BCAR1 facilitates the survival of lung adenocarcinoma cells by augmenting the unfolded protein response, autophagy, and the formation of vasculogenic mimicry.

BACKGROUND: Our objective was to elucidate the pivotal roles of BCAR1 in unfolded protein response (UPR), autophagy and vasculogenic mimicry (VM) formation, processes that essential for the metastasis of lung adenocarcinoma (LUAD) cells. METHODS: The morphological assessment of endoplasmic reticulum (ER) status and autolysosomes in H1975 and H1299 LUAD cells following BCAR1 knockout (KO) was conducted using transmission electron microscope. The expression of markers and cellular functions related to the UPR, autophagy, and VM formation were examined in LUAD cells tissues. Additionally, proteomic analysis of LUAD cells was performed via mass spectrometry, and the pertinent signaling pathways were analyzed using bioinformatics tools. RESULTS: BCAR1-KO inhibited autophagy and UPR induced triggered starvation in LUAD cells. Cleaved-ATF6a-mediated UPR and subsequent autophagy, enhanced by BCAR1, were confirmed using the UPR stimulator and blocker. High BCAR1 expression, along with elevated UPR and autophagy, predicts poor prognosis in LUAD patients. BCAR1-KO reduced tube formation and VM markers expressions in LUAD cells. Additionally, BCAR1 expression positively correlated with VM formation in BALB/c-nu mice xenografts and LUAD patient tissues. CONCLUSION: BCAR1 promotes LUAD metastasis by enhancing cancer cell survival in nutrient-poor environments through ATF6-mediated UPR activation and autophagy. As BCAR1 induces VM formation, metastatic lesions eventually colonize. Thus, BCAR1 is a promising anti-metastasis target.

Methods to analyze the mitochondrial unfolded protein response (UPRmt).

The mitochondrial unfolded protein response (UPRmt) is a mitochondria-to-nuclear signaling pathway that mediates the transcription of genes required to maintain mitochondrial function during development as well as during aging. In this chapter, we describe the approaches and techniques that we and others have used to elucidate the mechanism(s) by which cells detect mitochondrial stress or dysfunction and communicate with the nucleus to induce transcription of a protective stress response. We also describe approaches to evaluate the impact of UPRmt activation on mitochondrial function and mitochondrial biogenesis including imaging-based approaches as well as approaches to evaluate mitochondrial genome (mtDNA) copy number.

DELE1 maintains muscle proteostasis to promote growth and survival in mitochondrial myopathy.

Mitochondrial dysfunction causes devastating disorders, including mitochondrial myopathy, but how muscle senses and adapts to mitochondrial dysfunction is not well understood. Here, we used diverse mouse models of mitochondrial myopathy to show that the signal for mitochondrial dysfunction originates within mitochondria. The mitochondrial proteins OMA1 and DELE1 sensed disruption of the inner mitochondrial membrane and, in response, activated the mitochondrial integrated stress response (mt-ISR) to increase the building blocks for protein synthesis. In the absence of the mt-ISR, protein synthesis in muscle was dysregulated causing protein misfolding, and mice with early-onset mitochondrial myopathy failed to grow and survive. The mt-ISR was similar following disruptions in mtDNA maintenance (Tfam knockout) and mitochondrial protein misfolding (CHCHD10 G58R and S59L knockin) but heterogenous among mitochondria-rich tissues, with broad gene expression changes observed in heart and skeletal muscle and limited changes observed in liver and brown adipose tissue. Taken together, our findings identify that the DELE1 mt-ISR mediates a similar response to diverse forms of mitochondrial stress and is critical for maintaining growth and survival in early-onset mitochondrial myopathy.

Emc1 is essential for vision and zebrafish photoreceptor outer segment morphogenesis.

Inherited retinal diseases (IRDs) are a rare group of eye disorders characterized by progressive dysfunction and degeneration of retinal cells. In this study, we characterized the raifteirí (raf) zebrafish, a novel model of inherited blindness, identified through an unbiased ENU mutagenesis screen. A mutation in the largest subunit of the endoplasmic reticulum membrane protein complex, emc1 was subsequently identified as the causative raf mutation. We sought to elucidate the cellular and molecular phenotypes in the emc1-/- knockout model and explore the association of emc1 with retinal degeneration. Visual behavior and retinal electrophysiology assays demonstrated that emc1-/- mutants had severe visual impairments. Retinal histology and morphometric analysis revealed extensive abnormalities, including thinning of the photoreceptor layer, in addition to large gaps surrounding the lens. Notably, photoreceptor outer segments were drastically smaller, outer segment protein expression was altered and hyaloid vasculature development was disrupted. Transcriptomic profiling identified cone and rod-specific phototransduction genes significantly downregulated by loss of emc1. These data shed light on why emc1 is a causative gene in inherited retinal disease and how outer segment morphogenesis is regulated.

