Induction Of Endoplasmic Reticulum Stress Research Articles

Inhibition of ubiquitin specific peptidase 14 (USP14) promotes ER-phagy by inducing ER stress in human hepatoma HepG2 cells

ABSTRACT Autophagy plays a critical role in regulating the quality and quantity of cellular compartments; however, the mechanisms governing endoplasmic reticulum (ER) autophagy (ER-phagy) are still largely undefined. In this study, we identified several inhibitors of USP14, a ubiquitin-specific protease, including IU1 and b-AP15, as novel ER-phagy inducers through chemical library screening. While USP14 is known to act as an ER stress inducer, its precise role in ER-phagy remains unclear. Our findings demonstrate that treatment with either IU1 or b-AP15 induces ER-phagy by increasing ER stress in HepG2 cells. Similarly, depletion of USP14 augments ER-phagy and the ER stress response. The blockage of autophagy using an ULK1 inhibitor, SBI0206965, impedes ER-phagy. Moreover, inhibition of ER stress with tauroursodeoxycholic acid reverses ER-phagy by alleviating ER stress in HepG2 cells. We also found that suppression of the stress kinase JNK inhibited ER-phagy in IU1-treated cells. In conclusion, the results of this study suggest that the inhibition of USP14 accelerates ER-phagy by enhancing ER stress and JNK activation in HepG2 cells.

Synergistic lethality in chronic myeloid leukemia – targeting oxidative phosphorylation and unfolded protein response effectively complements tyrosine kinase inhibitor treatment

Chronic myeloid leukemia (CML) is effectively treated with tyrosine kinase inhibitors (TKIs), targeting the BCR::ABL1 oncoprotein. Still, resistance to therapy, relapse after treatment discontinuation, and side effects remain significant issues of long-term TKI treatment. Preliminary studies have shown that targeting oxidative phosphorylation (oxPhos) and the unfolded protein response (UPR) are promising therapeutic approaches to complement CML treatment. Here, we tested the efficacy of different TKIs, combined with the ATP synthase inhibitor oligomycin and the ER stress inducer thapsigargin in the CML cell lines K562, BV173, and KU812 and found a significant increase in cell death. Both, oligomycin and thapsigargin, triggered the upregulation of the UPR proteins ATF4 and CHOP, which was inhibited by imatinib. We observed comparable effects on cell death when combining TKIs with the ATP synthase inhibitor 8-chloroadenosine (8-Cl-Ado) as a potentially clinically applicable therapeutic agent. Stress-related apoptosis was triggered via a caspase cascade including the cleavage of caspase 3 and the inactivation of poly ADP ribose polymerase 1 (PARP1). The inhibition of PARP by olaparib also increased CML death in combination with TKIs. Our findings suggest a rationale for combining TKIs with 8-Cl-Ado or olaparib for future clinical studies in CML.

ALS/FTD-associated mutation in cyclin F inhibits ER-Golgi trafficking, inducing ER stress, ERAD and Golgi fragmentation

Amyotrophic lateral sclerosis (ALS) is a severely debilitating neurodegenerative condition that is part of the same disease spectrum as frontotemporal dementia (FTD). Mutations in the CCNF gene, encoding cyclin F, are present in both sporadic and familial ALS and FTD. However, the pathophysiological mechanisms underlying neurodegeneration remain unclear. Proper functioning of the endoplasmic reticulum (ER) and Golgi apparatus compartments is essential for normal physiological activities and to maintain cellular viability. Here, we demonstrate that ALS/FTD-associated variant cyclin FS621G inhibits secretory protein transport from the ER to Golgi apparatus, by a mechanism involving dysregulation of COPII vesicles at ER exit sites. Consistent with this finding, cyclin FS621G also induces fragmentation of the Golgi apparatus and activates ER stress, ER-associated degradation, and apoptosis. Induction of Golgi fragmentation and ER stress were confirmed with a second ALS/FTD variant cyclin FS195R, and in cortical primary neurons. Hence, this study provides novel insights into pathogenic mechanisms associated with ALS/FTD-variant cyclin F, involving perturbations to both secretory protein trafficking and ER-Golgi homeostasis.

Exploring the In Vitro and In Vivo Therapeutic Potential of BRAF and MEK Inhibitor Combination in NRAS-Mutated Melanoma

