Mitofusin 1 and 2 overexpression reduces AßO-mediated ER stress and apoptosis in N2a APPswe cells.

Alzheimer's disease (AD) is the most common neurodegenerative disorder, and amyloid beta oligomers (AßO), which are pathological markers of AD, are known to be highly toxic. AßO increase mitochondrial dysfunction, which is accompanied by a decrease in mitochondrial fusion. Although mitofusin (Mfn) 1 and Mfn2 are mitochondrial fusion proteins, Mfn2 is known to regulate endoplasmic reticulum (ER) function, as it is located in the ER. Several studies have shown that AßO exacerbates ER stress, however, the exact mechanism requires further elucidation. In this study, we used mouse neuroblastoma cells stably overexpressing the amyloid precursor protein (APP) with the Swedish mutation (N2a APPswe cells) to investigate the role of Mfn in ER stress. Our results revealed that  amyloid beta (Aß) caused cellular toxicity in N2a APPswe cells, upregulated ER stress-related proteins, and promoted ER expansion. The AßO-mediated ER stress was reduced when Mfn1 and Mfn2 were overexpressed. Moreover, Mfn1 and Mfn2 overexpressed resulted in reduced apoptosis of N2a APPswe cells. In conclusion, our results indicate that both Mfn1 and Mfn2 reduce ER stress and apoptosis. Our data provide a foundation for future studies on the roles of Mfn1 and Mfn2 in the molecular mechanisms underlying AßO-mediated ER stress and the pathogenesis of AD.

Long non-coding RNA-mediated modulation of endoplasmic reticulum stress under pathological conditions.

Endoplasmic reticulum (ER) stress, which ensues from an overwhelming protein folding capacity, activates the unfolded protein response (UPR) in an effort to restore cellular homeostasis. As ER stress is associated with numerous diseases, it is highly important to delineate the molecular mechanisms governing the ER stress to gain insight into the disease pathology. Long non-coding RNAs, transcripts with a length of over 200 nucleotides that do not code for proteins, interact with proteins and nucleic acids, fine-tuning the UPR to restore ER homeostasis via various modes of actions. Dysregulation of specific lncRNAs is implicated in the progression of ER stress-related diseases, presenting these molecules as promising therapeutic targets. The comprehensive analysis underscores the importance of understanding the nuanced interplay between lncRNAs and ER stress for insights into disease mechanisms. Overall, this review consolidates current knowledge, identifies research gaps and offers a roadmap for future investigations into the multifaceted roles of lncRNAs in ER stress and associated diseases to shed light on their pivotal roles in the pathogenesis of related diseases.

Identification of unique cell type responses in pancreatic islets to stress.

Diabetes involves the death or dysfunction of pancreatic ß-cells. Analysis of bulk sequencing from human samples and studies using in vitro and in vivo models suggest that endoplasmic reticulum and inflammatory signaling play an important role in diabetes progression. To better characterize cell type-specific stress response, we perform multiplexed single-cell RNA sequencing to define the transcriptional signature of primary human islet cells exposed to endoplasmic reticulum and inflammatory stress. Through comprehensive pair-wise analysis of stress responses across pancreatic endocrine and exocrine cell types, we define changes in gene expression for each cell type under different diabetes-associated stressors. We find that ß-, α-, and ductal cells have the greatest transcriptional response. We utilize stem cell-derived islets to study islet health through the candidate gene CIB1, which was upregulated under stress in primary human islets. Our findings provide insights into cell type-specific responses to diabetes-associated stress and establish a resource to identify targets for diabetes therapeutics.

Endoplasmic reticulum stress promotes hepatocellular carcinoma by modulating immunity: a study based on artificial neural networks and single-cell sequencing.

