Endoplasmic Reticulum Stress Response Pathway Research Articles

Mouse serum albumin induces neuronal apoptosis and tauopathies

The elderly frequently present impaired blood–brain barrier which is closely associated with various neurodegenerative diseases. However, how the albumin, the most abundant protein in the plasma, leaking through the disrupted BBB, contributes to the neuropathology remains poorly understood. We here demonstrated that mouse serum albumin-activated microglia induced astrocytes to A1 phenotype to remarkably increase levels of Elovl1, an astrocytic synthase for very long-chain saturated fatty acids, significantly promoting VLSFAs secretion and causing neuronal lippoapoptosis through endoplasmic reticulum stress response pathway. Moreover, MSA-activated microglia triggered remarkable tau phosphorylation at multiple sites through NLRP3 inflammasome pathway. Intracerebroventricular injection of MSA into the brains of C57BL/6J mice to a similar concentration as in patient brains induced neuronal apoptosis, neuroinflammation, increased tau phosphorylation, and decreased the spatial learning and memory abilities, while Elovl1 knockdown significantly prevented the deleterious effect of MSA. Overall, our study here revealed that MSA induced tau phosphorylation and neuron apoptosis based on MSA-activated microglia and astrocytes, respectively, showing the critical roles of MSA in initiating the occurrence of tauopathies and cognitive decline, and providing potential therapeutic targets for MSA-induced neuropathology in multiple neurodegenerative disorders.

Palmitate induces integrated stress response and lipoapoptosis in trophoblasts

Maternal obesity increases the risk of childhood obesity and programs the offspring to develop metabolic syndrome later in their life. Palmitate is the predominant saturated free fatty acid (FFA) that is transported across the placenta to the fetus. We have recently shown that saturated FFA in the maternal circulation as a result of increased adipose tissue lipolysis in third trimester of pregnancy induces trophoblast lipoapoptosis. Here, we hypothesized that palmitate induces integrated stress response by activating mitogen-activated protein kinases (MAPKs), endoplasmic reticulum (ER) stress and granular stress and lipoapoptosis in trophoblasts. Choriocarcinoma-derived third-trimester placental trophoblast-like cells (JEG-3 and JAR) referred as trophoblasts were exposed to various concentrations of palmitate (PA). Apoptosis was assessed by nuclear morphological changes and caspase 3/7 activity. Immunoblot and immunofluorescence analysis was performed to measure the activation of MAPKs, ER stress and granular stress response pathways. Trophoblasts exposed to pathophysiological concentrations of PA showed a concentration-dependent increase in trophoblast lipoapoptosis. PA induces a caspase-dependent trophoblast lipoapoptosis. Further, PA induces MAPK activation (JNK and ERK) via phosphorylation, and activation of ER stress as evidenced by an increased phosphorylation eIF2α & IRE1α. PA also induces the activation of stress granules formation. Two pro-apoptotic transcriptional mediators of PA-induced trophoblast lipoapoptosis, CHOP and FoxO3 have increased nuclear translocation. Mechanistically, PA-induced JNK is critical for trophoblast lipoapoptosis. However, PA-induced activation of ERK and stress granule formation were shown to be cell survival signals to combat subcellular stress due to PA exposure. In conclusion, PA induces the activation of integrated stress responses, among which small molecule inhibition of JNK demonstrated that activation of JNK is critical for PA-induced trophoblast lipoapoptosis and small molecule activation of stress granule formation significantly prevents PA-induced trophoblast lipoapoptosis.

Proposed mechanisms of tau: relationships to traumatic brain injury, Alzheimer's disease, and epilepsy.