Introduction: Patients with NRAS-mutant metastatic melanoma often have an aggressive disease requiring a fast-acting, effective therapy. The MEK inhibitor binimetinib shows an overall response rate of 15% in patients with NRAS-mutant melanoma, providing a backbone for combination strategies. Our previous studies demonstrated that in NRAS-mutant melanoma, the antitumor activity of the MEK inhibitor binimetinib was significantly potentiated by the BRAFV600E/K inhibitor encorafenib through the induction of ER stress, leading to melanoma cell death by apoptotic mechanisms. Encorafenib combined with binimetinib was well tolerated in a phase III trial showing potent antitumor activity in BRAF-mutant melanoma, making a rapid evaluation in NRAS-mutant melanoma imminently feasible. These data provide a mechanistic rationale for the evaluation of binimetinib combined with encorafenib in preclinical and clinical studies on NRAS-mutant metastatic melanoma. Methods: The combination of BRAFi plus MEKi was tested in a monolayer culture of patient-derived cell lines and in corresponding patient-derived tissue slice cultures of NRAS-mutant melanoma. To investigate the treatment in vivo, NSG (NOD. Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice were subcutaneously injected with three different BRAF wild-type melanoma models harboring oncogenic NRAS mutations and treated orally with encorafenib (6 mg/kg body weight, daily) with or without binimetinib (8 mg/kg body weight, twice daily). In parallel, an individual healing attempt was carried out by treating one patient with an NRAS-mutated tumor. Results: Encorafenib was able to enhance the inhibitory effect on cell growth of binimetinib only in the cell line SKMel147 in vitro. It failed to enhance the apoptotic effect found in two other NRAS-mutated cell lines. Encorafenib led to a hyperactivation of ERK which could be reduced with the combinational treatment. In two of the three patient-derived tissue slice culture models of NRAS-mutant melanomas, a slight tendency of a combinatorial effect was seen which was not significant. Encorafenib showed a slight induction of the ER stress genes ATF4, CHOP, and NUPR1. The combinational treatment was able to enhance this effect, but not significantly. In the mouse model, the combination therapy of encorafenib with binimetinib resulted in reduced tumor growth compared to the control and encorafenib groups; however, the best effect in terms of tumor growth inhibition was measured in the binimetinib therapy group. The therapy showed no effect in an individual healing attempt for a patient suffering from metastatic, therapy-refractory NRAS-mutated melanoma. Conclusion: In in vitro and ex vivo settings, the combination therapy was observed to elicit a response; however, it did not amplify the efficacy observed with binimetinib alone, whereas in a patient, the combinational treatment remained ineffective. The preclinical in vivo data showed no increased combinatorial effect. However, the in vivo effect of binimetinib as monotherapy was unexpectedly high in the tested regimen. Nevertheless, binimetinib proved to be advantageous in the treatment of melanoma in vivo and led to high rates of apoptosis in vitro; hence, it still seems to be a good base for combination with other substances in the treatment of patients with NRAS-mutant melanoma.

Gut-derived ammonia contributes to alcohol-related fatty liver development via facilitating ethanol metabolism and provoking ATF4-dependent de novo lipogenesis activation

Background & aimsDysbiosis contributes to alcohol-associated liver disease (ALD); however, the precise mechanisms remain elusive. Given the critical role of the gut microbiota in ammonia production, we herein aim to investigate whether and how gut-derived ammonia contributes to ALD. MethodsBlood samples were collected from human subjects with/without alcohol drinking. Mice were exposed to the Lieber-DeCarli isocaloric control or ethanol-containing diets with and without rifaximin (a nonabsorbable antibiotic clinically used for lowering gut ammonia production) supplementation for five weeks. Both in vitro (NH4Cl exposure of AML12 hepatocytes) and in vivo (urease administration for 5 days in mice) hyperammonemia models were employed. RNA sequencing and fecal amplicon sequencing were performed. Ammonia and triglyceride concentrations were measured. The gene and protein expression of enzymes involved in multiple pathways were measured. ResultsChronic alcohol consumption causes hyperammonemia in both mice and human subjects. In healthy livers and hepatocytes, ammonia exposure upregulates the expression of urea cycle genes, elevates hepatic de novo lipogenesis (DNL), and increases fat accumulation. Intriguingly, ammonia promotes ethanol catabolism and acetyl-CoA formation, which, together with ammonia, synergistically facilitates intracellular fat accumulation in hepatocytes. Mechanistic investigations uncovered that ATF4 activation, as a result of ER stress induction and general control nonderepressible 2 activation, plays a central role in ammonia-provoked DNL elevation. Rifaximin ameliorates ALD pathologies in mice, concomitant with blunted hepatic ER stress induction, ATF4 activation, and DNL activation. ConclusionsAn overproduction of ammonia by gut microbiota, synergistically interacting with ethanol, is a significant contributor to ALD pathologies.

Pan-Inhibition of Protein Disulfide Isomerase Caused Cell Death through Disrupting Cellular Proteostasis in Pancreatic Ductal Adenocarcinoma Cells.