INTRODUCTION: Hepatocellular carcinoma (HCC) is characterized by the complex pathogenesis, limited therapeutic methods, and poor prognosis. Endoplasmic reticulum stress (ERS) plays an important role in the development of HCC, therefore, we still need further study of molecular mechanism of HCC and ERS for early diagnosis and promising treatment targets. METHOD: The GEO datasets (GSE25097, GSE62232, and GSE65372) were integrated to identify differentially expressed genes related to HCC (ERSRGs). Random Forest (RF) and Support Vector Machine (SVM) machine learning techniques were applied to screen ERSRGs associated with endoplasmic reticulum stress, and an artificial neural network (ANN) diagnostic prediction model was constructed. The ESTIMATE algorithm was utilized to analyze the correlation between ERSRGs and the immune microenvironment. The potential therapeutic agents for ERSRGs were explored using the Drug Signature Database (DSigDB). The immunological landscape of the ERSRGs central gene PPP1R16A was assessed through single-cell sequencing and cell communication, and its biological function was validated using cytological experiments. RESULTS: An ANN related to the ERS model was constructed based on SRPX, THBS4, CTH, PPP1R16A, CLGN, and THBS1. The area under the curve (AUC) of the model in the training set was 0.979, and the AUC values in three validation sets were 0.958, 0.936, and 0.970, respectively, indicating high reliability and effectiveness. Spearman correlation analysis suggests that the expression levels of ERSRGs are significantly correlated with immune cell infiltration and immune-related pathways, indicating their potential as important targets for immunotherapy. Mometasone was predicted to be the most promising treatment drug based on its highest binding score. Among the six ERSRGs, PPP1R16A had the highest mutation rate, predominantly copy number mutations, which may be the core gene of the ERSRGs model. Single-cell analysis and cell communication indicated that PPP1R16A is predominantly distributed in liver malignant parenchymal cells and may reshape the tumor microenvironment by enhancing macrophage migration inhibitory factor (MIF)/CD74 + CXCR4 signaling pathways. Functional experiments revealed that after siRNA knockdown, the expression of PPP1R16A was downregulated, which inhibited the proliferation, migration, and invasion capabilities of HCCLM3 and Hep3B cells in vitro. CONCLUSION: The consensus of various machine learning algorithms and artificial intelligence neural networks has established a novel predictive model for the diagnosis of liver cancer associated with ERS. This study offers a new direction for the diagnosis and treatment of HCC.

Comprehensive analysis of endoplasmic reticulum stress related signature in head and neck squamous carcinoma.

Head and neck squamous carcinoma (HNSC) is a prevalent malignant disease, with the majority of patients being diagnosed at an advanced stage. Endoplasmic reticulum stress (ERS) is considered to be a process that promotes tumorigenesis and impacts the tumor microenvironment (TME) in various cancers. The study aims to investigate the predictive value of ERS in HNSC and explore the correlation between ERS-related genes and TME. A series of bioinformatics analyses were carried out based on mRNA and scRNA-seq data from the TCGA and GEO databases. We conducted RT-qPCR and western blot to validate the signature, and performed cell functional experiments to investigate the in vitro biological functions of the gene. We identified 63 ERS-related genes that were associated with outcome and stage in HNSC. A three-gene signature (ATF6, TRIB3, and UBXN6) was developed, which presents predictive value in the prognosis and immunotherapy response of HNSC patients. The high-risk group exhibited a worse prognosis but may benefit from immunotherapy. Furthermore, there was a significant correlation between the signature and immune infiltration. In the high-risk group, fibroblasts were more active in intercellular communication, and more T cells were observed at the end of the sequential phase. The genes in the ERS-related signature were overexpressed in HNSC cells, and the knockdown of TRIB3 significantly inhibited cell proliferation and migration. This study established a novel ERS-related signature that has potential implications for HNSC therapy and the understanding of TME.

Programmed cell death regulator BAP2 is required for IRE1-mediated unfolded protein response in Arabidopsis.

Environmental and physiological situations can challenge the balance between protein synthesis and folding capacity of the endoplasmic reticulum (ER) and cause ER stress, a potentially lethal condition. The unfolded protein response (UPR) restores ER homeostasis or actuates programmed cell death (PCD) when ER stress is unresolved. The cell fate determination mechanisms of the UPR are not well understood, especially in plants. Here, we integrate genetics and ER stress profiling with natural variation and quantitative trait locus analysis of 350 natural accessions of the model species Arabidopsis thaliana. Our analyses implicate a single nucleotide polymorphism to the loss of function of the general PCD regulator BON-ASSOCIATED PROTEIN2 (BAP2) in UPR outcomes. We establish that ER stress-induced BAP2 expression is antagonistically regulated by the UPR master regulator, inositol-requiring enzyme 1 (IRE1), and that BAP2 controls adaptive UPR amplitude in ER stress and ignites pro-death mechanisms in conditions of UPR insufficiency.