Role of SEL1L in the progression of solid tumors, with a special focus on its recent therapeutic potential.

Since suppressor/enhancer of Lin-12-like (SEL1L) was cloned in 1997, various pieces of evidence from lower species suggest it plays a significant role in protein degradation via the ubiquitin-proteasome system. The relevance of SEL1L in many aspects of malignant transformation and tumorigenic events has been the subject of research, which has shown compelling in vitro and in vivo findings relating its altered expression to changes in tumor aggressiveness. The Endoplasmic Reticulum (ER) in tumor cells is crucial for preserving cellular proteostasis by inducing the unfolded protein response (UPR), a stress response. A crucial component of the UPR is ER-associated degradation (ERAD), which guards against ER stress-induced apoptosis and the removal of unfolded or misfolded proteins by the ubiquitin-proteasome system. As a protein stabilizer of HMG-CoA reductase degradation protein 1 (HRD1), one of the main components of ERAD, SEL1L plays an important role in ER homeostasis. Notably, the expression levels of these two proteins fluctuate independently in various cancer types, yet changes in their expression affect the levels of other associated proteins during cancer pathogenesis. Recent studies have also outlined the function of SEL1L in cancer medication resistance. This review explores the value of targeting SEL1L as a novel treatment approach for cancer, focusing on the molecular processes of SEL1L and its involvement in cancer etiology.

The Novel Anti-Cancer Agent, SpiD3, Is Cytotoxic in CLL Cells Resistant to Ibrutinib or Venetoclax.

Background: B-cell receptor (BCR) signaling is a central driver in chronic lymphocytic leukemia (CLL), along with the activation of pro-survival pathways (e.g., NF-κB) and aberrant anti-apoptotic mechanisms (e.g., BCL2) culminating to CLL cell survival and drug resistance. Front-line targeted therapies such as ibrutinib (BTK inhibitor) and venetoclax (BCL2 inhibitor) have radically improved CLL management. Yet, persisting CLL cells lead to relapse in ~20% of patients, signifying the unmet need of inhibitor-resistant refractory CLL. SpiD3 is a novel spirocyclic dimer of analog 19 that displays NF-κB inhibitory activity and preclinical anti-cancer properties. Recently, we have shown that SpiD3 inhibits CLL cell proliferation and induces cytotoxicity by promoting futile activation of the unfolded protein response (UPR) pathway and generation of reactive oxygen species (ROS), resulting in the inhibition of protein synthesis in CLL cells. Methods: We performed RNA-sequencing using CLL cells rendered resistant to ibrutinib and venetoclax to explore potential vulnerabilities in inhibitor-resistant and SpiD3-treated CLL cells. Results: The transcriptomic analysis of ibrutinib- or venetoclax-resistant CLL cell lines revealed ferroptosis, UPR signaling, and oxidative stress to be among the top pathways modulated by SpiD3 treatment. By examining SpiD3-induced protein aggregation, ROS production, and ferroptosis in inhibitor-resistant CLL cells, our findings demonstrate cytotoxicity following SpiD3 treatment in cell lines resistant to current front-line CLL therapeutics. Conclusions: Our results substantiate the development of SpiD3 as a novel therapeutic agent for relapsed/refractory CLL disease.

The Involvement of Endoplasmic Reticulum Stress during the Interaction between Calcium Oxalate Crystals and Renal Tubular Epithelial Cells.

