bims-unfpre 2021-05-02 papers

bims-unfpre

Biomed News

on Unfolded protein response

Issue of 2021–05–02
twenty-one papers selected by
Susan Logue, University of Manitoba

Protomer alignment modulates specificity of RNA substrate recognition by Ire1.
A target-agnostic screen identifies approved drugs to stabilize the endoplasmic reticulum-resident proteome.
Endoplasmic reticulum chaperone GRP78/BiP is critical for mutant Kras-driven lung tumorigenesis.
Combined Transcriptomic and Proteomic Analysis of Perk Toxicity Pathways.
The Potential Role of Small-Molecule PERK Inhibitor LDN-0060609 in Primary Open-Angle Glaucoma Treatment.
CHOP Pro-Apoptotic Transcriptional Program in Response to ER Stress Is Hacked by Zika Virus.
Endoplasmic Reticulum Stress Contributes to Gefitinib-Induced Apoptosis in Glioma.
Targeting E7 antigen to the endoplasmic reticulum degradation pathway promotes a potent therapeutic antitumor effect.
The Stress of Lung Aging: Endoplasmic Reticulum and Senescence Tête-à-Tête.
The Epstein-Barr Virus Lytic Protein BMLF1 Induces Upregulation of GRP78 Expression through ATF6 Activation.
PERK activation by CCT020312 chemosensitizes colorectal cancer through inducing apoptosis regulated by ER stress.
The Road of Solid Tumor Survival: From Drug-Induced Endoplasmic Reticulum Stress to Drug Resistance.
FATP4 inactivation in cultured macrophages attenuates M1- and ER stress-induced cytokine release via a metabolic shift towards triacylglycerides.
The Unfolded Protein Response as a Compensatory Mechanism and Potential Therapeutic Target in PLN R14del Cardiomyopathy.
Oxidation of peroxiredoxin-4 induces oligomerization and promotes interaction with proteins governing protein folding and endoplasmic reticulum stress.
Establishment of a reporter system for monitoring activation of the ER stress transducer ATF6β.
ONC201 induces the unfolded protein response (UPR) in high- and low-grade ovarian carcinoma cell lines and leads to cell death regardless of platinum sensitivity.
Resveratrol Alleviates the Inhibitory Effect of Tunicamycin-Induced Endoplasmic Reticulum Stress on Expression of Genes Involved in Thyroid Hormone Synthesis in FRTL-5 Thyrocytes.
Different Expression of Mitochondrial and Endoplasmic Reticulum Stress Genes in Epicardial Adipose Tissue Depends on Coronary Atherosclerosis.
Traumatic Optic Neuropathy Is Associated with Visual Impairment, Neurodegeneration, and Endoplasmic Reticulum Stress in Adolescent Mice.
The oxidoreductase PYROXD1 uses NAD(P)+ as an antioxidant to sustain tRNA ligase activity in pre-tRNA splicing and unfolded protein response.

Elife. 2021 Apr 27. pii: e67425. [Epub ahead of print]10

Protomer alignment modulates specificity of RNA substrate recognition by Ire1.

Weihan Li, Kelly Crotty, Diego Garrido Ruiz, Mark Voorhies, Carlos Rivera, Anita Sil, R Dyche Mullins, Matthew P Jacobson, Jirka Peschek, Peter Walter.

  The unfolded protein response (UPR) maintains protein folding homeostasis in the endoplasmic reticulum (ER). In metazoan cells, the Ire1 branch of the UPR initiates two functional outputs-non-conventional mRNA splicing and selective mRNA decay (RIDD). By contrast, Ire1 orthologs from Saccharomyces cerevisiae and Schizosaccharomyces pombe are specialized for only splicing or RIDD, respectively. Previously, we showed that the functional specialization lies in Ire1's RNase activity, which is either stringently splice-site specific or promiscuous (W. Li et al., 2018). Here, we developed an assay that reports on Ire1's RNase promiscuity. We found that conversion of two amino acids within the RNase domain of S. cerevisiae Ire1 to their S. pombe counterparts rendered it promiscuous. Using biochemical assays and computational modeling, we show that the mutations rewired a pair of salt bridges at Ire1 RNase domain's dimer interface, changing its protomer alignment. Thus, Ire1 protomer alignment affects its substrates specificity.

Keywords:  S. cerevisiae; S. pombe; biochemistry; chemical biology

DOI:  https://doi.org/10.7554/eLife.67425
Cell Rep. 2021 Apr 27. pii: S2211-1247(21)00356-9. [Epub ahead of print]35(4): 109040

A target-agnostic screen identifies approved drugs to stabilize the endoplasmic reticulum-resident proteome.

Mark J Henderson, Kathleen A Trychta, Shyh-Ming Yang, Susanne Bäck, Adam Yasgar, Emily S Wires, Carina Danchik, Xiaokang Yan, Hideaki Yano, Lei Shi, Kuo-Jen Wu, Amy Q Wang, Dingyin Tao, Gergely Zahoránszky-Kőhalmi, Xin Hu, Xin Xu, David Maloney, Alexey V Zakharov, Ganesha Rai, Fumihiko Urano, Mikko Airavaara, Oksana Gavrilova, Ajit Jadhav, Yun Wang, Anton Simeonov, Brandon K Harvey.