A genome-wide CRISPR/Cas9 screen identifies calreticulin as a selective repressor of ATF6α.

Activating transcription factor 6 (ATF6) is one of three endoplasmic reticulum (ER) transmembrane stress sensors that mediate the unfolded protein response (UPR). Despite its crucial role in long-term ER stress adaptation, regulation of ATF6 alpha (α) signalling remains poorly understood, possibly because its activation involves ER-to-Golgi and nuclear trafficking. Here, we generated an ATF6α/Inositol-requiring kinase 1 (IRE1) dual UPR reporter CHO-K1 cell line and performed an unbiased genome-wide CRISPR/Cas9 mutagenesis screen to systematically profile genetic factors that specifically contribute to ATF6α signalling in the presence and absence of ER stress. The screen identified both anticipated and new candidate genes that regulate ATF6α activation. Among these, calreticulin (CRT), a key ER luminal chaperone, selectively repressed ATF6α signalling: Cells lacking CRT constitutively activated a BiP::sfGFP ATF6α-dependent reporter, had higher BiP levels and an increased rate of trafficking and processing of ATF6α. Purified CRT interacted with the luminal domain of ATF6α in vitro and the two proteins co-immunoprecipitated from cell lysates. CRT depletion exposed a negative feedback loop implicating ATF6α in repressing IRE1 activity basally and overexpression of CRT reversed this repression. Our findings indicate that CRT, beyond its known role as a chaperone, also serves as an ER repressor of ATF6α to selectively regulate one arm of the UPR.

The Missense Variant in the Signal Peptide of α-GLA Gene, c.13 A/G, Promotes Endoplasmic Reticular Stress and the Related Pathway's Activation.

Anderson-Fabry disease (AFD) is an X-linked multisystemic disorder with a heterogeneous phenotype, resulting from deficiency of the lysosomal enzyme α-galactosidase A (α-Gal A) and leading to globotriaosylceramide systemic accumulation. Lysosomal storage is not the unique player in organ failure and different mechanisms could drive tissue damage, including endoplasmic reticulum (ER) stress and its related signaling pathway's activation. We identified a new missense variant in the signal peptide of α-GLA gene, c.13 A/G, in a 55-year-old woman affected by chronic kidney disease, acroparesthesia, hypohidrosis, and deafness and exhibiting normal values of lysoGb3 and αGLA activity. The functional study of the new variant performed by its overexpression in HEK293T cells showed an increased protein expression of a key ER stress marker, GRP78, the pro-apoptotic BAX, the negative regulator of cell cycle p21, the pro-inflammatory cytokine, IL1ß, together with pNFkB, and the pro-fibrotic marker, N-cadherin. Transmission electron microscopy showed signs of ER injury and intra-lysosomal inclusions. The proband's PBMC exhibited higher expression of TGFß 1 and pNFkB compared to control. Our findings suggest that the new variant, although it did not affect enzymatic activity, could cause cellular damage by affecting ER homeostasis and promoting apoptosis, inflammation, and fibrosis. Further studies are needed to demonstrate the variant's contribution to cellular and tissue damage.

ER stress-induced transcriptional response reveals tolerance genes in yeast.