Our study aimed to elucidate the mechanisms behind the interaction between calcium oxalate (CaOx) crystals and renal tubular epithelial cells through transcriptome sequencing analysis. HK-2 cells were stimulated with or without CaOx monohydrate crystals and subjected to RNA-seq to assess the effects of CaOx crystals on gene expression changes, key pathways, and molecular players during this interaction. A total of 629 differentially expressed genes (DEGs) were identified between the control group and experimental group, with 491 genes up-regulated and 138 down-regulated. Functional enrichment analysis indicated that the DEGs were significantly associated with endoplasmic reticulum stress (ERS) and unfolded protein response. To validate our findings, we compared our results with the public dataset GSE73680 and confirmed the increased expression of two ERS-related DEGs, CHAC1 and FGF21, in renal papillary tissues from patients with CaOx stones. Collectively, these findings suggest that ERS plays a crucial role in the crystal-cell interaction and highlight the potential for developing therapeutic strategies aimed at reducing CaOx stone formation by targeting ERS-related molecules and pathways.

The Aspergillus flavus hacA Gene in the Unfolded Protein Response Pathway Is a Candidate Target for Host-Induced Gene Silencing.

Fungal HacA/Hac1 transcription factors play a crucial role in regulating the unfolded protein response (UPR). The UPR helps cells to maintain endoplasmic reticulum (ER) protein homeostasis, which is critical for growth, development, and virulence. The Aspergillus flavus hacA gene encodes a domain rich in basic and acidic amino acids (Bsc) and a basic leucine zipper (bZip) domain, and features a non-conventional intron (Nt20). In this study, CRISPR/Cas9 was utilized to dissect the Bsc-coding, bZip-coding, and Nt20 sequences to elucidate the relationship between genotype and phenotype. In the Bsc and bZip experimental sets, all observed mutations in both coding sequences were in frame, suggesting that out-of-frame mutations are lethal. The survival rate of transformants in the Nt20 experiment set was low, at approximately 7%. Mutations in the intron primarily consisted of out-of-frame insertions and deletions. In addition to the wild-type-like conidial morphology, the mutants exhibited varied colony morphologies, including sclerotial, mixed (conidial and sclerotial), and mycelial morphologies. An ER stress test using dithiothreitol revealed that the sclerotial and mycelial mutants were much more sensitive than the conidial mutants. Additionally, the mycelial mutants were unable to produce aflatoxin but still produced aspergillic acid and kojic acid. RNAi experiments targeting the region encompassing Bsc and bZip indicated that transformant survival rates generally decreased, with a small number of transformants displaying phenotypic changes. Defects in the hacA gene at the DNA and transcript levels affected the survival, growth, and development of A. flavus. Thus, this gene may serve as a promising target for future host-induced gene-silencing strategies aimed at controlling infection and reducing aflatoxin contamination in crops.

Inhibition of proteasome activity facilitates definitive endodermal specification of pluripotent stem cells by influencing YAP signalling.

AIMS: The knowledge of the molecular players that regulate the generation of endoderm cells is imperative to obtain homogenous population of pancreatic ß-cells from stem cells. The Ubiquitin proteasome system (UPS) has been envisaged as a crucial intracellular protein degradation system, but its role in the generation of ß-cells remains elusive. Hence, it would be appropriate to unravel the potential role of UPS in endoderm specification and utilize the understanding to generate ß-cells from pluripotent stem cells. MATERIALS AND METHODS: The pluripotent stem cells (mESCs, miPSCs and hIPSCs) were subjected to differentiation towards pancreatic ß-cells and assessed the proteasomal activity during endodermal differentiation. Pharmacologic agents MG132 and IU-1 were employed to inhibit and activate proteasomal activity respectively at the definitive endoderm stage to investigate its impact on the generation of ß-cells. The expression of stage-specific genes were analyzed at transcript and protein levels. We also explored the role of unfolded protein response and UPS-regulated signalling pathways in endodermal differentiation. KEY FINDINGS: We observed decreased proteasomal activity specifically during endoderm, but not during the generation of other lineages. Extraneous proteasomal inhibition enhanced the expression of endodermal genes while increasing the proteasomal activity hindered definitive endodermal differentiation. Proteasomal inhibition at the definitive endodermal stage culminated in an enriched generation of insulin-positive cells. Elevated endodermal gene expression was consistent in mESCs and hIPSCs upon proteasomal inhibition. Mechanistic insight revealed the proteasome-inhibited enhanced endodermal differentiation to be via modulating the YAP pathway. SIGNIFICANCE: Our study unravels the specific involvement of UPS in endoderm cell generation from pluripotent stem cells and paves the way for obtaining potential definitive endodermal cells for plausible cellular therapy in the future.

Unravelling the interplay between ER stress, UPR and the cGAS-STING pathway: Implications for osteoarthritis pathogenesis and treatment strategy.