Traumatic brain injury (TBI), Alzheimer's disease (AD), and epilepsy share proposed mechanisms of injury, including neuronal excitotoxicity, cascade signaling, and activation of protein biomarkers such as tau. Although tau is typically present intracellularly, in tauopathies, phosphorylated (p-) and hyper-phosphorylated (hp-) tau are released extracellularly, the latter leading to decreased neuronal stability and neurofibrillary tangles (NFTs). Tau cleavage at particular sites increases susceptibility to hyper-phosphorylation, NFT formation, and eventual cell death. The relationship between tau and inflammation, however, is unknown. In this review, we present evidence for an imbalanced endoplasmic reticulum (ER) stress response and inflammatory signaling pathways resulting in atypical p-tau, hp-tau and NFT formation. Further, we propose tau as a biomarker for neuronal injury severity in TBI, AD, and epilepsy. We present a hypothesis of tau phosphorylation as an initial acute neuroprotective response to seizures/TBI. However, if the underlying seizure pathology or TBI recurrence is not effectively treated, and the pathway becomes chronically activated, we propose a "tipping point" hypothesis that identifies a transition of tau phosphorylation from neuroprotective to injurious. We outline the role of amyloid beta (Aβ) as a "last ditch effort" to revert the cell to programmed death signaling, that, when fails, transitions the mechanism from injurious to neurodegenerative. Lastly, we discuss targets along these pathways for therapeutic intervention in AD, TBI, and epilepsy.

Antiviral effects of micafungin against pteropine orthoreovirus, an emerging zoonotic virus carried by bats

Bat-borne emerging zoonotic viruses cause major outbreaks, such as the Ebola virus, Nipah virus, and/or beta coronavirus. Pteropine orthoreovirus (PRV), whose spillover event occurred from fruits bats to humans, causes respiratory syndrome in humans widely in South East Asia. Repurposing approved drugs against PRV is an effective tool to confront future PRV pandemics. We screened 2,943 compounds in an FDA-approved drug library and identified eight hit compounds that reduce viral cytopathic effects on cultured Vero cells. Real-time quantitative PCR analysis revealed that six of eight hit compounds significantly inhibited PRV replication. Among them, micafungin used clinically as an antifungal drug, displayed a prominent antiviral effect on PRV. Secondly, the antiviral effects of micafungin on PRV infected human cell lines (HEK293T and A549), and their transcriptome changes by PRV infection were investigated, compared to four different bat-derived cell lines (FBKT1 (Ryukyu flying fox), DEMKT1 (Leschenault's rousette), BKT1 (Greater horseshoe bat), YUBFKT1 (Eastern bent-wing bats)). In two human cell lines, unlike bat cells that induce an IFN-γ response pathway, an endoplasmic reticulum stress response pathway was commonly activated. Additionally, micafungin inhibits viral release rather than suppressing PRV genome replication in human cells, although it was disturbed in Vero cells. The target of micafungin's action may vary depending on the animal species, but it must be useful for human purposes as a first choice of medical care.

Establishment of an endoplasmic reticulum stress-associated lncRNAs model to predict prognosis and immunological characteristics in hepatocellular carcinoma.

The endoplasmic reticulum stress (ERS) and unfolded protein response (UPR) pathways play an essential role in the pathophysiology of hepatocellular carcinoma (HCC), and activation of the UPR pathway is strongly associated with tumor growth. However, the function of ERS-associated long non-coding RNAs (lncRNAs) in HCC is less recognized. We have used TCGA (The Cancer Genome Atlas) to obtain clinical and transcriptome data for HCC patients and the GSEA (Gene Set Enrichment Analysis) molecular signature database to get the ERS gene. ERS-associated prognostic lncRNA was determined using univariate Cox regression study. Then, least absolute shrinkage and selection operator and multivariate Cox regression study were used to construct ERS-associated lncRNAs risk model. Next, we use Kaplan-Meier (KM) survival study, time-dependent receiver operating characteristic (ROC) curve, univariate and multivariate Cox regression study to validate and evaluate the risk model. GSEA reveals the underlying molecular mechanism of the risk model. In addition, differences in Immune cell Infiltration Study, half-maximal inhibitory concentration (IC50) and immune checkpoints blockade (ICB) treatment between high and low risk groups were analyzed. We constructed a risk model consisting of 6 ERS-associated lncRNAS (containingMKLN1-AS, LINC01224, AL590705.3, AC008622.2, AC145207.5, and AC026412.3). The KM survival study showed that the prognosis of HCC patients in low-risk group was better than that in high-risk group. ROC study, univariate and multivariate Cox regression study showed that the risk model had good predictive power for HCC patients. Our verification sample verified the aforesaid findings. GSEA suggests that several tumor- and metabolism-related signaling pathways are associated with risk groups. Simultaneously, we discovered that the risk models may help in the treatment of ICB and the selection of chemotherapeutic drugs. In this article, we created an ERS-associated lncRNAs risk model to help prognostic diagnosis and personalized therapy in HCC.