Saccharomyces cerevisiae is important for protein secretion studies, yet the complexities of protein synthesis and secretion under endoplasmic reticulum (ER) stress conditions remain not fully understood. ER stress, triggered by alterations in the ER protein folding environment, poses substantial challenges to cells, especially during heterologous protein production. In this study, we used RNA-seq to analyze the transcriptional responses of yeast strains to ER stress induced by reagents such as tunicamycin (Tm) or dithiothreitol (DTT). Our gene expression analysis revealed several crucial genes, such as HMO1 and BIO5, that are involved in ER-stress tolerance. Through metabolic engineering, the best engineered strain R23 with HMO1 overexpression and BIO5 deletion, showed enhanced ER stress tolerance and improved protein folding efficiency, leading to a 2.14-fold increase in α-amylase production under Tm treatment and a 2.04-fold increase in cell density under DTT treatment. Our findings contribute to the understanding of cellular responses to ER stress and provide a basis for further investigations into the mechanisms of ER stress at the cellular level.

M133S mutation possibly involve in the ER stress and mitophagy pathway in maintenance hemodialysis patients with occult hepatitis B infection.

Occult hepatitis B virus infection (OBI) is characterized by the presence of HBV DNA in the absence of detectable HBsAg. OBI is an important risk factor for cirrhosis and hepatocellular carcinoma, but its pathogenesis has not been fully elucidated. Mutations in the HBV preS/S genes can lead to impaired secretion of either HBsAg or S-protein resulting in the accumulation of defective viruses or S protein in cells. In our previous work, the M133S mutation was present in the HBV S gene of maintenance hemodialysis (MHD) patients with OBI. In this study, we investigated the potential role of amino acid substitutions in S proteins in S protein production and secretion through the construction of mutant S gene plasmids, structural prediction, transcriptome sequencing analysis, and in vitro functional studies. Protein structure prediction showed that the S protein M133S mutant exhibited hydrophilic modifications, with greater aggregation and accumulation of the entire structure within the membrane phospholipid bilayer. Differential gene enrichment analysis of transcriptome sequencing data showed that differentially expressed genes were mainly concentrated in protein processing in the endoplasmic reticulum (ER). The expression of heat shock family proteins and ER chaperone molecules was significantly increased in the wild-type and mutant groups, whereas the expression of mitochondria-associated proteins was decreased. Immunofluorescence staining and protein blotting showed that the endoplasmic reticulum-associated protein PDI, the autophagy marker LC3, and the lysosome-associated protein LAMP2 co-localized with the S proteins in the wild-type and mutant strains, and their expression was increased. The mitochondria-associated TOMM20 protein was also co-expressed with the S protein, but expression was significantly reduced in the mutant. The M133S mutation in the S gene is expressed as a defective and misfolded protein that accumulates in the endoplasmic reticulum causing secretion-impaired endoplasmic reticulum stress, which in turn triggers mitochondrial autophagy and recruits lysosomes to fuse with the autophagosome, leading to mitochondrial clearance. This study preliminarily demonstrated that the mutation of M133S in the S gene can cause OBI and is associated with disease progression, providing a theoretical basis for the diagnosis and treatment of OBI.

The Increased Burden of Rare Variants in Four Matrix Metalloproteinase-Related Genes in Childhood Glaucoma Suggests a Complex Genetic Inheritance of the Disease.

Childhood glaucoma encompasses congenital and juvenile primary glaucoma, which are heterogeneous, uncommon, and irreversible optic neuropathies leading to visual impairment with a poorly understood genetic basis. Our goal was to identify gene variants associated with these glaucoma types by assessing the mutational burden in 76 matrix metalloproteinase-related genes. We studied 101 childhood glaucoma patients with no identified monogenic alterations using next-generation sequencing. Gene expression was assessed through immunohistochemistry. Functional analysis of selected gene variants was conducted in cultured cells and in zebrafish. Patients presented a higher proportion of rare variants in four metalloproteinase-related genes, including CPAMD8 and ADAMTSL4, compared to controls. ADAMTSL4 protein expression was observed in the anterior segment of both the adult human and zebrafish larvae's eye, including tissues associated with glaucoma. In HEK-293T cells, expression of four ADAMTSL4 variants identified in this study showed that two variants (p.Arg774Trp and p.Arg98Trp) accumulated intracellularly, inducing endoplasmic reticulum stress. Additionally, overexpressing these ADAMTSL4 variants in zebrafish embryos confirmed partial loss-of-function effects for p.Ser719Leu and p.Arg1083His. Double heterozygous functional suppression of adamtsl4 and cpamd8 zebrafish orthologs resulted in reduced volume of both the anterior eye chamber and lens within the chamber, supporting a genetic interaction between these genes. Our findings suggest that accumulation of partial functional defects in matrix metalloproteinase-related genes may contribute to increased susceptibility to early-onset glaucoma and provide further evidence supporting the notion of a complex genetic inheritance pattern underlying the disease.