Osteoarthritis (OA) is a debilitating chronic degenerative disease affecting the whole joint organ leading to pain and disability. Cellular stress and injuries trigger inflammation and the onset of pathophysiological changes ensue after irreparable damage and inability to resolve inflammation, impeding the completion of the healing process. Extracellular matrix (ECM) degradation leads to dysregulated joint tissue metabolism. The reparative effort induces the proliferation of hypertrophic chondrocytes and matrix protein synthesis. Aberrant protein synthesis leads to endoplasmic reticulum (ER) stress and chondrocyte apoptosis with consequent cartilage matrix loss. These events in a vicious cycle perpetuate inflammation, hindering the restoration of normal tissue homeostasis. Recent evidence suggests that inflammatory responses and chondrocyte apoptosis could be caused by the activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signalling axis in response to DNA damage. It has been reported that there is a crosstalk between ER stress and cGAS-STING signalling in cellular senescence and other diseases. Based on recent evidence, this review discusses the role of ER stress, Unfolded Protein Response (UPR) and cGAS-STING pathway in mediating inflammatory responses in OA.

Activating transcription factor 4 plays a major role in shaping the transcriptional response to isoginkgetin in HCT116 cells.

Activating transcription factor 4 (ATF4) plays a central role in the integrated stress response (ISR) and one overlapping branch of the unfolded protein response (UPR). We recently reported that the splicing inhibitor isoginkgetin (IGG) induced ATF4 protein along with several known ATF4-regulated transcripts in a response that resembled the ISR and UPR. However, the contribution of ATF4-dependent and -independent transcriptional responses to IGG exposure was not known. Here we used RNA-sequencing in HCT116 colon cancer cells and an isogenic subline lacking ATF4 to investigate the contribution of ATF4 to IGG-induced changes in gene expression. Approximately 85% of the IGG-responsive DEGs in HCT116 cells were also differentially expressed in response to the ER stressor thapsigargin (Tg) and these were enriched for genes associated with the UPR and ISR. Most of these were positively regulated by IGG with impaired responses in the ATF4-deficient cells. Nonetheless, there were DEGs that responded similarly in both cell lines. The ATF4-independent IGG-induced DEGs included several metal responsive transcripts encoding metallothionines and a zinc transporter. Taken together, the predominant IGG response was ATF4-dependent in these cells and resembled the UPR and ISR while a second less prominent response involved the ATF4-independent regulation of metal responsive mRNAs.

The Tumor Suppressor TPD52-Governed Endoplasmic Reticulum Stress is Modulated by APCCdc20.

Aberrant regulation of unfolded protein response (UPR)/endoplasmic reticulum (ER) stress pathway is associated with cancer development, metastasis, and relapse, and the UPR signal transducer ATF6 has been proposed as a diagnostic and prognostic marker for many cancers. However, a causal molecular link between ATF6 activation and carcinogenesis is not established. Here, it is found that tumor protein D52 (TPD52) integrates ER stress and UPR signaling with the chaperone machinery by promoting S2P-mediated cleavage of ATF6. Although TPD52 has been generally considered as an oncogene, TPD52 is identified as a novel tumor suppressor in bladder cancer. Significantly, attenuation of the ER stress via depletion of TPD52 facilitated tumorigenesis in a subset of human carcinomas. Furthermore, the APCCdc20 E3 ligase is validated as the upstream regulator marking TPD52 for polyubiquitination-mediated proteolysis. In addition, inactivation of Cdc20 sensitized cancer cells to treatment with the ER stress inducer in a TPD52-dependent manner. Thus, the study suggests that TPD52 is a novel Cdc20 substrate that may modulate ER stress to prevent tumorigenesis.

Intravenous administration of muse cells improves cerebral ischemia outcome via immunomodulation in the spleen.

