bims-unfpre 2019-03-31 papers

bims-unfpre

Biomed News

on Unfolded protein response

Issue of 2019–03–31
twelve papers selected by
Susan Logue, Apoptosis Research Centre

The kinase PERK and the transcription factor ATF4 play distinct and essential roles in autophagy resulting from tunicamycin-induced ER stress.
HSP-4/BiP expression in secretory cells is regulated by a developmental program and not by the unfolded protein response.
Endoplasmic reticulum-associated degradation potentiates the infectivity of influenza A virus by regulating the host redox state.
Endoplasmic Reticulum Stress in Metabolic Liver Diseases and Hepatic Fibrosis.
The UPR-PERK pathway is not a promising therapeutic target for mutant SOD1-induced ALS.
Unfolded Protein Response Pathways Correlatively Modulate Endoplasmic Reticulum Stress Responses in Rat Retinal Müller Cells.
Upregulation of CHOP participates in caspase activation and virus release in human astrovirus-infected cells.
Cell surface expression of 78 kDa glucose regulated protein (GRP78) mediates diabetic nephropathy.
Salubrinal, a novel inhibitor of eIF-2α dephosphorylation, promotes erythropoiesis at early stage targeted by ufmylation pathway.
Pancreatic stone protein/regenerating protein is a potential biomarker for endoplasmic reticulum stress in beta cells.
Assays for protein retrotranslocation in ERAD.
CircRNA-012091/PPP1R13B-Mediated Lung Fibrotic Response in Silicosis via ER Stress and Autophagy.

J Biol Chem. 2019 Mar 29. pii: jbc.RA118.002829. [Epub ahead of print]

The kinase PERK and the transcription factor ATF4 play distinct and essential roles in autophagy resulting from tunicamycin-induced ER stress.

Morten Luhr, Maria Lyngaas Torgersen, Paula Szalai, Adnan Hashim, Andreas Brech, Judith Staerk, Nikolai Engedal.

  Endoplasmic reticulum (ER) stress is thought to activate autophagy via unfolded protein response (UPR)-mediated transcriptional up-regulation of autophagy machinery components, and modulation of microtubule-associated protein 1 light chain 3 (LC3). The upstream UPR constituents pancreatic EIF2-α kinase (PERK) and inositol-requiring enzyme 1 (IRE1) have been reported to mediate these effects, suggesting that UPR may stimulate autophagy via PERK and IRE1. However, how the UPR and its components affect autophagic activity has not been thoroughly examined. By analyzing the flux of LC3 through the autophagic pathway, as well as the sequestration and degradation of autophagic cargo, we here conclusively show that the classical ER stressor tunicamycin (TM) enhances autophagic activity in mammalian cells. PERK and its downstream factor, activating transcription factor 4 (ATF4), were crucial for this induction, but surprisingly, IRE1 constitutively suppressed autophagic activity. TM-induced autophagy required autophagy related 13 (ATG13), Unc-51-like autophagy-activating kinases 1/2 (ULK1/ULK2), and GABA type A receptor-associated proteins (GABARAPs), but interestingly, LC3 proteins appeared to be redundant. Strikingly, ATF4 was activated independently of PERK in both LNCaP and HeLa cells, and our further examination revealed that ATF4 and PERK regulated autophagy through separate mechanisms. Specifically, whereas ATF4 controlled transcription and was essential for autophagosome formation, PERK acted in a transcription-independent manner and was required at a post-sequestration step in the autophagic pathway. In conclusion, our results indicate that TM-induced UPR activates functional autophagy, and whereas IRE1 is a negative regulator, PERK and ATF4 are required at distinct steps in the autophagic pathway.