  Endoplasmic reticulum (ER) dysregulation is associated with pathologies including neurodegenerative, muscular, and diabetic conditions. Depletion of ER calcium can lead to the loss of resident proteins in a process termed exodosis. To identify compounds that attenuate the redistribution of ER proteins under pathological conditions, we performed a quantitative high-throughput screen using the Gaussia luciferase (GLuc)-secreted ER calcium modulated protein (SERCaMP) assay, which monitors secretion of ER-resident proteins triggered by calcium depletion. We identify several clinically used drugs, including bromocriptine, and further characterize them using assays to measure effects on ER calcium, ER stress, and ER exodosis. Bromocriptine elicits protective effects in cell-based models of exodosis as well as in vivo models of stroke and diabetes. Bromocriptine analogs with reduced dopamine receptor activity retain similar efficacy in stabilizing the ER proteome, indicating a non-canonical mechanism of action. This study describes a strategic approach to identify small-molecule drugs capable of improving ER proteostasis in human disease conditions.

Keywords:  ER calcium; ER proteome; ER retention sequence; ER stress; SERCaMP; bromocriptine; diabetes; endoplasmic reticulum; exodosis; stroke

DOI:  https://doi.org/10.1016/j.celrep.2021.109040
Oncogene. 2021 Apr 30.

Endoplasmic reticulum chaperone GRP78/BiP is critical for mutant Kras-driven lung tumorigenesis.

Daisy Flores Rangel, Louis Dubeau, Ryan Park, Priscilla Chan, Dat P Ha, Mario A Pulido, Daniel J Mullen, Ivetta Vorobyova, Beiyun Zhou, Zea Borok, Ite A Offringa, Amy S Lee.

Lung cancer is the leading cause of cancer mortality worldwide and KRAS is the most commonly mutated gene in lung adenocarcinoma (LUAD). The 78-kDa glucose-regulated protein GRP78/BiP is a key endoplasmic reticulum chaperone protein and a major pro-survival effector of the unfolded protein response (UPR). Analysis of the Cancer Genome Atlas database and immunostain of patient tissues revealed that compared to normal lung, GRP78 expression is generally elevated in human lung cancers, including tumors bearing the KRASG12D mutation. To test the requirement of GRP78 in human lung oncogenesis, we generated mouse models containing floxed Grp78 and Kras Lox-Stop-Lox G12D (KrasLSL-G12D) alleles. Simultaneous activation of the KrasG12D allele and knockout of the Grp78 alleles were achieved in the whole lung or selectively in lung alveolar epithelial type 2 cells known to be precursors for adenomas that progress to LUAD. Here we report that GRP78 haploinsufficiency is sufficient to suppress KrasG12D-mediated lung tumor progression and prolong survival. Furthermore, GRP78 knockdown in human lung cancer cell line A427 (KrasG12D/+) leads to activation of UPR and apoptotic markers and loss of cell viability. Our studies provide evidence that targeting GRP78 represents a novel therapeutic approach to suppress mutant KRAS-mediated lung tumorigenesis.

DOI: https://doi.org/10.1038/s41388-021-01791-9
Int J Mol Sci. 2021 Apr 27. pii: 4598. [Epub ahead of print]22(9):

Combined Transcriptomic and Proteomic Analysis of Perk Toxicity Pathways.

Rebeka Popovic, Ivana Celardo, Yizhou Yu, Ana C Costa, Samantha H Y Loh, L Miguel Martins.

  In Drosophila, endoplasmic reticulum (ER) stress activates the protein kinase R-like endoplasmic reticulum kinase (dPerk). dPerk can also be activated by defective mitochondria in fly models of Parkinson's disease caused by mutations in pink1 or parkin. The Perk branch of the unfolded protein response (UPR) has emerged as a major toxic process in neurodegenerative disorders causing a chronic reduction in vital proteins and neuronal death. In this study, we combined microarray analysis and quantitative proteomics analysis in adult flies overexpressing dPerk to investigate the relationship between the transcriptional and translational response to dPerk activation. We identified tribbles and Heat shock protein 22 as two novel Drosophila activating transcription factor 4 (dAtf4) regulated transcripts. Using a combined bioinformatics tool kit, we demonstrated that the activation of dPerk leads to translational repression of mitochondrial proteins associated with glutathione and nucleotide metabolism, calcium signalling and iron-sulphur cluster biosynthesis. Further efforts to enhance these translationally repressed dPerk targets might offer protection against Perk toxicity.

Keywords:  Drosophila; Drosophila protein kinase RNA (PKR)-like ER kinase (dPerk); ER stress; activating transcription factor 4 (ATF4); unfolded protein response

DOI:  https://doi.org/10.3390/ijms22094598
Int J Mol Sci. 2021 Apr 26. pii: 4494. [Epub ahead of print]22(9):

The Potential Role of Small-Molecule PERK Inhibitor LDN-0060609 in Primary Open-Angle Glaucoma Treatment.

Wioletta Rozpędek-Kamińska, Grzegorz Galita, Natalia Siwecka, Steven L Carroll, John Alan Diehl, Ewa Kucharska, Dariusz Pytel, Ireneusz Majsterek.

  Primary open-angle glaucoma (POAG) constitutes the most common type of glaucoma. Emerging evidence suggests that Endoplasmic Reticulum (ER) stress and the protein kinase RNA-like endoplasmic reticulum kinase (PERK)-mediated Unfolded Protein Response (UPR) signaling pathway play a key role in POAG pathogenesis. Thus, the main aim of the study was to evaluate the effectiveness of the PERK inhibitor LDN-0060609 in cellular model of glaucoma using primary human trabecular meshwork (HTM) cells. To evaluate the level of the ER stress marker proteins, Western blotting and TaqMan gene expression assay were used. The cytotoxicity was measured by XTT, LDH assays and Giemsa staining, whereas genotoxicity via comet assay. Changes in cell morphology were assessed by phase-contrast microscopy. Analysis of apoptosis was performed by caspase-3 assay and flow cytometry (FC), whereas cell cycle progression by FC. The results obtained have demonstrated that LDN-0060609 triggered a significant decrease of ER stress marker proteins within HTM cells with induced ER stress conditions. Moreover, LDN-0060609 effectively increased viability, reduced DNA damage, increased proliferation, restored normal morphology, reduced apoptosis and restored normal cell cycle distribution of HTM cells with induced ER stress conditions. Thereby, PERK inhibitors, such as LDN-0060609, may provide an innovative, ground-breaking treatment strategy against POAG.