[Differential expressions of endoplasmic reticulum stress-associated genes in aortic dissection and their correlation with immune cell infiltration].

OBJECTIVE: To explore differentially expressed endoplasmic reticulum stress-associated genes (ERSAGs) in aortic dissection (AD) and their correlations with immune cell infiltration to identify new therapeutic targets for AD. METHODS: Two AD mRNA expression datasets (GSE190635 and GSE98770) were downloaded from GEO database for analysis of differentially expressed genes between the aorta of AD patients and normal aorta using R software. ERSAGs dataset was downloaded from GeneCards website, and GeneMANIA database was used to analyze the protein-protein interaction network of the differentially expressed ERSAGs and the proteins interacting with these genes. Based on GSE98770 dataset we analyzed the distributions of 22 immune cells within the aortic wall of AD patients using CIBERSORT package of R software. Surgical aortic wall specimens were obtained from 10 AD patients and 10 non-AD patients for detecting AGER mRNA expression using qRTPCR, and the upstream transcriptional factors, miRNAs, and chemicals targeting AGER were analyzed using the TRRUST database and NetworkAnalyst database. RESULTS: Bioinformatic analysis suggested significant differential expression of AGER in AD, which interacted with 20 proteins involved in pattern recognition receptor signaling pathway, positive regulation of DNA-binding transcription factor activity, myeloid leukocyte migration, leukocyte migration, and regulation of the I-κB kinase/NF-κB signaling. In AD, AGER expression level was positively correlated with Treg cell abundance (r=0.59, P < 0.05). The results of qRT-PCR demonstrated significantly lower expression of AGER mRNA in AD than in non-AD patients (1.00±0.30 vs 1.76±0.68, P < 0.05). ROC curve analysis showed that at the cut-off value of 1.335, AGER had an AUC of 0.86 (95% CI: 0.67-1.00, P= 0.0073) for predicting AD. Three transcriptional factors, 3 miRNAs, and 27 chemicals were predicted in the AGER regulatory network. CONCLUSION: AGER is lowly expressed in the aorta of AD patients and may influence the occurrence of AD through Treg cells.

Tazarotene-induced Gene 1 Induces Melanoma Cell Death by Triggering Endoplasmic Reticulum Stress Response.

BACKGROUND: This study investigated the mechanism by which tazarotene-induced gene 1 (TIG1) inhibits melanoma cell growth. The main focus was to analyze downstream genes regulated by TIG1 in melanoma cells and its impact on cell growth. METHODS: The effects of TIG1 expression on cell viability and death were assessed using water-soluble tetrazolium 1 (WST-1) mitochondrial staining and lactate dehydrogenase release assays. RNA sequencing and Western blot analysis were employed to investigate the genes regulated by TIG1 in melanoma cells. Additionally, the correlation between TIG1 expression and its downstream genes was analyzed in a melanoma tissue array. RESULTS: TIG1 expression in melanoma cells was associated with decreased cell viability and increased cell death. RNA-sequencing (RNA-seq), quantitative reverse transcription PCR (reverse RT-QPCR), and immunoblots revealed that TIG1 expression induced the expression of Endoplasmic Reticulum (ER) stress response-related genes such as Homocysteine-responsive endoplasmic reticulum-resident ubiquitin-like domain member 1 (HERPUD1), Binding immunoglobulin protein (BIP), and DNA damage-inducible transcript 3 (DDIT3). Furthermore, analysis of the melanoma tissue array revealed a positive correlation between TIG1 expression and the expression of HERPUD1, BIP, and DDIT3. Additionally, attenuation of the ER stress response in melanoma cells weakened the impact of TIG1 on cell growth. CONCLUSIONS: TIG1 expression effectively hinders the growth of melanoma cells. TIG1 induces the upregulation of ER stress response-related genes, leading to an increase in caspase-3 activity and subsequent cell death. These findings suggest that the ability of retinoic acid to prevent melanoma formation may be associated with the anticancer effect of TIG1.