Keywords:  GABA type A receptor-associated protein (GABARAP); activating transcription factor 4 (ATF4); autophagic sequestration; autophagy; endoplasmic reticulum stress (ER stress); microtubule-associated protein 1 light chain 3 (LC3); pancreatic EIF2-α kinase (PERK); protein degradation; signal transduction; unfolded protein response (UPR)

DOI:  https://doi.org/10.1074/jbc.RA118.002829
PLoS Biol. 2019 Mar 25. 17(3): e3000196

HSP-4/BiP expression in secretory cells is regulated by a developmental program and not by the unfolded protein response.

Ji Zha, Mingjie Ying, Jasmine Alexander-Floyd, Tali Gidalevitz.

Differentiation of secretory cells leads to sharp increases in protein synthesis, challenging endoplasmic reticulum (ER) proteostasis. Anticipatory activation of the unfolded protein response (UPR) prepares cells for the onset of secretory function by expanding the ER size and folding capacity. How cells ensure that the repertoire of induced chaperones matches their postdifferentiation folding needs is not well understood. We find that during differentiation of stem-like seam cells, a typical UPR target, the Caenorhabditis elegans immunoglobulin heavy chain-binding protein (BiP) homologue Heat-Shock Protein 4 (HSP-4), is selectively induced in alae-secreting daughter cells but is repressed in hypodermal daughter cells. Surprisingly, this lineage-dependent induction bypasses the requirement for UPR signaling. Instead, its induction in alae-secreting cells is controlled by a specific developmental program, while its repression in the hypodermal-fated cells requires a transcriptional regulator B-Lymphocyte-Induced Maturation Protein 1 (BLMP-1/BLIMP1), involved in differentiation of mammalian secretory cells. The HSP-4 induction is anticipatory and is required for the integrity of secreted alae. Thus, differentiation programs can directly control a broad-specificity chaperone that is normally stress dependent to ensure the integrity of secreted proteins.

DOI: https://doi.org/10.1371/journal.pbio.3000196
Free Radic Biol Med. 2019 Mar 21. pii: S0891-5849(19)30029-2. [Epub ahead of print]

Endoplasmic reticulum-associated degradation potentiates the infectivity of influenza A virus by regulating the host redox state.

Kwang Il Jung, Nary Shin, Dong-Hyun Ko, Chul Woong Pyo, Sang-Yun Choi.

  During influenza A virus (IAV) infection, significant effects of oxidative stress often emerge due to the disruption of the redox balance. Reactive oxygen species (ROS) generated during IAV infection have been known to exert various effects on both the virus and host tissue. However, the mechanisms underlying the accumulation of ROS and their physiological significance in IAV infection have been extensively studied but remain to be fully understood. Here, we show that the levels of Sp1, a key controller of Cu-Zn superoxide dismutase (SOD1) gene expression, and SOD1 are mainly dependent upon the activity of X-box-binding protein 1 (XBP1), which is a downstream factor of the endoplasmic reticulum (ER) transmembrane sensor inositol-requiring enzyme 1 (IRE1) during ER stress. In IRE1-deficient mouse embryo fibroblasts (MEFs) or A549 human lung cells treated with XBP1 siRNA, IAV-induced Sp1 loss was mitigated. However, overexpression of the spliced form of XBP1 in IRE1-deficient MEFs resulted in a further decrease in Sp1 levels, whereas the unspliced form showed no significant differences. Treatment with proteasome inhibitor MG132 markedly inhibited the IRE1/XBP1-mediated loss of Sp1 and SOD, suggesting the involvement of proteasome-dependent ER-associated degradation (ERAD). The increase in SOD1 levels with the expression of siRNA-targeting p97, a central component of the ubiquitin-proteasome system, supports the major role of the ERAD process in IAV-mediated SOD1 loss. In addition, ROS generation due to IAV infection was attenuated in cells lacking either IRE1 or JNK. These results reveal the important roles of both IRE1/XBP1-mediated ERAD and the JNK pathway in IAV infection. Interestingly, the increase in ROS due to IAV infection is correlated with the increase in the virus titer in vitro and in vivo. However, 4-phenylbutyrate (4-PBA), an inhibitor of ER stress signaling, weakened the effect of IAV infection on SOD1 loss in a dose-dependent manner. Furthermore, the treatment of mice with 4-PBA efficiently attenuated ROS generation and ER stress in lung tissue and eventually lowered the IAV titer. These results strongly suggest that the ERAD process plays a major role in IAV infection, thus making it a potential target for antiviral drug therapy.