Keywords:  PERK; PERK inhibitor; apoptosis; eIF2α; endoplasmic reticulum stress; glaucoma; glaucoma treatment; unfolded protein response

DOI:  https://doi.org/10.3390/ijms22094494
Int J Mol Sci. 2021 Apr 03. pii: 3750. [Epub ahead of print]22(7):

CHOP Pro-Apoptotic Transcriptional Program in Response to ER Stress Is Hacked by Zika Virus.

Jonathan Turpin, Daed El-Safadi, Grégorie Lebeau, Etienne Frumence, Philippe Desprès, Wildriss Viranaïcken, Pascale Krejbich-Trotot.

  Zika virus (ZIKV) is an emerging mosquito-borne flavivirus considered as a threat to human health due to large epidemics and serious clinical outcomes such as microcephaly in new-borns. Like all flaviviruses, ZIKV relies on the cellular machinery to complete its viral cycle, with the endoplasmic reticulum (ER) being the critical site of viral replication factories. The sudden high protein load in the ER induces an ER stress to which the cell responds with an appropriate unfolded protein response (UPR) in an attempt to restore its disturbed homeostasis. When the restoration fails, the cell signalling leads to a programmed cell death by apoptosis with the upregulation of the UPR-induced C/EBP homologous protein (CHOP) which acts as the main trigger for this fatal outcome. Our previous studies have shown the ability of ZIKV to manipulate various cellular responses in order to optimize virus production. ZIKV is able to delay apoptosis to its benefit and although ER stress is induced, the UPR is not complete. Here we discovered that ZIKV impairs the expression of CHOP/DDIT3, the main factor responsible of ER-stress driven apoptosis. Surprisingly, the mechanism does not take place at the transcriptional level but at the translational level.

Keywords:  CHOP; ER stress; Zika virus; apoptosis; unfolded protein response

DOI:  https://doi.org/10.3390/ijms22073750
Int J Mol Sci. 2021 Apr 11. pii: 3934. [Epub ahead of print]22(8):

Endoplasmic Reticulum Stress Contributes to Gefitinib-Induced Apoptosis in Glioma.

Cheng-Yi Chang, Ping-Ho Pan, Chih-Cheng Wu, Su-Lan Liao, Wen-Ying Chen, Yu-Hsiang Kuan, Wen-Yi Wang, Chun-Jung Chen.

  Adequate stress on the Endoplasmic Reticulum (ER) with the Unfolded Protein Response (UPR) could maintain glioma malignancy. Uncontrolled ER stress, on the other hand, predisposes an apoptosis-dominant UPR program. We studied here the proapoptotic actions of the Epidermal Growth Factor Receptor (EGFR) inhibitor gefitinib, with the focus on ER stress. The study models were human H4 and U87 glioma cell lines. We found that the glioma cell-killing effects of gefitinib involved caspase 3 apoptotic cascades. Three branches of ER stress, namely Activating Transcription Factor-6 (ATF6), Protein Kinase R (PKR)-Like ER Kinase (PERK), and Inositol-Requiring Enzyme 1 (IRE1), were activated by gefitinib, along with the elevation of intracellular free Ca2+, Reactive Oxygen Species (ROS), and NADPH Oxidase2/4 (NOX2/4). Specifically, elevated IRE1 phosphorylation, Tumor Necrosis Factor (TNF) Receptor-Associated Factor-2 (TRAF2) expression, Apoptosis Signal-Regulating Kinase-1 (Ask1) phosphorylation, c-Jun N-Terminal Kinase (JNK) phosphorylation, and Noxa expression appeared in gefitinib-treated glioma cells. Genetic, pharmacological, and biochemical studies further indicated an active ROS/ER stress/Ask1/JNK/Noxa axis causing the glioma apoptosis induced by gefitinib. The findings suggest that ER-stress-based therapeutic targeting could be a promising option in EGFR inhibitor glioma therapy, and may ultimately achieve a better patient response.

Keywords:  EGFR inhibitors; ER stress; Noxa; apoptosis; glioma

DOI:  https://doi.org/10.3390/ijms22083934
J Drug Target. 2021 Apr 29. 1-9

Targeting E7 antigen to the endoplasmic reticulum degradation pathway promotes a potent therapeutic antitumor effect.

David Hernán Martínez-Puente, Rodolfo Garza-Morales, José Juan Pérez-Trujillo, Aracely García-García, Arnulfo Villanueva-Olivo, Humberto Rodríguez-Rocha, Laura Mireya Zavala-Flores, Odila Saucedo-Cárdenas, Roberto Montes de Oca-Luna, María de Jesús Loera-Arias.