The protein disulfide isomerase (PDI) family is a group of thioredoxin endoplasmic reticulum (ER)-resident enzymes and molecular chaperones that play crucial roles in the correct folding of proteins. PDIs are upregulated in multiple cancer types and are considered a novel target for cancer therapy. In this study, we found that a potent pan-PDI inhibitor, E64FC26, significantly decreased the proliferation of pancreatic ductal adenocarcinoma (PDAC) cells. As expected, E64FC26 treatment increased ER stress and the unfolded protein response (UPR), as evidenced by upregulation of glucose-regulated protein, 78-kDa (GRP78), phosphorylated (p)-PKR-like ER kinase (PERK), and p-eukaryotic initiation factor 2α (eIF2α). Persistent ER stress was found to lead to apoptosis, ferroptosis, and autophagy, all of which are dependent on lysosomal functions. First, there was little cleaved caspase-3 in E64FC26-treated cells according to Western blotting, but a higher dose of E64FC26 was needed to induce caspase activity. Then, E64FC26-induced cell death could be reversed by adding the iron chelator, deferoxamine, and the reactive oxygen species scavengers, ferrostatin-1 and N-acetylcysteine. Furthermore, the autophagosome-specific marker, light chain 3B (LC3B)-II, increased, but the autolysosome marker, sequestosome 1 (SQSTM1)/p62, was not degraded in E64FC26-treated cells. Using the FUW mCherry-LC3 plasmid and acridine orange staining, we also discovered a lower number of acidic vesicles, such as autolysosomes and mature lysosomes, in E64FC26-treated cells. Finally, E64FC26 treatment increased the cathepsin L precursor (pre-CTSL) but decreased mature CTSL expression according to Western blotting, indicating a defective lysosome. These results suggested that the PDI inhibitor, E64FC26, might initially impede proper folding of proteins, and then induce ER stress and disrupt proteostasis, subsequently leading to lysosomal defects. Due to defective lysosomes, the extents of apoptosis and ferroptosis were limited, and fusion with autophagosomes was blocked in E64FC26-treated cells. Blockade of autolysosomal formation further led to the autophagic cell death of PDAC cells.

Alcohol induces ER stress and apoptosis by inducing oxidative stress and disruption of calcium homeostasis in glial cells

Alcohol has teratogenic effects that can cause developmental abnormalities and alter anatomical and functional characteristics of the developed brain and other organs. Glial cells play a crucial role in alcohol metabolism and protect neurons from toxic effects of alcohol. However, chronic alcohol exposure can lead to uncontrollable levels of reactive oxygen species, resulting in the death of glial cells and exposing neuronal cells to the toxic effects of alcohol. The exact molecular mechanism of alcohol-induced glial cell death has not been fully explored. This study reported that different concentrations of alcohol induce different expressions of ER stress markers in glial cells, focusing on the role of endoplasmic reticulum (ER) stress. Alcohol-induced concentration-dependent toxicity in both cells also induced oxidative stress, leading to mitochondrial damage. The expression of p53 and apoptotic proteins was significantly up-regulated after alcohol exposure, while Bcl2 (anti-apoptotic) was down-regulated. The signalling pathway for ER stress was activated and up-regulated marker proteins in a concentration-dependent manner. Cells pre-treated with BAPTA-AM and NAC showed significant resistance against alcohol assault compared to other cells. These in vitro findings will prove valuable for defining the mechanism by which alcohol modulates oxidative stress, mitochondrial and ER damage leading to glial cell death.

TMEM16A deficiency in alveolar type 2 epithelial cells protected against endoplasmic reticulum stress-induced ferroptosis during acute lung injury

Transmembrane protein 16A (TMEM16A) is one of the members of the ten-member family of “transmembrane protein 16”, playing critical roles in infection and solid organ injury. Acute lung injury (ALI) is a devastating disease which could be triggered by sepsis, trauma, and ischemia reperfusion. However, molecular mechanisms contributing to ALI are poorly understood at presently. In this study, we investigated the role of TMEM16A in sepsis-induced ALI using TMEM16A-deficient mice. Sepsis-induced ALI model was established by intratracheal injection of lipopolysaccharide (LPS). Our results showed that LPS stimulation significantly upregulated the expression levels of TMEM16A in lung tissues and in alveolar epithelial type II (AT2) cells. Knockout of TMEM16A in AT2 cells significantly improved pulmonary function and alleviated lung pathological injury in LPS-treated mice. Meanwhile, TMEM16A deficiency also inhibited endoplasmic reticulum (ER) stress and ferroptosis in AT2 cells from LPS-treated mice. In vitro experiments further demonstrated that ER stress and ferroptosis were inhibited after TMEM16A was knocked out. Furthermore, we used ER stress inducer thapsigargin to induce ER stress in TMEM16A-null AT2 cells and found that the induction of ER stress abolished the inhibition of ferroptosis by TMEM16A deficiency in LPS-treated AT2 cells. Finally, we disclosed that pharmacological inhibition of TMEM16A by shikonin also showed similar therapeutic effect on LPS-induced ALI in vivo. In conclusion, TMEM16A deficiency in AT2 cells could alleviate sepsis-induced ALI by decreasing ER stress-induced ferroptosis during ALI.