Keywords:  ER stress; Influenza; ROS; SOD1

DOI:  https://doi.org/10.1016/j.freeradbiomed.2019.03.021
Semin Liver Dis. 2019 Mar 25.

Endoplasmic Reticulum Stress in Metabolic Liver Diseases and Hepatic Fibrosis.

Jessica L Maiers, Harmeet Malhi.

Endoplasmic reticulum (ER) stress is a major contributor to liver disease and hepatic fibrosis, but the role it plays varies depending on the cause and progression of the disease. Furthermore, ER stress plays a distinct role in hepatocytes versus hepatic stellate cells (HSCs), which adds to the complexity of understanding ER stress and its downstream signaling through the unfolded protein response (UPR) in liver disease. Here, the authors focus on the current literature of ER stress in nonalcoholic and alcoholic fatty liver diseases, how ER stress impacts hepatocyte injury, and the role of ER stress in HSC activation and hepatic fibrosis. This review provides insight into the complex signaling and regulation of the UPR, parallels and distinctions between different liver diseases, and how ER stress may be targeted as an antisteatotic or antifibrotic therapy to limit the progression of liver disease.

DOI: https://doi.org/10.1055/s-0039-1681032
Neurobiol Dis. 2019 Mar 25. pii: S0969-9961(19)30077-4. [Epub ahead of print]

The UPR-PERK pathway is not a promising therapeutic target for mutant SOD1-induced ALS.

Yulia Dzhashiashvili, Chase P Monckton, Harini S Shah, Rejani B Kunjamma, Brian Popko.

  Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease, characterized by motor neuron death in the brain and spinal cord. Mutations in the Cu/Zn superoxide dismutase (SOD1) gene account for ~20% of all familial ALS forms, corresponding to 1%-2% of all ALS cases. One of the suggested mechanisms by which mutant SOD1 (mtSOD1) exerts its toxic effects involves intracellular accumulation of abnormal mtSOD1 aggregates, which trigger endoplasmic reticulum (ER) stress and activate its adaptive signal transduction pathways, including the unfolded protein response (UPR). PERK, an eIF2α kinase, is central to the UPR and is the most rapidly activated pathway in response to ER stress. Previous reports using mtSOD1 transgenic mice indicated that genetic or pharmacological enhancement of the UPR-PERK pathway may be effective in treating ALS. We investigated the response to PERK haploinsufficiency, and the response to deficiency of its downstream effectors GADD34 and CHOP, in five distinct lines of mtSOD1 mice. We demonstrate that, in contrast to a previously published study, PERK haploinsufficiency has no effect on disease in all mtSOD1 strains examined. We also show that deficiency of GADD34, which enhances the UPR by prolonging the phosphorylation of eIF2α,does not ameliorate disease in these mtSOD1 mouse strains. Finally, we demonstrate that genetic ablation of CHOP transcription factor, which is known to be pro-apoptotic, does not ameliorate disease in mtSOD1 mice. Cumulatively, our studies reveal that neither genetic inhibition of the UPR via ablation of PERK, nor genetic UPR enhancement via ablation of GADD34, is beneficial for mtSOD1-induced motor neuron disease. Therefore, the PERK pathway is not a likely target for therapeutic intervention in ALS.

Keywords:  Amyotrophic lateral sclerosis; CHOP; Endoplasmic reticulum stress; GADD34; Motor neurons; PERK; SOD1 mice; Unfolded protein response

DOI:  https://doi.org/10.1016/j.nbd.2019.03.024
J Ophthalmol. 2019 ;2019 9028483

Unfolded Protein Response Pathways Correlatively Modulate Endoplasmic Reticulum Stress Responses in Rat Retinal Müller Cells.