  It has been previously reported that targeting and retaining antigens in the endoplasmic reticulum (ER) can induce an ER stress response. In this study, we evaluated the antitumor effect of E7 antigen fused to an ERresident protein, cyclooxygenase-2, which possesses a 19-aminoacid cassette that directs it to the endoplasmic reticulum-associated protein degradation (ERAD) pathway. The featured DNA constructs, COX2-E7 and COX2-E7ΔERAD, with a deletion in the 19-aminoacid cassette, were used to evaluate the importance of this sequence. In vitro analysis of protein expression and ER localisation were verified. We observed that both constructs induced an ER stress response. This finding correlated with the antitumor effect in mice injected with TC-1 cells and treated with different DNA constructs by biolistic vaccination. Immunisation with COX2-E7 and COX2-E7ΔERAD DNA constructs induced a significant antitumor effect in mice, without a significant difference between them, although the COX2-E7 construct induced a significant E7-specific immune response. These results demonstrate that targeting the E7 antigen to the ERAD pathway promotes a potent therapeutic antitumor effect. This strategy could be useful for the design of other antigen-specific therapies.

Keywords:  COX-2; DNA vaccine; E7; ER stress; GRP78/BiP; TC-1; endoplasmic reticulum; endoplasmic reticulum-associated protein degradation (ERAD); unfolded protein response (UPR)

DOI:  https://doi.org/10.1080/1061186X.2021.1919124
Physiology (Bethesda). 2021 May 01. 36(3): 150-159

The Stress of Lung Aging: Endoplasmic Reticulum and Senescence Tête-à-Tête.

M L Koloko Ngassie, C A Brandsma, R Gosens, Y S Prakash, J K Burgess.

  Beyond the structural changes, features including the dysregulation of endoplasmic reticulum (ER) stress response and increased senescence characterize the lung aging. ER stress response and senescence have been reported to be induced by factors like cigarette smoke. Therefore, deciphering the mechanisms underlying ER and senescent pathways interaction has become a challenge. In this review we highlight the known and unknown regarding ER stress response and senescence and their cross talk in aged lung.

Keywords:  COPD; cellular senescence; endoplasmic reticulum stress; lung aging; unfolded protein response

DOI:  https://doi.org/10.1152/physiol.00039.2020
Int J Mol Sci. 2021 Apr 14. pii: 4024. [Epub ahead of print]22(8):

The Epstein-Barr Virus Lytic Protein BMLF1 Induces Upregulation of GRP78 Expression through ATF6 Activation.

Lee-Wen Chen, Shie-Shan Wang, Chien-Hui Hung, Ya-Hui Hung, Chun-Liang Lin, Pey-Jium Chang.

  The unfolded protein response (UPR) is an intracellular signaling pathway essential for alleviating the endoplasmic reticulum (ER) stress. To support the productive infection, many viruses are known to use different strategies to manipulate the UPR signaling network. However, it remains largely unclear whether the UPR signaling pathways are modulated in the lytic cycle of Epstein-Barr virus (EBV), a widely distributed human pathogen. Herein, we show that the expression of GRP78, a central UPR regulator, is up-regulated during the EBV lytic cycle. Our data further revealed that knockdown of GRP78 in EBV-infected cell lines did not substantially affect lytic gene expression; however, GRP78 knockdown in these cells markedly reduced the production of virus particles. Importantly, we identified that the early lytic protein BMLF1 is the key regulator critically contributing to the activation of the grp78 gene promoter. Mechanistically, we found that BMLF1 can trigger the proteolytic cleavage and activation of the UPR senor ATF6, which then transcriptionally activates the grp78 promoter through the ER stress response elements. Our findings therefore provide evidence for the connection between the EBV lytic cycle and the UPR, and implicate that the BMLF1-mediated ATF6 activation may play critical roles in EBV lytic replication.

Keywords:  ATF6; BMLF1; Epstein-Barr virus; GRP78; lytic replication; unfolded protein response

DOI:  https://doi.org/10.3390/ijms22084024
Biochem Biophys Res Commun. 2021 Apr 21. pii: S0006-291X(21)00423-X. [Epub ahead of print]557 316-322

PERK activation by CCT020312 chemosensitizes colorectal cancer through inducing apoptosis regulated by ER stress.

Yunpeng Lei, Lirui He, Chang Yan, Yuchen Wang, Guoqing Lv.

  Endoplasmic reticulum (ER) stress is a significant mechanism for chemoresistance to colorectal cancer (CRC) treatment. The RNA-like endoplasmic reticulum kinase (PERK) is critical for ER stress induction. In the present study, we attempted to explore whether PERK activator CCT020312 (CCT) could be effective for CRC treatment, and reveal the underlying mechanisms. We first found that CCT dose- and time-dependently reduced CRC cell proliferation. Importantly, it markedly improved the chemosensitivity of CRC cells that were drug-sensitive or -resistant to taxol treatment, as evidenced by the significantly decreased cell viability. Moreover, CCT at the non-toxic concentration exhibited obviously synergistic effects with taxol to induce apoptosis and cell cycle arrest in G2/M phase in vitro. In addition, we showed that CCT alone considerably induced ER stress in CRC cells through a dose- and time-dependent fashion. Meanwhile, CCT combined with taxol caused significant ER stress through improving phosphorylated PERK, eukaryotic translation initiation factor 2α (eIF2ɑ), C/EBP homologous protein (CHOP) and glucose-regulated protein 78 (GRP78). More studies showed that the interaction between PERK and GRP78 was a potential target for CCT to perform its regulatory events. Intriguingly, PERK knockdown markedly abolished the regulatory role of CCT and taxol cotreatments in cell proliferation suppression and apoptosis induction, indicating the importance of PERK for CCT to perform its anti-cancer bioactivity. Our in vivo experiments confirmed that CCT plus taxol dramatically reduced tumor growth in CRC xenografts. Together, all these results suggested that promoting PERK activation by CCT may be an effective therapeutic strategy to improve CRC to taxol treatment.