Ischemic stroke is a leading cause of disability and death globally. Stem cell therapies are emerging as a frontier for enhancing post-stroke recovery, with Muse cells-a subclass of pluripotent stem cells-demonstrating considerable promise. Muse cells are notable not only for their potential in cell replacement but also for their role in modulating immune responses following cerebral infarction. In the present study, we administered Muse cells intravenously to mice after inducing a stroke via distal middle cerebral artery occlusion. We evaluated motor outcomes, splenocyte populations, cytokine profiles, and gene expression 2 weeks after inducing stroke. Additionally, comparisons were drawn between outcomes in splenectomized mice and those receiving adoptive splenocyte transfer to discern the specific influence of the spleen on treatment efficacy. Our findings revealed that Muse cell therapy facilitates motor recovery, an effect that is compromised in the absence of the spleen. Spleens in treated mice exhibited a shift in neutrophil counts, increased cytokine activity, and a notable uptick in the expression of genes related to protein folding. These insights affirm the potential therapeutic effect of Muse cells in post-stroke treatment strategies, with their efficacy attributed, at least in part, to immunomodulatory pathways involving the spleen.

Genetic knock-in of EIF2AK3 variants reveals differences in PERK activity in mouse liver and pancreas under endoplasmic reticulum stress.

Common single-nucleotide variants (SNVs) of eukaryotic translation initiation factor 2 alpha kinase 3 (EIF2AK3) slightly increase the risk of disorders in the periphery and the central nervous system. EIF2AK3 encodes protein kinase RNA-like endoplasmic reticulum kinase (PERK), a key regulator of ER stress. Three exonic EIF2AK3 SNVs form the PERK-B haplotype, which is present in 28% of the global population. Importantly, the precise impact of these SNVs on PERK activity remains elusive. In this study, we demonstrate that PERK-B SNVs do not alter PERK expression or basal activity in vitro and in the novel triple knock-in mice expressing the exonic PERK-B SNVs in vivo. However, the kinase activity of PERK-B protein is higher than that of PERK-A in a cell-free assay and in mouse liver homogenates. Pancreatic tissue in PERK-B/B mice also exhibit increased susceptibility to apoptosis under acute ER stress. Monocyte-derived macrophages from PERK-B/B mice exhibit higher PERK activity than those from PERK-A/A mice, albeit with minimal functional consequences at acute timepoints. The subtle PERK-B-driven effects observed in liver and pancreas during acute stress implicate PERK as a contributor to disease susceptibility. The novel PERK-B mouse model provides valuable insights into ER stress-induced PERK activity, aiding the understanding of the genetic basis of disorders associated with ER stress.

Endoplasmic reticulum stress: The underlying mechanism of chronic pain.

Chronic pain (CP) affects over 30 % of the global population, imposing significant financial burdens on individuals and society. However, existing treatments for CP offer limited efficacy and troublesome side effects, primarily owing to a lack of knowledge of its precise underlying mechanism. Pathological stimuli disrupt the intricate process of protein folding and endoplasmic reticulum (ER) homeostasis. This disruption leads to the accumulation of misfolded or unfolded proteins in the ER, generating a condition termed ER stress. Emerging data have indicated that ER stress, occurring in the peripheral and central nervous systems, contributes to the development and maintenance of CP. This review aimed to comprehensively explore the intersection of ER stress and CP within the lower and upper nervous systems and highlight the cell-specific contributions of the unfolded protein response in different CP types. We provide a comprehensive synthesis of evidence from animal models, examining neuronal and non-neuronal mechanisms and discuss the damaging ER stress-linked inflammation, autophagy, oxidative stress, and apoptosis, which collectively drive disease progression and contribute to a neurotoxic environment. However, the mechanisms through which ER stress influences the most advanced centre-of-pain projections in the brain remain unclear. Further investigation in this area is crucial to elucidate the relationship between ER stress and CP and facilitate the development of novel therapeutic drugs for this intractable dilemma.

Reticulophagy and viral infection.