Abstract 6306: Pharmacological deuteration of SCID mice using the water-isotopologue deuterium oxide (D2O) inhibits tumor growth in bioluminescent models of human malignant melanoma and pancreatic ductal adenocarcinoma

Abstract Since its initial discovery as a natural isotopologue of dihydrogen oxide (1H2O), extensive research has focused on the biophysical, biochemical, and pharmacological effects of deuterated water [2H2O (D2O, also referred to as ‘heavy water’)]. Here, using diverse panels of cultured human malignant melanoma and pancreatic ductal adenocarcinoma (PDAC) cells we have profiled (i) D2O-induced phenotypic anti-proliferative and apoptogenic effects, (ii) redox- and proteotoxicity-directed stress response gene expression, and (iii) phosphoprotein-signaling related to endoplasmic reticulum (ER) and MAP-kinase stress response pathways. Differential RT-qPCR array analysis revealed early modulation of stress response gene expression elicited by D2O (90%; ≤6 h) confirmed by independent RT-qPCR analysis. Immunoblot-analysis revealed rapid (< 6 h) onset of D2O-induced MAP-kinase signaling (p-JNK) together with ER stress response upregulation (p-eIF2α, ATF4, XBP1s, DDIT3/CHOP) attributable to deuteration-induced alteration of hydrogen bonding triggering proteotoxic stress. Next, we tested the chemotherapeutic efficacy of D2O-based drinking water supplementation in bioluminescent murine models of malignancy (A375-luc melanoma and BxPC-3-luc orthotopic PDAC xenografts in SCID mice). Time course of systemic deuteration (30% D2O in drinking water fed continuously for up to 21 days) was established using time-resolved whole-body proton magnetic resonance imaging (MRI) and isotope-ratio mass spectrometry (IR-MS)-based plasma (D/H)-analysis. In both models, D2O supplementation significantly suppressed tumor growth and metastasis with downregulated expression of PCNA/Ki67 while increasing tumor levels of DDIT3/CHOP, HO-1, and p-eIF2α. Taken together, these data demonstrate for the first time that pharmacological induction of systemic deuteration by oral administration of D2O, associated with induction of cellular ER stress, reduces tumor burden and metastasis in murine models of human malignant melanoma and PDAC. Citation Format: Jana Jandova, Jean-Philippe Galons, David L. Dettman, Georg T. Wondrak. Pharmacological deuteration of SCID mice using the water-isotopologue deuterium oxide (D2O) inhibits tumor growth in bioluminescent models of human malignant melanoma and 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 6306.

The p53 endoplasmic reticulum stress-response pathway evolved in humans but not in mice via PERK-regulated p53 mRNA structures

Cellular stress conditions activate p53-dependent pathways to counteract the inflicted damage. To achieve the required functional diversity, p53 is subjected to numerous post-translational modifications and the expression of isoforms. Little is yet known how p53 has evolved to respond to different stress pathways. The p53 isoform p53/47 (p47 or ΔNp53) is linked to aging and neural degeneration and is expressed in human cells via an alternative cap-independent translation initiation from the 2nd in-frame AUG at codon 40 (+118) during endoplasmic reticulum (ER) stress. Despite an AUG codon in the same location, the mouse p53 mRNA does not express the corresponding isoform in either human or mouse-derived cells. High-throughput in-cell RNA structure probing shows that p47 expression is attributed to PERK kinase-dependent structural alterations in the human p53 mRNA, independently of eIF2α. These structural changes do not take place in murine p53 mRNA. Surprisingly, PERK response elements required for the p47 expression are located downstream of the 2nd AUG. The data show that the human p53 mRNA has evolved to respond to PERK-mediated regulation of mRNA structures in order to control p47 expression. The findings highlight how p53 mRNA co-evolved with the function of the encoded protein to specify p53-activities under different cellular conditions.