Keywords:  Apoptosis; CCT020312; Colorectal cancer; ER stress; PERK

DOI:  https://doi.org/10.1016/j.bbrc.2021.03.041
Front Mol Biosci. 2021 ;8 620514

The Road of Solid Tumor Survival: From Drug-Induced Endoplasmic Reticulum Stress to Drug Resistance.

Shulong Cao, Jingyi Tang, Yichun Huang, Gaofeng Li, Zhuoya Li, Wenqi Cai, Yuning Yuan, Junlong Liu, Xuqun Huang, Haiyuan Zhang.

  Endoplasmic reticulum stress (ERS), which refers to a series of adaptive responses to the disruption of endoplasmic reticulum (ER) homeostasis, occurs when cells are treated by drugs or undergo microenvironmental changes that cause the accumulation of unfolded/misfolded proteins. ERS is one of the key responses during the drug treatment of solid tumors. Drugs induce ERS by reactive oxygen species (ROS) accumulation and Ca2+ overload. The unfolded protein response (UPR) is one of ERS. Studies have indicated that the mechanism of ERS-mediated drug resistance is primarily associated with UPR, which has three main sensors (PERK, IRE1α, and ATF6). ERS-mediated drug resistance in solid tumor cells is both intrinsic and extrinsic. Intrinsic ERS in the solid tumor cells, the signal pathway of UPR-mediated drug resistance, includes apoptosis inhibition signal pathway, protective autophagy signal pathway, ABC transporter signal pathway, Wnt/β-Catenin signal pathway, and noncoding RNA. Among them, apoptosis inhibition is one of the major causes of drug resistance. Drugs activate ERS and its downstream antiapoptotic proteins, which leads to drug resistance. Protective autophagy promotes the survival of solid tumor cells by devouring the damaged organelles and other materials and providing new energy for the cells. ERS induces protective autophagy by promoting the expression of autophagy-related genes, such as Beclin-1 and ATG5-ATG12. ABC transporters pump drugs out of the cell, which reduces the drug-induced apoptosis effect and leads to drug resistance. In addition, the Wnt/β-catenin signal pathway is also involved in the drug resistance of solid tumor cells. Furthermore, noncoding RNA regulates the ERS-mediated survival and death of solid tumor cells. Extrinsic ERS in the solid tumor cells, such as ERS in immune cells of the tumor microenvironment (TME), also plays a crucial role in drug resistance by triggering immunosuppression. In immune system cells, ERS in dendritic cells (DCs) and myeloid-derived suppressor cells (MDSCs) influences the antitumor function of normal T cells, which results in immunosuppression. Meanwhile, ERS in T cells can also cause impaired functioning and apoptosis, leading to immunosuppression. In this review, we highlight the core molecular mechanism of drug-induced ERS involved in drug resistance, thereby providing a new strategy for solid tumor treatment.

Keywords:  drug resistance; endoplasmic reticulum stress; immunosuppression; solid tumor; unfolded protein response

DOI:  https://doi.org/10.3389/fmolb.2021.620514
Biochem J. 2021 Apr 26. pii: BCJ20210155. [Epub ahead of print]

FATP4 inactivation in cultured macrophages attenuates M1- and ER stress-induced cytokine release via a metabolic shift towards triacylglycerides.

Yuling Zhang, Ning Wu, Hongying Gan-Schreier, Feng Xu, Sabine Tuma-Kellner, Simone Staffer, Jessica Seessle, Uta Merle, Walee Chamulitrat.

  Fatty acid transport protein 4 (FATP4) belongs to a family of acyl-CoA synthetases which activate long-chain fatty acids into acyl-CoAs subsequently used in specific metabolic pathways. Patients with FATP4 mutations and Fatp4-null mice show thick desquamating skin and other complications, however, FATP4 role on macrophage functions has not been studied. We here determined whether the levels of macrophage glycerophospholipids, sphingolipids including ceramides, triacylglycerides, and cytokine release could be altered by FATP4 inactivation. Two in vitro experimental systems were studied: FATP4-knockdown in THP-1-derived macrophages undergoing M1 (LPS+IFNγ) or M2 (IL-4) activation and bone marrow-derived macrophages (BMDMs) from macrophage-specific Fatp4-knockout (Fatp4M-/-) mice undergoing tunicamycin (TM)-induced ER stress. FATP4-deficient macrophages showed a metabolic shift towards triacylglycerides and were protected from M1- or TM-induced release of pro-inflammatory cytokines and cellular injury. Fatp4M-/- BMDMs showed specificity in attenuating TM-induced activation of inositol-requiring enzyme1α, but not other unfolded protein response pathways. Under basal conditions, FATP4/Fatp4 deficiency decreased the levels of ceramides and induced an upregulation of mannose receptor CD206 expression. The deficiency led to an attenuation of IL-8 release in THP-1 cells as well as TNF-α and IL-12 release in BMDMs. Thus, FATP4 functions as an acyl-CoA synthetase in macrophages and its inactivation suppresses the release of pro-inflammatory cytokines by shifting fatty acids towards the synthesis of specific lipids.

Keywords:  ER stress; ceramides; fatty acid transport protein 4; lipidomics; polarized macrophages; triacylglycerides

DOI:  https://doi.org/10.1042/BCJ20210155
Circulation. 2021 Apr 30.

The Unfolded Protein Response as a Compensatory Mechanism and Potential Therapeutic Target in PLN R14del Cardiomyopathy.