The endoplasmic reticulum (ER) chaperone GRP78 is secreted during ER Stress and alleviates endothelial cell inflammation

Abstract Introduction Glucose-Regulated Protein 78kD (GRP78) is a chaperone and the main regulator of the ER-stress response which is triggered by a variety of conditions that disturb folding of proteins in the ER. Upon ER Stress, GRP78 activates the unfolded protein response (UPR), which aims to clear unfolded proteins and restore ER homeostasis. Recently, extracellular secretion of GRP78 was described. However, the pathophysiological relevance of secreted GRP78 in atherosclerosis and endothelial cell inflammation remains to be elucidated. Aim of this study is to investigate the role of GRP78 secretion in endothelial cells. Methods and Results First, we sought to investigate if vascular cells secrete GRP78 during ER Stress. Human coronary artery endothelial cells (HCAEC) were treated with the ER stress inductor tunicamycin for up to 48h. After ER Stress induction, Western Blot and ELISA experiments detected an increased intracellular GRP78 expression. Intriguingly, prolonged ER Stress also promoted extracellular secretion of GRP78. Unbiased proteomic analysis of the HCAEC secretome confirmed that after ER-Stress induction, GRP78 is one of the most highly upregulated extracellular proteins (2.43-fold). Co-incubation with Brefeldin A, an inhibitor of ER-Golgi protein transport, abolished extracellular secretion (Fig.1). Hence, ER-Stress-induced GRP78 secretion is an actively regulated process. Next, the effect of GRP78 containing conditioned medium (CM) on HCAEC was analyzed. For control, HCAEC were co-incubated with BFA, or GRP78-knockdown was performed by siRNA. Treatment with GRP78 containing CM decreased GRP78 mRNA expression in target cells (0.35-fold vs. control [+BFA], p<0.0001). Furthermore, it increased viability ( BFA: 93.0 % vs. 83%, p=0.002; GRP78-siRNA: 94% vs. 85%, p=0.01) and decreased the rate of apoptosis (e.g. BFA: 1.7-fold vs. 5.1, p=0.02). Moreover, expression of markers of vascular inflammation and ER Stress (e.g., NF-κB and CHOP) were decreased when compared to CM without GRP78 (Fig.2). Furthermore, increased H2O2-generation was measured (BFA: 1.4-fold, p>0.001, GRP78-siRNA:1.3-fold, p=0.004) (Fig. 2). To confirm that the beneficial effects of the CM are in fact caused by GRP78, we treated HCAEC with recombinant GRP78 and observed an increased cell viability in a dose-dependent manner (1000 ng/ml: p=0.02 vs. control). CRIPTO is a membrane-bound co-receptor for growth factors and was previously linked to GRP78-signalling. siRNA-Knockdown of CRIPTO reduces viability and leads to cell apoptosis, irrespective of GRP78-treatment. Thus, GRP78-mediated effects may be mediated by CRIPTO. Conclusion Endothelial ER Stress promotes GRP78 secretion. Presence of GRP78 in conditioned medium ameliorates subsequent ER Stress and endothelial inflammation, which play a critical role in atherogenesis. Further investigations are important to shed light on the mechanism of release and the effect on target cells.

IRE1-mediated degradation of pre-miR-301a promotes apoptosis through upregulation of GADD45A

The unfolded protein response is a survival signaling pathway that is induced during various types of ER stress. Here, we determine IRE1’s role in miRNA regulation during ER stress. During induction of ER stress in human bronchial epithelial cells, we utilized next generation sequencing to demonstrate that pre-miR-301a and pre-miR-106b were significantly increased in the presence of an IRE1 inhibitor. Conversely, using nuclear-cytosolic fractionation on ER stressed cells, we found that these pre-miRNAs were decreased in the nuclear fractions without the IRE1 inhibitor. We also found that miR-301a-3p targets the proapoptotic UPR factor growth arrest and DNA-damage-inducible alpha (GADD45A). Inhibiting miR-301a-3p levels or blocking its predicted miRNA binding site in GADD45A’s 3’ UTR with a target protector increased GADD45A mRNA expression. Furthermore, an elevation of XBP1s expression had no effect on GADD45A mRNA expression. We also demonstrate that the introduction of a target protector for the miR-301a-3p binding site in GADD45A mRNA during ER stress promoted cell death in the airway epithelial cells. In summary, these results indicate that IRE1’s endonuclease activity is a two-edged sword that can splice XBP1 mRNA to stabilize survival or degrade pre-miR-301a to elevate GADD45A mRNA expression to lead to apoptosis.9J7o_YRPucfgg8rDu5QLJgVideo

The vitamin-k-cycle-enzyme VKORC1L1, but not its isoenzyme VKORC1, reduces inflammation in coronary endothelial cells