Potential roles of the endoplasmic reticulum stress pathway in amyotrophic lateral sclerosis.

The endoplasmic reticulum (ER) is a major organelle involved in protein quality control and cellular homeostasis. ER stress results from structural and functional dysfunction of the organelle, along with the accumulation of misfolded proteins and changes in calcium homeostasis, it leads to ER stress response pathway such as unfolded protein response (UPR). Neurons are particularly sensitive to the accumulation of misfolded proteins. Thus, the ER stress is involved in neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, prion disease and motor neuron disease (MND). Recently, the complex involvement of ER stress pathways has been demonstrated in experimental models of amyotrophic lateral sclerosis (ALS)/MND using pharmacological and genetic manipulation of the unfolded protein response (UPR), an adaptive response to ER stress. Here, we aim to provide recent evidence demonstrating that the ER stress pathway is an essential pathological mechanism of ALS. In addition, we also provide therapeutic strategies that can help treat diseases by targeting the ER stress pathway.

Runx1/3-driven adaptive endoplasmic reticulum stress pathways contribute to neurofibromagenesis.

Neurofibromatosis type 1 (NF1) patients are predisposed to develop plexiform neurofibromas (PNFs). Three endoplasmic reticulum (ER) stress response pathways restore cellular homeostasis. The unfolded protein response (UPR) sensors contribute to tumor initiation in many cancers. We found that all three UPR pathways were activated in mouse and human PNFs, with protein kinase RNA [PKR]-like ER kinase (PERK) the most highly expressed. We tested if neurofibroma cells adapt to ER stress, leading to their growth. Pharmacological or genetic inhibition of PERK reduced mouse neurofibroma-sphere number, and genetic inhibition in PERK in Schwann cell precursors (SCPs) decreased tumor-like lesion numbers in a cell transplantation model. Further, in a PNF mouse model, deletion of PERK in Schwann cells (SCs) and SCPs reduced tumor size, number, and increased survival. Mechanistically, loss of Nf1 activated PERK-eIF2α-ATF4 signaling and increased ATF4 downstream target gene p21 translocation from nucleus to cytoplasm. This nucleus-cytoplasm translocation was mediated by exportin-1. Runx transcriptionally activated ribosome gene expression and increased protein synthesis to allow SCs to adapt to ER stress and tumor formation. We propose that targeting proteostasis might provide cytotoxic and/or potentially durable novel therapy for PNFs.

Systemic deuteration of SCID mice using the water-isotopologue deuterium oxide (D2 O) inhibits tumor growth in an orthotopic bioluminescent model of human pancreatic ductal adenocarcinoma.

Since its initial discovery as a natural isotopologue of dihydrogen oxide (1 H2 O), extensive research has focused on the biophysical, biochemical, and pharmacological effects of deuterated water (2 H2 O [D2 O, also referred to as "heavy water"]). Using a panel of cultured human pancreatic ductal adenocarcinoma (PDAC) cells we have profiled (i) D2 O-induced phenotypic antiproliferative and apoptogenic effects, (ii) redox- and proteotoxicity-directed stress response gene expression, and (iii) phosphoprotein-signaling related to endoplasmic reticulum (ER) and MAP-kinase stress response pathways. Differential array analysis revealed early modulation of stress response gene expression in both BxPC-3 and PANC-1 PDAC cells elicited by D2 O (90%; ≤6 h; upregulated: HMOX1, NOS2, CYP2E1, CRYAB, DDIT3, NFKBIA, PTGS1, SOD2, PTGS2; downregulated: RUNX1, MYC, HSPA8, HSPA1A) confirmed by independent RT-qPCR analysis. Immunoblot-analysis revealed rapid (≤6 h) onset of D2 O-induced MAP-kinase signaling (p-JNK, p-p38) together with ER stress response upregulation (p-eIF2α, ATF4, XBP1s, DDIT3/CHOP). Next, we tested the chemotherapeutic efficacy of D2 O-based drinking water supplementation in an orthotopic PDAC model employing firefly luciferase-expressing BxPC-3-FLuc cells in SCID mice. First, feasibility and time course of systemic deuteration (30% D2 O in drinking water; 21 days) were established using time-resolved whole-body proton magnetic resonance imaging and isotope-ratio mass spectrometry-based plasma (D/H)-analysis. D2 O-supplementation suppressed tumor growth by almost 80% with downregulated expression of PCNA, MYC, RUNX1, and HSP70 while increasing tumor levels of DDIT3/CHOP, HO-1, and p-eIF2α. Taken together, these data demonstrate for the first time that pharmacological induction of systemic deuteration significantly reduces orthotopic tumor burden in a murine PDAC xenograft model.