Dries A M Feyen, Isaac Perea-Gil, Renee G C Maas, Magdalena Harakalova, Alexandra A Gavidia, Jennifer Arthur Ataam, Ting-Hsuan Wu, Aryan Vink, Jiayi Pei, Nirmal Vadgama, Albert J Suurmeijer, Wouter P Te Rijdt, Michelle Vu, Prashila L Amatya, Maricela Prado, Yuan Zhang, Logan Dunkenberger, Joost P G Sluijter, Karim Sallam, Folkert W Asselbergs, Mark Mercola, Ioannis Karakikes.

  Background: Phospholamban (PLN) is a critical regulator of calcium cycling and contractility in the heart. The loss of arginine at position 14 in PLN (R14del) is associated with dilated cardiomyopathy (DCM) with a high prevalence of ventricular arrhythmias. How the R14 deletion causes DCM is poorly understood and there are no disease-specific therapies. Methods: We used single-cell RNA sequencing to uncover PLN R14del disease-mechanisms in human induced pluripotent stem cells (hiPSC-CMs). We utilized both 2D and 3D functional contractility assays to evaluate the impact of modulating disease relevant pathways in PLN R14del hiPSC-CMs. Results: Modeling of the PLN R14del cardiomyopathy with isogenic pairs of hiPSC-CMs recapitulated the contractile deficit associated with the disease in vitro. Single-cell RNA sequencing revealed the induction of the unfolded protein response pathway (UPR) in PLN R14del compared to isogenic control hiPSC-CMs. The activation of UPR was also evident in the hearts from PLN R14del patients. Silencing of each of the three main UPR signaling branches (IRE1, ATF6, or PERK) by siRNA exacerbated the contractile dysfunction of PLN R14del hiPSC-CMs. We explored the therapeutic potential of activating the UPR with a small molecule activator, BiP protein Inducer X (BiX). PLN R14del hiPSC-CMs treated with BiX showed a dose-dependent amelioration of the contractility deficit of in both 2D cultures and 3D engineered heart tissues without affecting calcium homeostasis. Conclusions: Together, these findings suggest that the UPR exerts a protective effect in the setting of PLN R14del cardiomyopathy and that modulation of the UPR might be exploited therapeutically.

Keywords:  UPR; disease modeling; induced pluripotent stem cells; single-cell RNA sequencing

DOI:  https://doi.org/10.1161/CIRCULATIONAHA.120.049844
J Biol Chem. 2021 Apr 22. pii: S0021-9258(21)00453-1. [Epub ahead of print] 100665

Oxidation of peroxiredoxin-4 induces oligomerization and promotes interaction with proteins governing protein folding and endoplasmic reticulum stress.

Evan A Elko, Allison M Manuel, Sheryl White, Ester Zito, Albert van der Vliet, Vikas Anathy, Yvonne M W Janssen-Heininger.

  Peroxiredoxins catalyze the reduction of hydrogen peroxide (H2O2). Peroxiredoxin 4 (PRDX4) is the only peroxiredoxin located within the endoplasmic reticulum (ER), and is the most highly expressed H2O2 scavenger in the ER. PRDX4 has emerged as an important player in numerous diseases such as fibrosis and metabolic syndromes, and its over-oxidation is a potential indicator of ER redox stress. It is unclear how over-oxidation of PRDX4 governs its oligomerization state and interacting partners. Herein we addressed these questions via non-reducing Western blots, mass spectrometry and site-directed mutagenesis. We report that the oxidation of PRDX4 in lung epithelial cells treated with tertbutyl hydroperoxide (TBuOOH) caused a shift of PRDX4 from monomer/dimer to high molecular weight (HMW) species, which contain PRDX4 modified with sulfonic acid residues (PRDX4-SO3), as well as of a complement of ER-associated proteins, including protein disulfide isomerases important in protein folding, thioredoxin domain containing protein 5, and heat shock protein A5, a key regulator of the ER stress response. Mutation of any of the four cysteines in PRDX4 altered the HMW species in response to TBuOOH, as well as the secretion of PRDX4. We also demonstrate that the expression of ER oxidoreductase 1 (ERO1a), which generates H2O2 in the ER, increased PRDX4 HMW formation and secretion. These results suggest a link between SO3 modification in the formation of HMW PRDX4 complexes in cells, while the association of key regulators of ER homeostasis with HMW oxidized PRDX4 point to a putative role of PRDX4 in regulating ER stress responses.

Keywords:  Airway Epithelial Cells; ER stress; Lung; Peroxiredoxin 4

DOI:  https://doi.org/10.1016/j.jbc.2021.100665
Biochem Biophys Res Commun. 2021 Apr 21. pii: S0006-291X(21)00658-6. [Epub ahead of print]558 1-7

Establishment of a reporter system for monitoring activation of the ER stress transducer ATF6β.

Le Thi Hien, Sung Hoon Back.

  ATF6 has two isoforms, ATF6α and ATF6β, which are ubiquitously expressed type II transmembrane glycoproteins in the endoplasmic reticulum (ER). While the regulatory mechanisms and transcriptional roles of ATF6α in response to ER stress have been well-studied, those of its paralogue ATF6β are less understood. Moreover, there is no specific cell-based reporter assay to monitor ATF6β activation. Here, we developed a new cell-based reporter system that can monitor activation of endogenous ATF6β. This system expresses a chimeric protein containing a synthetic transcription factor followed by the transmembrane domain and C-terminal luminal domain of ATF6β. Under ER stress conditions, the chimeric protein was cleaved by regulated intramembrane proteolysis (RIP) to liberate the N-terminal synthetic transcription factor, which induced luciferase expression in the HeLa Luciferase Reporter cell line. This new stable reporter cell line will be an innovative tool to investigate RIP of ATF6β.