Abstract Introduction Vascular inflammation is a crucial contributor to atherosclerosis, in which oxidative stress, endoplasmic reticulum (ER) stress and transition of endothelial to mesenchymal cells (EndMT) are of critical importance. Vitamin-K-antagonists (VKA) promote vascular dysfunction, while vitamin K intake was proposed to reduce incidence of coronary artery disease. Key enzyme for regeneration of vitamin K is the Vitamin K epoxide reductase complex subunit 1 (VKORC1), which also represents the pharmacological target of VKA. VKOR-like1 (VKORC1L1) is an isoenzyme of VKORC1, which resides at the ER-membrane, exerts antioxidative properties and is involved in vitamin K maintenance. Aim of this study was to investigate the role of VKORC1 and VKORC1L1 in inflammation of human coronary artery endothelial cells (HCAEC). Methods and Results In silico analyses of the proteome of human coronary atherosclerosis revealed increased expression of VKORC1L1, but not of VKORC1. In vitro induction of oxidative stress (H2O2) and ER-Stress (tunicamycin) in HCAEC promoted time- and dose-dependent enhanced expression of VKORC1L1, without altering VKORC1 expression (H2O2: 2.3-fold vs. 0.8-fold after 40 minutes, p=0.04; tunicamycin: 1 μg/ml: 1.43-fold vs. 0.8-fold, p<0.01). Moreover, inducing EndMT by treatment with a differentiation medium (containing TGF-ß2, IL-1ß) resulted in a reduction of VKORC1L1, but not of VKORC1 (Fig.1). To analyze the relevance of the VKOR-enzymes on endothelial cell function, VKORC1 and VKORC1L1 were downregulated by siRNA. VKORC1L1 downregulation increased formation of reactive oxygen species (ROS) (DCFDA signal: 0.98-fold vs. 1.31-fold, p = 0.03) and reduced proliferation (EdU signal: 0.56 ± 0.02 vs. control, p < 0.01), whereas viability of HCAEC remained unchanged. ELISA and qPCR experiments revealed enhanced expression of a variety of markers of vascular inflammation after VKORC1L1 (e.g., IL-6: 3.42-fold ± 0.53, NF-κB: 1.95-fold ± 0.13, VCAM-1: 1.59-fold ± 0.12, vs. control). Additionally, expression of ER-stress markers was increased (e.g., GRP78; 1.32-fold ± 0.04 vs. control, p<0.01) (Fig.1). In contrast, VKORC1 downregulation significantly promoted proliferation (EdU signal: 3.9 ± 1.7 vs. control, p<0.01) as well as viability (alamarBlue® absorbance: 1.07 ± 0.08 vs. control, p=0.04), and decreased ROS (0.88 ± 0.17 vs. control, p=0.03). Pro-inflammatory and ER-stress proteins were either unaltered or decreased after VKORC1 knockdown (e.g., GRP78: 0.89-fold, VCAM-1: 0.68-fold) (Fig.2). To further elucidate these findings, we will perform overexpression experiments and stable knockdown of the VKOR-enzymes. Conclusion VKORC1 and its isoenzyme VKORC1L1 exhibit divergent effects on endothelial cell inflammation. Further studies are warranted to shed light on the regulation of the enzymes to improve our understanding regarding vascular side effects of VKA treatment and beneficial effects of vitamin K supplementation.

Establishment of an in vivo murine model of immune checkPoint inhibitors cardiotoxicity: emerging role of vascular permeability in pembrolizumab-induced cardiotoxicity

Abstract Introduction Advances in oncology have led to the discovery of immune checkpoint inhibitors (ICIs) that exhibit remarkable results in cancer patients’ survival and tumour regression. Despite their antitumor potential, some life-threatening immune-related adverse effects (AEs) including cardiotoxicity have been reported, while the pathomechanism of the cardiac AEs is obscure. Purpose We herein investigated the cardiotoxic effects of ipilimumab (IPI, anti-CTLA-4), pembrolizumab (PEM, anti-PD-1) and avelumab (AVE, anti-PD-L1) in primary murine ventricular cardiomyocytes (mAVCs) and spleenocytes in vitro and established the mechanism and progression of Pem’s cardiotoxicity in an in vivo murine model. Methods Primary mAVCs and spleenocytes were isolated and incubated with IPI, PEM and AVE (0-100 μg/ml) for 24h. Cell viability was assessed by MTT assay. Subsequently conditioned media from spleenocytes treated with the aforementioned ICIs was transferred onto the mAVCs for 24h. The experiments were repeated for molecular analyses. For the confirmation of PEM binding on the murine PD-1, human and murine PD-1 extracellular domains (ED) were biotechnologically produced and PEM binding was confirmed by circular dichroism (CD). C57BL6/J male mice were randomized into i. IgG4 and ii. Pem groups and treated for 2 (n = 5/group) and 5 weeks (n = 9/group). IgG4 and Pem were administered intraperitoneally once weekly at 2mg/ml. Pem dose was directly translated from humans. Mice underwent weekly echocardiography analysis, while at the endpoint, mice were sacrificed for myocardial sampling and histology analyses. Results IPI, PEM and AVE did not induce direct cytotoxicity on primary mAVCs, whereas IPI was excluded from the study due to unexpected spleenocyte cytotoxicity. Incubation of mAVCs with PEM and AVE conditioned media, revealed that only PEM could induce Immune cell (IC)-dependent cytotoxicity at 50 and 100 μg/ml and establishment of inflammation and autophagy in vitro. CD experiments showed that PEM binds on the murine PD-1 ED, which supports for the first time the use of the murine model for the extraction of translational results. In vivo Pem led to %Fractional shortening (FS%) decline, which was significantly reduced versus baseline at 2 weeks and further depressed at 5 weeks. At two weeks, Pem increased ICAM-1 expression as well as iNOS and beclin-1 expression in the myocardium, which was accompanied by intra-cardiac IC infiltration. At five weeks, Pem further increased vascular permeability, as shown by e-selectin and ICAM-1 upregulation and led to increase of cardiac autophagy and inflammation markers. Conclusions Herein we established for the first time a murine in vivo model of PEM cardiotoxicity. PEM induces an IC-mediated mAVCs cytotoxicity, via induction of inflammation, autophagy and ER-stress. Increased vascular permeability and dysregulated endothelial homeostasis seem to orchestrate early-on Pem’s cardiotoxicity manifestation in vivo.