Hypoxia in the Blue Mussel Mytilus chilensis Induces a Transcriptome Shift Associated with Endoplasmic Reticulum Stress, Metabolism, and Immune Response.

The increase in hypoxia events, a result of climate change in coastal and fjord ecosystems, impacts the health and survival of mussels. These organisms deploy physiological and molecular responses as an adaptive mechanism to maintain cellular homeostasis under environmental stress. However, the specific effects of hypoxia on mussels of socioeconomic interest, such as Mytilus chilensis, are unknown. Using RNA-seq, we investigated the transcriptomic profiles of the gills, digestive gland, and adductor muscle of M. chilensis under hypoxia (10 days at 2 mg L-1) and reoxygenation (10 days at 6 mg L-1). There were 15,056 differentially expressed transcripts identified in gills, 11,864 in the digestive gland, and 9862 in the adductor muscle. The response varied among tissues, showing chromosomal changes in Chr1, Chr9, and Chr10 during hypoxia. Hypoxia regulated signaling genes in the Toll-like, mTOR, citrate cycle, and apoptosis pathways in gills, indicating metabolic and immunological alterations. These changes suggest that hypoxia induced a metabolic shift in mussels, reducing reliance on aerobic respiration and increasing reliance on anaerobic metabolism. Furthermore, hypoxia appeared to suppress the immune response, potentially increasing disease susceptibility, with negative implications for the mussel culture industry and natural bed populations. This study provides pivotal insights into metabolic and immunological adaptations to hypoxia in M. chilensis, offering candidate genes for adaptive traits.

Expression of Stress-Induced Genes in Bronchoalveolar Lavage Cells and Lung Fibroblasts from Healthy and COPD Subjects.

Chronic obstructive pulmonary disease (COPD) is commonly caused from smoking cigarettes that induce biological stress responses. Previously we found disorganized endoplasmic reticulum (ER) in fibroblasts from COPD with different responses to chemical stressors compared to healthy subjects. Here, we aimed to investigate differences in stress-related gene expressions within lung cells from COPD and healthy subjects. Bronchoalveolar lavage (BAL) cells were collected from seven COPD and 35 healthy subjects. Lung fibroblasts were derived from 19 COPD and 24 healthy subjects and exposed to cigarette smoke extract (CSE). Gene and protein expression and cell proliferation were investigated. Compared to healthy subjects, we found lower gene expression of CHOP in lung fibroblasts from COPD subjects. Exposure to CSE caused inhibition of lung fibroblast proliferation in both groups, though the changes in ER stress-related gene expressions (ATF6, IRE1, PERK, ATF4, CHOP, BCL2L1) and genes relating to proteasomal subunits mostly occurred in healthy lung fibroblasts. No differences were found in BAL cells. In this study, we have found that lung fibroblasts from COPD subjects have an atypical ER stress gene response to CSE, particularly in genes related to apoptosis. This difference in response to CSE may be a contributing factor to COPD progression.

Identification of endoplasmic reticulum stress genes in human stroke based on bioinformatics and machine learning.

After ischemic stroke (IS), secondary injury is intimately linked to endoplasmic reticulum (ER) stress and body-brain crosstalk. Nonetheless, the underlying mechanism systemic immune disorder mediated ER stress in human IS remains unknown. In this study, 32 candidate ER stress-related genes (ERSRGs) were identified by overlapping MSigDB ER stress pathway genes and DEGs. Three Key ERSRGs (ATF6, DDIT3 and ERP29) were identified using LASSO, random forest, and SVM-RFE. IS patients with different ERSRGs profile were clustered into two groups using consensus clustering and the difference between 2 group was further explored by GSVA. Through immune cell infiltration deconvolution analysis, and middle cerebral artery occlusion (MCAO) mouse scRNA analysis, we found that the expression of 3 key ERSRGs were closely related with peripheral macrophage cell ER stress in IS and this was further confirmed by RT-qPCR experiment. These ERS genes might be helpful to further accurately regulate the central nervous system and systemic immune response through ER stress and have potential application value in clinical practice in IS.