Acute Pharmacological Inhibition of Protein Kinase R-Like Endoplasmic Reticulum Kinase Signaling After Spinal Cord Injury Spares Oligodendrocytes and Improves Locomotor Recovery.

Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) is a major signal transducer of the endoplasmic reticulum stress response (ERSR) pathway. Outcomes of PERK activation range from abrogating ER stress to induction of cell death, dependent on its level, duration, and cellular context. Current data demonstrate that after mouse spinal cord injury (SCI), acute inhibition of PERK (0-72 h) with the small molecule inhibitor GSK2656157 reduced ERSR while improving white matter sparing and hindlimb locomotion recovery. GSK2656157-treated mice showed increased numbers of oligodendrocytes at the injury epicenter. Moreover, GSK2656157 protected cultured primary mouse oligodendrocyte precursor cells from ER stress-induced cytotoxicity. These findings suggest that in the context of SCI, excessive acute activation of PERK contributes to functionally relevant white matter damage. Pharmacological inhibition of PERK is a potential strategy to protect central nervous system (CNS) white matter following acute injuries, including SCI.

African Swine Fever Virus Host-Pathogen Interactions.

African swine fever virus is a complex double-stranded DNA virus that exhibits tropism for cells of the mononuclear phagocytic system. Virus replication is a multi-step process that involves the nucleus of the host cell as well the formation of large perinuclear sites where progeny virions are assembled prior to transport to, and budding through, the plasma membrane. Like many viruses, African swine fever virus reorganises the cellular architecture to facilitate its replication and has evolved multiple mechanisms to avoid the potential deleterious effects of host cell stress response pathways. However, how viral proteins and virus-induced structures trigger cellular stress pathways and manipulate the subsequent responses is still relatively poorly understood. African swine fever virus alters nuclear substructures, modulates autophagy, apoptosis and the endoplasmic reticulum stress response pathways. The viral genome encodes for at least 150 genes, of which approximately 70 are incorporated into the virion. Many of the non-structural genes have not been fully characterised and likely play a role in host range and modifying immune responses. As the field moves towards approaches that take a broader view of the effect of expression of individual African swine fever genes, we summarise how the different steps in virus replication interact with the host cell and the current state of knowledge on how it modulates the resulting stress responses.

A KO mouse model for the lncRNA Lhx1os produces motor neuron alterations and locomotor impairment

Here, we describe a conserved motor neuron-specific long non-coding RNA, Lhx1os, whose knockout in mice produces motor impairment and postnatal reduction of mature motor neurons (MNs). The ER stress-response pathway result specifically altered with the downregulation of factors involved in the unfolded protein response (UPR). Lhx1os was found to bind the ER-associated PDIA3 disulfide isomerase and to affect the expression of the same set of genes controlled by this protein, indicating that the two factors act in conjunction to modulate the UPR. Altogether, the observed phenotype and function of Lhx1os indicate its important role in the control of MN homeostasis and function.