Quantitative Measurement of Transthyretin Mistargeting by Proximity Labeling and Parallel Reaction Monitoring.

Proximity labeling is a powerful approach for characterizing subcellular proteomes. We recently demonstrated that proximity labeling can be used to identify mistrafficking of secretory proteins, such as occurs during pre-emptive quality control (pre-QC) following endoplasmic reticulum (ER) stress. This assay depends on protein quantification by immunoblotting and densitometry, which sometimes suffers from poor sensitivity. Here, we integrate parallel reaction monitoring (PRM) mass spectrometry to enable a more quantitative platform, and assess how chemical ER stressors impact pre-QC of the model secretory protein transthyretin in HEK293T cells. We find that some drug treatments affect labeling efficiency, which can be controlled for by normalizing to APEX2 auto-labeling. While some chemical ER stress inducers including Brefeldin A and thapsigargin induce pre-QC, tunicamycin and dithiothreitol do not, indicating ER stress alone is not sufficient. This finding contrasts with the canonical model of pre-QC induction, and establishes the utility of our platform.

Abstract 16819: Activation of Mitochondrial Calpain 1 Contributes to Complex I Damage During ER Stress

Introduction: ER (Endoplasmic reticulum) stress is increased in failing hearts. The increased ER stress impairs complex I in cardiac mitochondria (MITO). Calpain 1 (CPN1) and calpain 2 (CPN2) are calcium-dependent cysteine proteases located in cytosol and MITO (mCPN1 and mCPN2). Activation of mCPN1 contributes to complex I damage during ischemia-reperfusion (IR) and aging. Activation of the mCPN2 causes complex I damage following IR. However. the role of mCPN1 and mCPN2 activation in ER stress-mediated complex I damage remains unclear. Hypothesis: ER stress leads to complex I damage by activating mCPN1 and mCPN2. Methods: Wild type (WT) and conditional cardiomyocyte specific calpain 4 (CPN4) knockout (KO) mice were used. CPN4 is a small regulatory subunit of CPN1 and 2, and KO of CPN4 eliminates CPN1 and CPN2 activities. Tunicamycin (TUNI, 0.4 mg/kg) was used to induce ER stress in both WT and KO mice through one time IP injection. DMSO was used as vehicle treatment. Mice were sacrificed for cardiac mitochondrial isolation after 72 hours of vehicle or TUNI treatment. Results: ER stress was increased in both WT and KO mice, as shown by increased BIP in TUNI-treated mice (Figure, Panel A). Compared to vehicle, the rate of oxidative phosphorylation (OXPHOS) was decreased in TUNI-treated WT when glutamate + malate was used as complex I substrate (Panel B). In contrast, TUNI treatment did not alter OXPHOS with complex I substrate in KO (Panel B). Direct measurement of complex I activity showed that TUNI treatment only decreased complex I in MITO from WT but not from KO (Panel C). Proteomic study showed that the contents of complex I subunits including NDUFV2 and ND5 were decreased in WT but not in KO mice (Panel D); confirmed by western blotting (data not shown). These results indicate that activation of mCPN1 and mCPN2 contributes to the degradation of complex I subunits during ER stress. Conclusion: Induction of acute ER stress using TUNI damages complex I by activating mCPN1 and mCPN2.

Abstract 11344: Targeted Deletion of Arrdc4 Mitigates Cardiac Injury in Insulin-Deficient Diabetes

Introduction: Diabetic cardiomyopathy is an important contributor to mortality in the diabetic population. However, the pathogenesis of diabetic cardiomyopathy is unclear. Hypothesis: We recently reported that the interaction between glucose transporter 1 (GLUT1) and its adaptor protein Arrdc4 mediates metabolic stress in the ischemic heart. Here, we tested whether this mechanism plays a role in diabetic cardiomyopathy. Methods: To determine glucose responsiveness, mouse neonatal cardiomyocytes were treated with high glucose (25 mM). To investigate cardiac phenotype in vivo, we engineered Arrdc4-knockout (KO) mice by CRISPR/Cas9 technology and created an insulin-deficient diabetic model by intraperitoneal injection of streptozotocin. Results: Arrdc4 expression was robustly stimulated by D-glucose (324±10%, p<0.01) in cardiomyocytes. The glucose responsiveness was mediated by MondoA, a nutrient-sensing transcriptional factor, through a cytoplasmic-nuclear shuttling mechanism. Confocal live imaging showed that Arrdc4 triggered endocytic trafficking of GLUT1 into lysosomes. In the animal model, levels of insulin depletion were comparable between the genotypes. Nevertheless, Arrdc4-KO diabetic mice had enhanced glucose uptake in multiple organs including the heart (139±13%, p<0.01) compared to wild-type (WT) diabetic mice. Arrdc4-KO diabetic hearts had lower induction of oxidative stress, ER stress, and autophagy by hyperglycemia, leading to better mitochondrial function and less cardiomyocyte apoptosis (70±5%, p<0.01) than WT diabetic hearts. Although resting echocardiography could not detect early signs of left ventricular dysfunction, stress hemodynamic analysis uncovered the masked mechanical dysfunction by diabetes and found that Arrdc4-KO diabetic mice retained a greater cardiac response to beta-adrenergic stimulation than wild-type mice (149±5%, p<0.01). Conclusion: The metabolic rearrangements by Arrdc4 deletion improve glucose transport due to the absence of its inhibition towards GLUT1 function, protecting the heart against diabetic damage. These results provide a novel link between GLUT1 and Arrdc4 as a fundamental mechanism of the regulation of glucose homeostasis in diabetic cardiomyopathy.