Endoplasmic reticulum stress-dependent regulation of the expression of serine hydroxymethyltransferase 2 in glioblastoma cells.

Objective. Serine hydroxymethyltransferase (SHMT2) plays a multifunctional role in mitochondria (folate-dependent tRNA methylation, translation, and thymidylate synthesis). The endoplasmic reticulum stress, hypoxia, and glucose and glutamine supply are significant factors of malignant tumor growth including glioblastoma. Previous studies have shown that the knockdown of the endoplasmic reticulum to nucleus signaling 1 (ERN1) pathway of endoplasmic reticulum stress strongly suppressed glioblastoma cell proliferation and modified the sensitivity of these cells to hypoxia and glucose or glutamine deprivations. The present study aimed to investigate the regulation of the SHMT2 gene in U87MG glioblastoma cells by ERN1 knockdown, hypoxia, and glucose or glutamine deprivations with the intent to reveal the role of ERN1 signaling in sensitivity of this gene expression to hypoxia and nutrient supply. Methods. The control U87MG glioblastoma cells (transfected by an empty vector) and ERN1 knockdown cells with inhibited ERN1 endoribonuclease and protein kinase (dnERN1) or only ERN1 endoribonuclease (dnrERN1) were used. Hypoxia was introduced by dimethyloxalylglycine (500 ng/ml for 4 h). For glucose and glutamine deprivations, cells were exposed in DMEM without glucose and glutamine, respectively for 16 h. RNA was extracted from cells and reverse transcribed. The expression level of the SHMT2 gene was studied by real-time qPCR and normalized to ACTB. Results. It was found that inhibition of ERN1 endoribonuclease and protein kinase in glioblastoma cells led to a down-regulation of SHMT2 gene expression in U87MG cells. At the same time, the expression of this gene did not significantly change in cells with inhibited ERN1 endoribonuclease, but tunicamycin strongly increased its expression. Moreover, the expression of the SHMT2 gene was not affected in U87MG cells after silencing of XBP1. Hypoxia up-regulated the expression level of the SHMT2 gene in both control and ERN1 knockdown U87MG cells. The expression of this gene was significantly up-regulated in glioblastoma cells under glucose and glutamine deprivations and ERN1 knockdown significantly increased the sensitivity of the SHMT2 gene to these nutrient deprivation conditions. Conclusion. The results of the present study demonstrate that the expression of the SHMT2 gene responsible for serine metabolism and formation of folate one-carbon is controlled by ERN1 protein kinase and induced by hypoxia as well as glutamine and glucose deprivation conditions in glioblastoma cells and reflects the ERN1-mediated reprogramming of sensitivity this gene expression to nutrient deprivation.

MicroRNA-15b promotes cardiac ischemia injury by the inhibition of Mitofusin 2/PERK pathway.

MicroRNA and mitofusin-2 (Mfn2) play an important role in the myocardial apoptosis induced by acute myocardial infarction (AMI). However, the target relationship and underlying mechanism associated with interorganelle interaction between endoplasmic reticulum (ER) and mitochondria under ischemic condition is not completely clear. MI-induced injury, Mfn2 expression, Mfn2-mediated mitochondrial function and ER stress, and target regulation by miRNA-15b (miR-15b) were evaluated by animal MI and cellular hypoxic models with advanced molecular techniques. The results confirmed that Mfn2 was down-regulated and miR-15b was up-regulated upon the target binding profile under ischemic/hypoxic condition. Our data showed that miR-15b caused cardiac apoptotic injury that was reversed by rAAV9-anti-miR-15b or AMO-15b. The damage effect of miR-15b on Mfn2 expression and mitochondrial function was observed and rescued by rAAV9-anti-miR-15b or AMO-15b. The targeted regulation of miR-15b on Mfn2 was verified by luciferase reporter and microRNA-masking. Importantly, miR-15b-mediated Mfn2 suppression activated PERK/CHOP pathway, by which leads to ER stress and mitochondrial dysfunction, and cardiac apoptosis eventually. In conclusion, our research, for the first time, revealed the missing molecular link in Mfn2 and apoptosis and elucidated that pro-apoptotic miR-15b plays crucial roles during the pathogenesis of AMI through down-regulation of Mfn2 and activation of PERK-mediated ER stress. These findings may provide an opportunity to develop new therapies for prophylaxis and treatment of ischemic heart disease.

