsa_circ_0090081 and EIF4A3 were highly expressed in GC, and hsa_circ_0090081 was associated with poor prognosis. Data revealed that hsa_circ_0090081 inhibition restrained GC cell proliferation, invasion, and migration. Additionally, EIF4A3 could bind to the pre-mRNA of PHEX (linear form of hsa_circ_0090081) to enhance hsa_circ_0090081 expression in GC cells. Moreover, EIF4A3 overexpression nullified the malignant phenotypic suppression caused by hsa_circ_0090081 silencing in GC cells. Furthermore, EVs secreted by GC cells delivered hsa_circ_0090081 to facilitate the malignant progression of targeted GC cells.ConclusionThis study showed that hsa_circ_0090081 was enhanced by EIF4A3 to play a promotive role in GC development. The results may help understand the mechanism of EIF4A3 and EV-delivered hsa_circ_0090081 and offer a valuable GC therapeutic target.

Acute exposure to polystyrene nanoplastics induces unfolded protein response and global protein ubiquitination in lungs of mice

Inhaling microplastics (MPs) and nanoplastics (NPs) in the air can damage lung function. Xenobiotics in the body can cause endoplasmic reticulum (ER) stress, and the unfolded protein response (UPR) activation alleviates ER stress. Degradation of unfolded or misfolded proteins is an important pathway for recovering cellular homeostasis. The UPR and protein degradation induced by MPs/NPs in lung tissues are not well understood. Here, we investigated the UPR and protein ubiquitination in the lungs of mice exposed to polystyrene (PS)-NPs and their possible molecular mechanisms leading to protein ubiquitination. Mice were intratracheally administered with 5.6, 17, and 51 mg/kg PS-NPs once for 24 h. Exposure to PS-NPs elevated protein ubiquitination in the lungs of mice in a dose-dependent manner. PS-NPs activated three branches of UPR including inositol-requiring protein 1α (IRE1α), eukaryotic translation initiator factor 2α (eIF2α), and activating transcription factor 6α (ATF6α) in the lungs of mice. However, activated IRE1α did not trigger X-box binding protein 1 (XBP1) mRNA splicing. Exposure to PS-NPs induced an increase in the levels of E3 ubiquitin ligase hydroxymethyl glutaryl-coenzyme A reductase degradation protein 1 (HRD1) and carboxy terminus of Hsc70 interacting protein (CHIP) in the lungs of mice and BEAS-2B cells. ATF6α siRNA inhibited the levels of HRD1 and CHIP proteins induced by PS-NPs in BEAS-2B cells. These results suggest that ATF6α plays a critical role in increasing ubiquitination of unfolded or misfolded proteins by alleviating PS-NPs induced ER stress through UPR to achieve ER homeostasis in the lungs of mice.