Abstract 15060: Inhibition of Endoplasmic Reticulum Stress and YAP/TAZ Signaling Protects Endothelial Function in Diabetes

Introduction: Previous studies have demonstrated the role of Hippo pathway with its major effectors, Yes-associated protein (YAP) and transcription activator with PDZ binding motif (TAZ) in atherosclerosis. However, the involvement of YAP/TAZ signaling in diabetes-associated endothelial dysfunction remains unexplored. Hypothesis: We hypothesized that activation of YAP/TAZ signaling links with endoplasmic reticulum (ER) stress and contributes to endothelial dysfunction. Methods: Male C57BL/6 mice were fed with high-fat diet, 45% kcal% fat for 15 weeks to induce obesity and diabetes. ER stress alleviator 4-phenylbutyric acid was orally administered for 4 weeks at 100 mg/kg/day. Vascular reactivity of mouse aortas was assessed by wire myograph. Human umbilical vein endothelial cells (HUVECs) were cultured with risk factors such as high glucose and ER stress inducer tunicamycin. Western blot and immunofluorescent staining were performed to evaluate the expressions of protein targets. Results: YAP/TAZ signaling was activated accomplished with ER stress in aortas from obese diabetic mice and in endothelial cells treated with risk factors. Chronic treatment with 4-phenylbutyric acid ameliorated endothelium-dependent relaxations as well as inhibited ER stress, YAP/TAZ signaling and oxidative stress in aortas. Phosphorylation of YAP at Ser127 was reduced in mouse aortic arch which was downregulated by ER stress alleviator. In HUVECs, ER stress alleviators significantly inhibited YAP/TAZ signaling and increased NO bioavailability but YAP inhibitor did not suppress ER stress. Conclusions: ER stress activates YAP/TAZ signaling, resulting in endothelial dysfunction in diabetes. Inhibition of YAP/TAZ is vasoprotective and exhibits therapeutic potential against diabetic vascular complication.

Animal Models for the Study of Gaucher Disease.

In Gaucher disease (GD), a relatively common sphingolipidosis, the mutant lysosomal enzyme acid β-glucocerebrosidase (GCase), encoded by the GBA1 gene, fails to properly hydrolyze the sphingolipid glucosylceramide (GlcCer) in lysosomes, particularly of tissue macrophages. As a result, GlcCer accumulates, which, to a certain extent, is converted to its deacylated form, glucosylsphingosine (GlcSph), by lysosomal acid ceramidase. The inability of mutant GCase to degrade GlcSph further promotes its accumulation. The amount of mutant GCase in lysosomes depends on the amount of mutant ER enzyme that shuttles to them. In the case of many mutant GCase forms, the enzyme is largely misfolded in the ER. Only a fraction correctly folds and is subsequently trafficked to the lysosomes, while the rest of the misfolded mutant GCase protein undergoes ER-associated degradation (ERAD). The retention of misfolded mutant GCase in the ER induces ER stress, which evokes a stress response known as the unfolded protein response (UPR). GD is remarkably heterogeneous in clinical manifestation, including the variant without CNS involvement (type 1), and acute and subacute neuronopathic variants (types 2 and 3). The present review discusses animal models developed to study the molecular and cellular mechanisms underlying GD.