All viruses are obligate intracellular parasites that use host machinery to synthesize viral proteins. In infected eukaryotes, viral secreted and transmembrane proteins are synthesized at the endoplasmic reticulum (ER). Many viruses refashion ER membranes into bespoke factories where viral products accumulate while evading host pattern recognition receptors. ER processes are tightly regulated to maintain cellular homeostasis, so viruses must either conform to ER regulatory mechanisms or subvert them to ensure efficient viral replication. Reticulophagy is a catabolic process that directs lysosomal degradation of ER components. There is accumulating evidence that reticulophagy serves as a form of antiviral defense; we call this defense "xERophagy" to acknowledge its relationship to xenophagy, the catabolic degradation of microorganisms by macroautophagy/autophagy. In turn, viruses can subvert reticulophagy to suppress host antiviral responses and support efficient viral replication. Here, we review the evidence for functional interplay between viruses and the host reticulophagy machinery.Abbreviations: AMFR: autocrine motility factor receptor; ARF4: ADP-ribosylation factor 4; ARL6IP1: ADP-ribosylation factor-like 6 interacting protein 1; ATL3: atlastin GTPase 3; ATF4: activating transcription factor 4; ATF6: activating transcription factor 6; BPIFB3: BPI fold containing family B, member 3; CALCOCO1: calcium binding and coiled coil domain 1; CAMK2B: calcium/calmodulin-dependent protein kinase II, beta; CANX: calnexin; CDV: canine distemper virus; CCPG1: cell cycle progression 1; CDK5RAP3/C53: CDK5 regulatory subunit associated protein 3; CIR: cargo-interacting region; CoV: coronavirus; CSNK2/CK2: casein kinase 2; CVB3: coxsackievirus B3; DAPK1: death associated protein kinase 1; DENV: dengue virus; DMV: double-membrane vesicles; EBOV: Ebola virus; EBV: Epstein-Barr Virus; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; EMCV: encephalomyocarditis virus; EMV: extracellular microvesicle; ER: endoplasmic reticulum; ERAD: ER-associated degradation; ERN1/IRE1: endoplasmic reticulum to nucleus signalling 1; EV: extracellular vesicle; EV71: enterovirus 71; FIR: RB1CC1/FIP200-interacting region; FMDV: foot-and-mouth disease virus; HCMV: human cytomegalovirus; HCV: hepatitis C virus; HMGB1: high mobility group box 1; HSPA5/BiP: heat shock protein 5; IFN: interferon; IFNG/IFN-γ: interferon gamma; KSHV: Kaposi's sarcoma-associated herpesvirus; LIR: MAP1LC3/LC3-interacting region; LNP: lunapark, ER junction formation factor; MAP1LC3: microtubule-associated protein 1 light chain 3; MAP3K5/ASK1: mitogen-activated protein kinase kinase kinase 5; MAPK/JNK: mitogen-activated protein kinase; MeV: measles virus; MHV: murine hepatitis virus; NS: non-structural; PDIA3: protein disulfide isomerase associated 3; PRR: pattern recognition receptor; PRRSV: porcine reproductive and respiratory syndrome virus; RB1CC1/FIP200: RB1-inducible coiled-coil 1; RETREG1/FAM134B: reticulophagy regulator 1; RHD: reticulon homology domain; RTN3: reticulon 3; RTN3L: reticulon 3 long; sAIMs: shuffled Atg8-interacting motifs; SARS-CoV: severe acute respiratory syndrome coronavirus; SINV: Sindbis virus; STING1: stimulator of interferon response cGAMP interactor 1; SVV: Seneca Valley virus; SV40: simian virus 40; TEX264: testis expressed gene 264 ER-phagy receptor; TFEB: transcription factor EB; TRAF2: TNF receptor-associated factor 2; UIM: ubiquitin-interacting motif; UFM1: ubiquitin-fold modifier 1; UPR: unfolded protein response; VAPA: vesicle-associated membrane protein, associated protein A; VAPB: vesicle-associated membrane protein, associated protein B and C; VZV: varicella zoster virus; WNV: West Nile virus; XBP1: X-box binding protein 1; XBP1s: XBP1 spliced; xERophagy: xenophagy involving reticulophagy; ZIKV: Zika virus.

Viral Infections and Their Ability to Modulate Endoplasmic Reticulum Stress Response Pathways.

In eukaryotic cells, the endoplasmic reticulum is particularly important in post-translational modification of proteins before they are released extracellularly or sent to another endomembrane system. The correct three-dimensional folding of most proteins occurs in the ER lumen, which has an oxidative environment that is essential for the formation of disulfide bridges, which are important in maintaining protein structure. The ER is a versatile organelle that ensures the correct structure of proteins and is essential in the synthesis of lipids and sterols, in addition to offering support in the maintenance of intracellular calcium. Consequently, the cells needed to respond to demands caused by physiological conditions and pathological disturbances in the organelle homeostasis, leading to proper functioning of the cell or even programmed cell death. Disturbances to the ER function trigger a response to the accumulation of unfolded or misfolded proteins, known as the unfolded protein response. Such disturbances include abiotic stress, pharmacological agents, and intracellular pathogens, such as viruses. When misfolded proteins accumulate in the ER, they can undergo ubiquitination and proteasomal degradation through components of the ER-associated degradation system. Once a prolonged activity of the UPR pathway occurs, indicating that homeostasis cannot be reestablished, components of this pathway induce cell death by apoptosis. Here, we discuss how viruses have evolved ways to counteract UPR responses to maximize replication. This evolutionary viral ability is important to understand cell pathology and should be taken into account when designing therapeutic interventions and vaccines.