AMELX Mutations and Genotype-Phenotype Correlation in X-Linked Amelogenesis Imperfecta.

AMELX mutations cause X-linked amelogenesis imperfecta (AI), known as AI types IE, IIB, and IIC in Witkop's classification, characterized by hypoplastic (reduced thickness) and/or hypomaturation (reduced hardness) enamel defects. In this study, we conducted whole exome analyses to unravel the disease-causing mutations for six AI families. Splicing assays, immunoblotting, and quantitative RT-PCR were conducted to investigate the molecular and cellular effects of the mutations. Four AMELX pathogenic variants (NM_182680.1:c.2T>C; c.29T>C; c.77del; c.145-1G>A) and a whole gene deletion (NG_012494.2:g.307534_403773del) were identified. The affected individuals exhibited enamel malformations, ranging from thin, poorly mineralized enamel with a "snow-capped" appearance to severe hypoplastic defects with minimal enamel. The c.145-1G>A mutation caused a -1 frameshift (NP_001133.1:p.Val35Cysfs*5). Overexpression of c.2T>C and c.29T>C AMELX demonstrated that mutant amelogenin proteins failed to be secreted, causing elevated endoplasmic reticulum stress and potential cell apoptosis. This study reveals a genotype-phenotype relationship for AMELX-associated AI: While amorphic mutations, including large deletions and 5' truncations, of AMELX cause hypoplastic-hypomaturation enamel with snow-capped teeth (AI types IIB and IIC) due to a complete loss of gene function, neomorphic variants, including signal peptide defects and 3' truncations, lead to severe hypoplastic/aplastic enamel (AI type IE) probably caused by "toxic" cellular effects of the mutant proteins.

RNASeq highlights ATF6 pathway regulators for CHO cell engineering with different impacts of ATF6ß and WFS1 knockdown on fed-batch production of IgG1.

Secretion levels required of industrial Chinese hamster ovary (CHO) cell lines can challenge endoplasmic reticulum (ER) homeostasis, and ER stress caused by accumulation of misfolded proteins can be a bottleneck in biomanufacturing. The unfolded protein response (UPR) is initiated to restore homeostasis in response to ER stress, and optimization of the UPR can improve CHO cell production of therapeutic proteins. We compared the fed-batch growth, production characteristics, and transcriptomic response of an immunoglobulin G1 (IgG1) producer to its parental, non-producing host cell line. We conducted differential gene expression analysis using high throughput RNA sequencing (RNASeq) and quantitative polymerase chain reaction (qPCR) to study the ER stress response of each cell line during fed-batch culture. The UPR was activated in the IgG1 producer compared to the host cell line and our analysis of differential expression profiles indicated transient upregulation of ATF6α target mRNAs in the IgG1 producer, suggesting two upstream regulators of the ATF6 arm of the UPR, ATF6ß and WFS1, are rational engineering targets. Although both ATF6ß and WFS1 have been reported to negatively regulate ATF6α, this study shows knockdown of either target elicits different effects in an IgG1-producing CHO cell line. Stable knockdown of ATF6ß decreased cell growth without decreasing titer; however, knockdown of WFS1 decreased titer without affecting growth. Relative expression measured by qPCR indicated no direct relationship between ATF6ß and WFS1 expression, but upregulation of WFS1 in one pool was correlated with decreased growth and upregulation of ER chaperone mRNAs. While knockdown of WFS1 had negative impacts on UPR activation and product mRNA expression, knockdown of ATF6ß improved the UPR specifically later in fed-batch leading to increased overall productivity.