Therapeutic Repurposing of Brincidofovir in Natural Killer/T-Cell Lymphoma Reveals Potent Induction of Replication Stress, Sting Pathway Activation and Immunogenic Cell Death

Introduction Natural killer/T-cell lymphoma (NKTCL) is an aggressive subtype of non-Hodgkin lymphoma associated with Epstein-Barr virus (EBV) infection. Brincidofovir (BCV), a lipid conjugated form of cidofovir, is a biologically active acyclic nucleoside phosphonate known for both antiviral and antiproliferative properties. Methods We investigated BCV in a panel of NKTCL cell lines (n=11) and mouse xenograft models. The mechanism of action was examined using bulk and single cell RNA sequencing (10X Chromium 3' Gene Expression Profiling), and validated in orthogonal assays. Results BCV evoked a dose- and time-dependent inhibition of cell viability in all NKTCL cell lines, independent of EBV presence. Exquisite sensitivity was observed in 4 cell lines KAI-3, NK-S1, NK-92 and KHYG-1 (IC50 range, 36.0 to 303.6 ng/ml), accompanied by increase in sub-G1 fraction and S-phase arrest on cell cycle analyses. Bulk RNA sequencing of BCV-treated cell lines (KAI-3 and NK-S1) revealed pervasive downregulation of MYC target pathways, along with upregulation of immune-related and DNA repair pathways. Single cell RNA sequencing revealed distinct temporal cell states evoked by BCV. In KAI-3 cells, a significant shift in transcriptomic cell states occurred upon BCV treatment for 48h, with appearance of cell clusters enriched for type I interferon and cytokine signaling pathways, as well as activation of p53 and apoptosis pathways. Cell cycle and DNA repair pathways were also evident. At 72h post-BCV treatment, type II interferon signaling and endoplasmic reticulum stress response pathways were prominent. Similarly in NK-S1 cells, cell clusters enriched for immune signals, including interleukin-18 and cytokine pathways emerged, as did cell cycle, DNA repair, and apoptosis pathways. Western blot demonstrated increase in replication stress markers (p-CHK1 S317 and S345, RRM2, p-RPA2 S33), p-H2AX (DNA double strand break) and p-TBK1 (STING pathway activation). In keeping with this result, BCV triggered micronuclei formation, DNA fragmentation, and cytoplasmic DNA release. Gene expression levels of type I (IFNA, IFNB1) and II (IFNY) interferons and cytokines (CCL5, CXCL10) were upregulated following BCV treatment, as did the proportion of calreticulin-expressing cells and amount of extracellular HMGB1 released, indicative of immunogenic cell death. Notably, nuclear and membranous protein expression of the immune checkpoint protein PD-L1 also markedly increased. Intraperitoneal administration of BCV (40 mg/kg, twice per week) inhibited tumor volume in NOD/SCID mouse xenografts of NKTCL, compared with vehicle alone (p=0.0005, two-tailed t-test). On immunohistochemistry, PD-L1 was significantly overexpressed in BCV-treated tumor xenografts compared with control, with a 1.9-fold higher median H-score (p=0.0007, Mann-Whitney test). Conclusions Taken together, these results demonstrate a novel repurposed role of BCV in the treatment of NKTCL. The activation of DNA damage-induced STING pathway and immune-related signals suggests a further potential for combination with checkpoint immunotherapy.

Tissue Proteogenomic Landscape Reveals the Role of Uncharacterized SEL1L3 in Progression and Immunotherapy Response in Lung Adenocarcinoma.

The fundamental pursuit to complete the human proteome atlas and the unmet clinical needs in lung adenocarcinoma have prompted us to study the functional role of uncharacterized proteins and explore their implications in cancer biology. In this study, we characterized SEL1L3, a previously uncharacterized protein encoded from chromosome 4 as a dysregulated protein in lung adenocarcinoma from the large-scale tissue proteogenomics data set established using the cohort of Taiwan Cancer Moonshot. SEL1L3 was expressed in abundance in the tumor parts compared with paired adjacent normal tissues in 90% of the lung adenocarcinoma patients in our cohorts. Moreover, survival analysis revealed the association of SEL1L3 with better clinical outcomes. Intriguingly, silencing of SEL1L3 imposed a reduction in cell viability and activation of ER stress response pathways, indicating a role of SEL1L3 in the regulation of cell stress. Furthermore, the immune profiles of patients with higher SEL1L3 expression were corroborated with its active role in immunophenotype and favorable clinical outcomes in lung adenocarcinoma. Taken together, our study revealed that SEL1L3 might play a vital role in the regulation of cell stress, interaction with cancer cells and the immune microenvironment. Our research findings provide promising insights for further investigation of its molecular signaling network and also suggest SEL1L3 as a potential emerging adjuvant for immunotherapy in lung adenocarcinoma.