Abstract 17307: Pre-Existing Endoplasmic Reticulum (ER) Stress Inhibits Sars-Co-V2 Entry Into Host Cells Through Modulation of AKT Signaling and Unfolded Protein Response (UPR)

Introduction: It is well known that SARS-Co-V2 infection can induce ER stress-associated activation of unfolded protein response (UPR) in various host cells which may contribute to the pathogenesis of COVID-19. However, the interplay between SARS-Co-V2 infection and UPR signaling in pre-existing ER stress associated pathological conditions has not been well elucidated. Hypothesis: We hypothesized that modulation of a pre-existing ER stress in host cells could attenuate susceptibility to SARS-CoV-2 infection by activating a range of cellular defense. Methods: Increasing concentrations of Tunicamycin (Tm) and Thapsigargin (Tg) have been used to induce ER stress in Huh-7 cells. After 6h treatment, cells were infected with SARS-CoV-2 pseudotyped particles (SARS-CoV-2pp) for 48 p.i. SARS-Co-V2-2pp entry was measured using Bright Glo TM luciferase assay. Cell viability was measured by cell titer Glo ® luminescent cell viability assay. The mRNA and protein expression of UPR markers were evaluated using RT-qPCR and Western blot methods. Results: Tm (5 μg/ml) and Tg (1 μM) efficiently inhibited SARS-CoV-2pp entry into cells without any cytotoxic effect. Chemical ER stress-induced inhibition of SARS-CoV-2pp entry was associated with significant reduction of ACE2 expression in Tg-infected cells but not with Tm. Strikingly, Tm and Tg revealed differential effects in modulating the expression of ER stress genes in infected cells. Both Tm and Tg significantly reduced the expression of stress-inducible ER chaperone GRP78/BIP in infected cells. In contrast, the IRE1-XBP1s and PERK-eIF2α-ATF4-CHOP signaling pathways were downregulated in Tg-infected cells only. Additionally, insulin-mediated glucose uptake, phosphorylation of Ser 307 IRS-1 and downstream p-AKT were enhanced in Tg-infected cells without any change in ERK phosphorylation. Conclusions: These findings suggest that pre-existing ER stress could modulate a specific UPR response in infected cells capable of counteracting the stress-inducible elements signaling, thereby depriving SARS-Co-V2 of essential components for their entry and replication. Pharmacological manipulation of ER stress in host cells might provide new therapeutic strategies to alleviate SARS-CoV-2 infection.

Acetylshikonin induces apoptosis through the endoplasmic reticulum stress-activated PERK/eIF2α /CHOP axis in oesophageal squamous cell carcinoma.

Acetylshikonin (AS) is an active component of Lithospermum erythrorhizon Sieb. et Zucc that exhibits activity against various cancers; however, the underlying mechanisms of AS against oesophageal squamous carcinoma (ESCC) need to be elusive. The research explores the anti-cancer role and potential mechanism of AS on ESCC invitro and invivo, providing evidences for AS treatment against ESCC. In this study, we firstly demonstrated that AS treatment effectively inhibits cell viability and proliferation of ESCC cells. In addition, AS significantly induces G1/S phage arrest and promotes apoptosis in ESCC cell lines. Further studies reveal that AS induces ER stress, as observed by dose- and time-dependently increased expression of BIP, PDI, PERK, phosphorylation of eIF2α , CHOP and splicing of XBP1. CHOP knockdown or PERK inhibition markedly rescue cell apoptosis induced by AS. Moreover, AS treatment significantly inhibits ESCC xenograft growth in nude mice. Elevated expression of BIP and CHOP is also observed in xenograft tumours. Taken together, AS inhibits proliferation and induces apoptosis through ER stress-activated PERK/eIF2α /CHOP pathway in ESCC, which indicates AS represents a promising candidate for ESCC treatment.

An innovative nanoformulation utilizing tumor microenvironment-responsive PEG-polyglutamic coating and dynamic charge adjustment for specific targeting of ER stress inducer, microRNA, and immunoadjuvant in pancreatic cancer: In vitro investigations

Pancreatic ductal adenocarcinoma (PDAC) is a significant obstacle to lowering global cancer deaths. CB-5083, a novel valosin-containing protein (VCP)/p97 inhibitor that disrupts proteasomal degradation and induces endoplasmic reticulum stress (ERS) accumulation, was evaluated as an inducer of immunogenic cell death (ICD) in PDAC treatment. Furthermore, miR-142 enhances checkpoint blockade and promotes M1 repolarization, while Toll-like receptor 7/8 agonist resiquimod (R) acts as an immunoadjuvant to amplify the immune response to miR-142. This research signifies the first integration of CB, miR-142, and R in solid lipid nanoparticles (SLNs) modified with peptides targeting PD-L1, EGFR, and ER, which were shelled by the PEG-polyglutamic (PGA) coating that detaches in response to the acidic pH values in the tumor microenvironment (TME). The modified SLNs exhibited pH-sensitive cytotoxicity against Panc-02 cells, preserving normal cells and preventing hemolysis. The innovative approach simultaneously modulated pathways, including VCP/Bip/K48-Ub/ATF6, IRE1α/XBPs/LC3II, PD-L1/TGF-β/IL-10/CD206/MSR1/Arg1, and TNF-α/IFN-γ/IL-6/iNOS/COX-2. Combined treatment blocked VCP, arrested the cell cycle, inhibited EMT, triggered ERS-mediated autophagy/apoptosis, and stimulated robust ICD via the release of damage-associated molecular patterns. This adaptable nanoformulation, displaying pH-sensitive PEG-PGA de-coating and precisely targeting EGFR, PD-L1, and ER, serves to hinder EMT and immune evasion, subsequently amplifying ICD in PDAC cells and the TME.