Keywords:  ATF6β; ER stress; GAL4 binding site; Luciferase; Regulated intramembrane proteolysis; Reporter cell line

DOI:  https://doi.org/10.1016/j.bbrc.2021.04.052
Cancer Med. 2021 May 01.

ONC201 induces the unfolded protein response (UPR) in high- and low-grade ovarian carcinoma cell lines and leads to cell death regardless of platinum sensitivity.

Marufa Rumman, Steven Buck, Lisa Polin, Sijana Dzinic, Julie Boerner, Ira S Winer.

   OBJECTIVES: Treatment of both platinum resistant high grade (HG) and low-grade (LG) ovarian cancer (OVCA) poses significant challenges as neither respond well to conventional chemotherapy leading to morbidity and mortality. Identification of novel agents that can overcome chemoresistance is therefore critical. Previously, we have demonstrated that OVCA has basal upregulated unfolded protein response (UPR) and that targeting cellular processes leading to further and persistent upregulation of UPR leads to cell death. ONC201 is an orally bioavailable Dopamine Receptor D2 inhibitor demonstrating anticancer activity and was found to induce UPR. Given its unique properties, we hypothesized that ONC201 would overcome platinum resistance in OVCA.
METHODS: Cisplatin sensitive and resistant HG OVCA and two primary LG OVCA cell lines were studied. Cell viability was determined using MTT assay. Cell migration was studied using wound healing assay. Apoptosis and mitochondrial membrane potential were investigated using flow cytometry. Analysis of pathway inhibition was performed by Western Blot. mRNA expression of UPR related genes were measured by qPCR. In vivo studies were completed utilizing axillary xenograft models. Co-testing with conventional chemotherapy was performed to study synergy.
RESULTS: ONC201 significantly inhibited cell viability and migration in a dose dependent manner with IC50's from 1-20 µM for both cisplatin sensitive and resistant HG and LG-OVCA cell lines. ONC201 lead to upregulation of the pro-apoptotic arm of the UPR, specifically ATF-4/CHOP/ATF3 and increased the intrinsic apoptosispathway. The compensatory, pro-survival PI3K/AKT/mTOR pathway was downregulated. In vivo, weekly dosing of single agent ONC201 decreased xenograft tumor size by ~50% compared to vehicle. ONC201 also demonstrated significant synergy with paclitaxel in a highly platinum resistant OVCA cell-line (OV433).
CONCLUSIONS: Our findings demonstrate that ONC201 can effectively overcome chemoresistance in OVCA cells by blocking pro-survival pathways and inducing the apoptotic arm of the UPR. This is a promising, orallybioavailable therapeutic agent to consider in clinical trials for patients with both HG and LG OVCA.

Keywords:  ONC201; apoptosis; high-grade ovarian cancer; low-grade ovarian cancer; unfolded protein response

DOI:  https://doi.org/10.1002/cam4.3858
Int J Mol Sci. 2021 Apr 22. pii: 4373. [Epub ahead of print]22(9):

Resveratrol Alleviates the Inhibitory Effect of Tunicamycin-Induced Endoplasmic Reticulum Stress on Expression of Genes Involved in Thyroid Hormone Synthesis in FRTL-5 Thyrocytes.

Gaiping Wen, Klaus Eder, Robert Ringseis.

  Recently, ER stress induced by tunicamycin (TM) was reported to inhibit the expression of key genes involved in thyroid hormone synthesis, such as sodium/iodide symporter (NIS), thyroid peroxidase (TPO) and thyroglobulin (TG), and their regulators such as thyrotropin receptor (TSHR), thyroid transcription factor-1 (TTF-1), thyroid transcription factor-2 (TTF-2) and paired box gene 8 (PAX-8), in FRTL-5 thyrocytes. The present study tested the hypothesis that resveratrol (RSV) alleviates this effect of TM in FRTL-5 cells. While treatment of FRTL-5 cells with TM alone (0.1 µg/mL) for 48 h strongly induced the ER stress-sensitive genes heat shock protein family A member 5 (HSPA5) and DNA damage inducible transcript 3 (DDIT3) and repressed NIS, TPO, TG, TSHR, TTF-1, TTF-2 and PAX-8, combined treatment with TM (0.1 µg/mL) and RSV (10 µM) for 48 h attenuated this effect of TM. In conclusion, RSV alleviates TM-induced ER stress and attenuates the strong impairment of expression of genes involved in thyroid hormone synthesis and their regulators in FRTL-5 thyrocytes exposed to TM-induced ER stress. Thus, RSV may be useful for the treatment of specific thyroid disorders, provided that strategies with improved oral bioavailability of RSV are applied.

Keywords:  FRTL-5; NIS; endoplasmic reticulum stress; iodide uptake; resveratrol; thyroid

DOI:  https://doi.org/10.3390/ijms22094373
Int J Mol Sci. 2021 Apr 26. pii: 4538. [Epub ahead of print]22(9):

Different Expression of Mitochondrial and Endoplasmic Reticulum Stress Genes in Epicardial Adipose Tissue Depends on Coronary Atherosclerosis.

Helena Kratochvílová, Miloš Mráz, Barbora J Kasperová, Daniel Hlaváček, Jakub Mahrík, Ivana Laňková, Anna Cinkajzlová, Zdeněk Matloch, Zdeňka Lacinová, Jaroslava Trnovská, Peter Ivák, Peter Novodvorský, Ivan Netuka, Martin Haluzík.