Emerging links between endoplasmic reticulum stress responses and acute kidney injury.

All cell types must maintain homeostasis under periods of stress. To prevent the catastrophic effects of stress, all cell types also respond to stress by inducing protective pathways. Within the cell, the endoplasmic reticulum (ER) is exquisitely stress-sensitive, primarily because this organelle folds, posttranslationally processes, and sorts one-third of the proteome. In the 1990s, a specialized ER stress response pathway was discovered, the unfolded protein response (UPR), which specifically protects the ER from damaged proteins and toxic chemicals. Not surprisingly, UPR-dependent responses are essential to maintain the function and viability of cells continuously exposed to stress, such as those in the kidney, which have high metabolic demands, produce myriad protein assemblies, continuously filter toxins, and synthesize ammonia. In this mini-review, we highlight recent articles that link ER stress and the UPR with acute kidney injury (AKI), a disease that arises in ∼10% of all hospitalized individuals and nearly half of all people admitted to intensive care units. We conclude with a discussion of prospects for treating AKI with emerging drugs that improve ER function.

Dissociation of ERMES clusters plays a key role in attenuating the endoplasmic reticulum stress

In yeast, ERMES, which mediates phospholipid transport between the ER and mitochondria, forms a limited number of oligomeric clusters at ER-mitochondria contact sites in a cell. Although the number of the ERMES clusters appears to be regulated to maintain proper inter-organelle phospholipid trafficking, its underlying mechanism and physiological relevance remain poorly understood. Here, we show that mitochondrial dynamics control the number of ERMES clusters. Moreover, we find that ER stress causes dissociation of the ERMES clusters independently of Ire1 and Hac1, canonical ER-stress response pathway components, leading to a delay in the phospholipid transport from the ER to mitochondria. Our biochemical and genetic analyses strongly suggest that the impaired phospholipid transport contributes to phospholipid accumulation in the ER, expanding the ER for ER stress attenuation. We thus propose that the ERMES dissociation constitutes an overlooked pathway of the ER stress response that operates in addition to the canonical Ire1/Hac1-dependent pathway.

Preliminary study of the intranuclear function of Sma and Mad related protein 5 gene in Litopenaeus vannamei

Litopenaeus vannamei Smad5 (LvSmad5) in cytoplasm has been proved to be involved in environmental stress response. As LvSmad5 could also locate in nucleus under specific stress, it was conjectured that LvSmad5 might participate in environmental stress response. While, the experimental evidence is still lacking. In this study, cytosolic LvSmad5 mutant or nuclear LvSmad5 mutant was expressed in Drosophila S2 cells, and then transcriptomic analysis of mentioned cells was performed using Illumina HiSeq based RNA-Seq, to reveal the function of LvSmad5 in nucleus. By comparing the two groups of cDNA libraries from S2 cells with cytosolic or nucleus LvSmad5 mutant, 86 differentially expressed genes as well as 765 differentially expressed transcripts were found. It was revealed that genes in the ER-stress response pathway, such as unfolded protein response and ER-associated degradation (ERAD) were enriched. Additionally, some kinds of metabolic reprogramming occurred in S2 cells with over-expressing nuclear LvSmad5, for significant changes in the expression of some metabolism-related genes. To test our infer that nuclear LvSmad5 was engaged in environmental stress response, homologous gene of Drosophila translocation in renal carcinoma on chromosome 8 in L.vannamei (LvTRC8) was chosen for further investigation. And studies about LvTRC8, a member of ERAD showed that it was induced by ER-stress or heat shock treatment. Suppressed the expression of LvTRC8 increased the cumulative mortality of shrimp upon stress. In some degree, these results support our speculation that nuclear LvSmad5 are involved in the environmental stress response of L. vannamei in fact.

PERK promotes immunosuppressive M2 macrophage phenotype by metabolic reprogramming and epigenetic modifications through the PERK-ATF4-PSAT1 axis.