Therapeutic potential of 4-phenylbutyric acid against methylmercury-induced neuronal cell death in mice.

Methylmercury (MeHg) is an environmental neurotoxin that induces damage to the central nervous system and is the causative agent in Minamata disease. The mechanisms underlying MeHg neurotoxicity remain largely unknown, and there is a need for effective therapeutic agents, such as those that target MeHg-induced endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), which is activated as a defense mechanism. We investigated whether intraperitoneal administration of the chemical chaperone, 4-phenylbutyric acid (4-PBA), at 120 mg/kg/day can alleviate neurotoxicity in the brains of mice administered 50 ppm MeHg in drinking water for 5 weeks. 4-PBA significantly reduced MeHg-induced ER stress, neuronal apoptosis, and neurological symptoms. Furthermore, 4-PBA was effective even when administered 2 weeks after the initiation of exposure to 30 ppm MeHg in drinking water. Our results strongly indicate that ER stress and the UPR are key processes involved in MeHg toxicity, and that 4-PBA is a novel therapeutic candidate for MeHg-induced neurotoxicity.

AGR2 knockdown induces ER stress and mitochondria fission to facilitate pancreatic cancer cell death.

Anterior gradient 2 (AGR2) is often overexpressed in many human cancers, including pancreatic ductal adenocarcinoma (PDAC). Elevated AGR2 expression is known to play a critical role in tumor development, progression, and metastasis and positively correlates with poor patient survival. However, the relationship between AGR2 expression and tumor growth is not fully understood. Our study aims to investigate the impact of AGR2 knockdown on the survival of two pancreatic cancer cell lines, HPAF-II and PANC-1, that exhibit high AGR2 expression. This study revealed that the knockdown of AGR2 expression through an inducible shRNA-mediated approach reduced the proliferative ability and colony-forming potential of PDAC cells compared to scramble controls. Significantly, knocking down AGR2 led to the inhibition of multiple protein biosynthesis pathways and induced ER stress through unfolded protein response (UPR) activation. AGR2 knockdown induced ER stress and increased mitochondrial fission, while mitochondrial fusion remained unaffected. Ultimately, apoptotic cell death was heightened in AGR2 knockdown PDAC cells compared to the controls. Overall, these data reveal a new axis involving AGR2-ER stress-associated mitochondrial fission that could be targeted to improve PDAC patient outcomes.

CEAM is a mitochondrial-localized, amyloid-like motif-containing microprotein expressed in human cardiomyocytes.

Microproteins synthesized through non-canonical translation pathways are frequently found within mitochondria. However, the functional significance of these mitochondria-localized microproteins in energy-intensive organs such as the heart remains largely unexplored. In this study, we demonstrate that the long non-coding RNA CD63-AS1 encodes a mitochondrial microprotein. Notably, in ribosome profiling data of human hearts, there is a positive correlation between the expression of CD63-AS1 and genes associated with cardiomyopathy. We have termed this microprotein CEAM (CD63-AS1 encoded amyloid-like motif containing microprotein), reflecting its sequence characteristics. Our biochemical assays show that CEAM forms protease-resistant aggregates within mitochondria, whereas deletion of the amyloid-like motif transforms CEAM into a soluble cytosolic protein. Overexpression of CEAM triggers mitochondrial stress responses and adversely affect mitochondrial bioenergetics in cultured cardiomyocytes. In turn, the expression of CEAM is reciprocally inhibited by the activation of mitochondrial stresses induced by oligomycin. When expressed in mouse hearts via adeno-associated virus, CEAM impairs cardiac function. However, under conditions of pressure overload-induced cardiac hypertrophy, CEAM expression appears to offer a protective benefit and mitigates the expression of genes associated with cardiac remodeling, presumably through a mechanism that suppresses stress-induced translation reprogramming. Collectively, our study uncovers a hitherto unexplored amyloid-like microprotein expressed in the human cardiomyocytes, offering novel insights into myocardial hypertrophy pathophysiology.