  The aim of our study was to analyze mitochondrial and endoplasmic reticulum (ER) gene expression profiles in subcutaneous (SAT) and epicardial (EAT) adipose tissue, skeletal muscle, and myocardium in patients with and without CAD undergoing elective cardiac surgery. Thirty-eight patients, 27 with (CAD group) and 11 without CAD (noCAD group), undergoing coronary artery bypass grafting and/or valvular surgery were included in the study. EAT, SAT, intercostal skeletal muscle, and right atrium tissue and blood samples were collected at the start and end of surgery; mRNA expression of selected mitochondrial and ER stress genes was assessed using qRT-PCR. The presence of CAD was associated with decreased mRNA expression of most of the investigated mitochondrial respiratory chain genes in EAT, while no such changes were seen in SAT or other tissues. In contrast, the expression of ER stress genes did not differ between the CAD and noCAD groups in almost any tissue. Cardiac surgery further augmented mitochondrial dysfunction in EAT. In our study, CAD was associated with decreased expression of mitochondrial, but not endoplasmic reticulum stress genes in EAT. These changes may contribute to the acceleration of coronary atherosclerosis.

Keywords:  cardiac surgery; coronary artery disease; diabetes mellitus; endoplasmic reticulum stress; epicardial fat; gene expression; inflammation; mitochondrial dysfunction

DOI:  https://doi.org/10.3390/ijms22094538
Cells. 2021 Apr 23. pii: 996. [Epub ahead of print]10(5):

Traumatic Optic Neuropathy Is Associated with Visual Impairment, Neurodegeneration, and Endoplasmic Reticulum Stress in Adolescent Mice.

Shelby M Hetzer, Fernanda Guilhaume-Correa, Dylan Day, Alicia Bedolla, Nathan K Evanson.

  Traumatic brain injury (TBI) results in a number of impairments, often including visual symptoms. In some cases, visual impairments after head trauma are mediated by traumatic injury to the optic nerve, termed traumatic optic neuropathy (TON), which has few effective options for treatment. Using a murine closed-head weight-drop model of head trauma, we previously reported in adult mice that there is relatively selective injury to the optic tract and thalamic/brainstem projections of the visual system. In the current study, we performed blunt head trauma on adolescent C57BL/6 mice and investigated visual impairment in the primary visual system, now including the retina and using behavioral and histologic methods at new time points. After injury, mice displayed evidence of decreased optomotor responses illustrated by decreased optokinetic nystagmus. There did not appear to be a significant change in circadian locomotor behavior patterns, although there was an overall decrease in locomotor behavior in mice with head injury. There was evidence of axonal degeneration of optic nerve fibers with associated retinal ganglion cell death. There was also evidence of astrogliosis and microgliosis in major central targets of optic nerve projections. Further, there was elevated expression of endoplasmic reticulum (ER) stress markers in retinas of injured mice. Visual impairment, histologic markers of gliosis and neurodegeneration, and elevated ER stress marker expression persisted for at least 30 days after injury. The current results extend our previous findings in adult mice into adolescent mice, provide direct evidence of retinal ganglion cell injury after head trauma and suggest that axonal degeneration is associated with elevated ER stress in this model of TON.

Keywords:  ER stress; adolescent head trauma; head trauma; mice; traumatic optic neuropathy

DOI:  https://doi.org/10.3390/cells10050996
Mol Cell. 2021 Apr 23. pii: S1097-2765(21)00312-9. [Epub ahead of print]

The oxidoreductase PYROXD1 uses NAD(P)+ as an antioxidant to sustain tRNA ligase activity in pre-tRNA splicing and unfolded protein response.

Igor Asanović, Emilia Strandback, Alena Kroupova, Djurdja Pasajlic, Anton Meinhart, Pai Tsung-Pin, Nemanja Djokovic, Dorothea Anrather, Thomas Schuetz, Marcin Józef Suskiewicz, Sirelin Sillamaa, Thomas Köcher, Rebecca Beveridge, Katarina Nikolic, Alexander Schleiffer, Martin Jinek, Markus Hartl, Tim Clausen, Josef Penninger, Peter Macheroux, Stefan Weitzer, Javier Martinez.

  The tRNA ligase complex (tRNA-LC) splices precursor tRNAs (pre-tRNA), and Xbp1-mRNA during the unfolded protein response (UPR). In aerobic conditions, a cysteine residue bound to two metal ions in its ancient, catalytic subunit RTCB could make the tRNA-LC susceptible to oxidative inactivation. Here, we confirm this hypothesis and reveal a co-evolutionary association between the tRNA-LC and PYROXD1, a conserved and essential oxidoreductase. We reveal that PYROXD1 preserves the activity of the mammalian tRNA-LC in pre-tRNA splicing and UPR. PYROXD1 binds the tRNA-LC in the presence of NAD(P)H and converts RTCB-bound NAD(P)H into NAD(P)+, a typical oxidative co-enzyme. However, NAD(P)+ here acts as an antioxidant and protects the tRNA-LC from oxidative inactivation, which is dependent on copper ions. Genetic variants of PYROXD1 that cause human myopathies only partially support tRNA-LC activity. Thus, we establish the tRNA-LC as an oxidation-sensitive metalloenzyme, safeguarded by the flavoprotein PYROXD1 through an unexpected redox mechanism.

Keywords:  NADH; NADPH; PYROXD1; RtcB; UPR; copper; metalloenzyme; myopathy; oxidative stress; oxidoreductase; pre-tRNA splicing; tRNA ligase complex

DOI:  https://doi.org/10.1016/j.molcel.2021.04.007