The endoplasmic reticulum (ER) is a specialized organelle that participates in multiple cellular functions including protein folding, maturation, trafficking, and degradation to maintain homeostasis. However, hostile conditions in the tumor microenvironment (TME) disturb ER homeostasis. To overcome these conditions, cells activate ER stress response pathways, which are shown to augment the suppressive phenotypes of immune cells; however, the molecular mechanisms underpinning this process remain elusive. Here, we discuss a recent study by Raines et al, that suggests the role of the helper T-cell 2 (TH2) cytokine interleukin-4 (IL-4), and the TME in facilitating a protein kinase RNA-like ER kinase (PERK)-signaling cascade in macrophages, which promotes immunosuppressive M2 macrophage activation and proliferation. Further, the authors showed that PERK signaling promotes both mitochondrial respirations to fulfill cellular energy requirements and signaling through ATF4, which regulate phosphoserine aminotransferase 1 (PSAT1) activity to mediate the serine biosynthesis pathway. These results highlight a previously uncharacterized role for PERK in cellular metabolism and epigenetic modification in M2 macrophages, and thus offers a new therapeutic strategy for overcoming the immunosuppressive effects in the TME.

Abstract 5811: Ovarian cancer cell fate is regulated by the balance between saturated and unsaturated fatty acids

Abstract Background: Fat represents an important source of energy for ovarian cancer (OC) cells and is supplied either through import from the tumor milieu or via de novo lipogenesis. During fast tumor growth, when nutrients are scarce, lipogenesis becomes the primary source of fatty acids. Stearoyl-CoA desaturase (SCD), a rate-limiting enzyme in this pathway converts saturated (SFAs) into unsaturated fatty acids (UFAs) and is highly expressed in OC. The goal of this study was to determine how the balance between SFAs and UFAs tightly controlled by SCD, regulates OC cell survival and tumorigenicity. Methods: SCD was knocked down by shRNA or inhibited by using the small molecule CAY10566 and global effects on the lipidome and transcriptome were examined by targeted and untargeted lipidomics, stimulated Raman spectroscopy (SRS), and RNA sequencing. In OC cells in which SCD was blocked or knocked down, the effects of exogenous SFAs and UFAs on cell survival and endoplasmic reticulum (ER) stress pathway were assessed by using annexin V staining, XBP1 splicing assay and Western blotting of PERK/eIF2α/ATF4. Tumorigenicity was assessed by using an intraperitoneal (i.p.) xenograft model in nude mice. Results: RNA-seq analysis of SCD knockdown cells cultured under low serum conditions revealed activation of ER stress response pathways. Targeted lipidomics and SRS microscopy showed downregulation of UFAs vs. SFAs, while untargeted lipidomics discovered decreased fatty acyl chain plasticity among phosphatidylcholines. Activation of IRE1α/XBP1 and PERK/eIF2α/ATF4 axes was observed in cells accumulating SFAs. Stiff and disorganized ER membrane was detected by electron microscopy and Raman spectroscopy. Annexin V staining demonstrated apoptosis of OC cells under long-term mild ER stress or short-time severe ER stress induced by increased levels of SFAs. ER stress and apoptosis were rescued by addition of UFAs. In vivo SCD knockdown suppressed tumor growth and treatment with the SCD inhibitor CAY10566 reduced the number of metastases and the volume of ascites in mice fed with SFA enriched diet, but not in mice fed a balanced diet. Conclusion: Our data support that OC cells are highly susceptible to unbalanced intracellular SFAs/UFAs, undergoing ER stress and apoptosis in the presence of excessive intracellular SFAs. SCD inhibition coupled with a diet rich in saturated fats decreased cancer progression in vivo. These results support SCD as a key regulator of cancer cell fate during metabolic stress in growing tumors and point to new treatment strategies targeting lipid metabolism. Citation Format: Guangyuan Zhao, Yuying Tan, Horacio Cardenas, David Vayngart, Yinu Wang, Christina Ferreira, Ji-Xin Cheng, Daniela Matei. Ovarian cancer cell fate is regulated by the balance between saturated and unsaturated fatty acids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5811.