Shengyu Wu, Xiaolu Zhu, Biechuan Guo, Tian Zheng, Jiangbo Ren, Wen Zeng, Xiaomin Chen, Min Ke.

Background: Endoplasmic reticulum stress (ERS) in the retinal Müller cells is a key factor contributing to the retinal inflammation and vascular leakage in diabetic retinopathy (DR). This study was to investigate the underlying mechanisms through which the 3 main unfolded protein response (UPR) pathways regulate ERS and to examine the expression levels of vascular endothelial growth factor (VEGF) in Müller cells in vitro.
Methods: Rat Müller cell lines were stimulated with high glucose to mimic a diabetic environment in vitro. PKR-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6) were downregulated or upregulated with shRNA or overexpression plasmids. The transfected Müller cells were cultivated in high glucose medium for 48 hours. Expression of glucose-regulated protein 78 (GRP78), activating transcription factor 4 (ATF4), X-box binding protein 1 (XBP1), ATF6, and VEGF was examined with immunofluorescence and western blot.
Results: Our data indicated that ERS was found in both high glucose and osmotic control groups. Overexpression or downregulation of UPR pathways effectively increased or reduced the production of GRP78, ATF4, XBP1, ATF6, and VEGF, respectively. These 3 signaling pathways had similar regulatory effects on VEGF.
Conclusion: The 3 UPR-mediated inflammatory pathways were dependent on each other. Inhibition any of these signaling pathways in UPR might be a potential therapeutic target for DR.

DOI: https://doi.org/10.1155/2019/9028483
J Gen Virol. 2019 Mar 26.

Upregulation of CHOP participates in caspase activation and virus release in human astrovirus-infected cells.

Tomoyasu Isobe, Shoichiro Tange, Hidetaka Tasaki, Kumiko Kanamori, Akiko Kato, Akira Nakanishi.

Human astroviruses (HAstVs), non-enveloped RNA viruses with positive-sense RNA genomes, are an important cause of acute gastroenteritis in young children, although the processes that produce infectious virions are not clearly defined. To track the viral replication complex (RC) upon HAstV1 infection, the subcellular distribution of double-stranded (ds) RNA and of ORF1b, a viral RNA polymerase, was examined by immunocytochemistry. Foci that were positive for dsRNA and for ORF1b were co-localized, and both foci were also co-localized with resident proteins of the endoplasmic reticulum (ER). Focusing on the association between the HAstV RC and ER, we examined the expression of unfolded protein response (UPR) markers and found that targets of eukaryotic translation initiation factor 2α (eIF2α)-activating transcription factor 4 (ATF4), including CCAAT/enhancer-binding protein homologous protein (CHOP), a proapoptotic transcription factor, were upregulated at the late phase in HAstV-infected cells. Consistently, eIF2α was phosphorylated at the late phase of HAstV infection. The formation of foci resembling stress granules, another known downstream response to eIF2α phosphorylation, was also observed at the same period. Phosphorylation of eIF2α was attenuated in protein kinase R (PKR)-knockdown cells, suggesting that, unlike the canonical ER stress response, PKR was involved in eIF2α phosphorylation in response to HAstV infection. Studies have indicated that immature HAstV capsid protein is processed by caspases, and caspase cleavage is integral to particle release. Inhibition of CHOP upregulation reduced caspase activation and the release of HAstV RNA from cells during HAstV infection. Our results suggest that the eIF2α-ATF4-CHOP pathway participates in HAstV propagation.

DOI: https://doi.org/10.1099/jgv.0.001250
J Biol Chem. 2019 Mar 26. pii: jbc.RA118.006939. [Epub ahead of print]

Cell surface expression of 78 kDa glucose regulated protein (GRP78) mediates diabetic nephropathy.

Richard Van Krieken, Neel Mehta, Tony Wang, Mengyu Zheng, Renzhong Li, Bo Gao, Ehab Ayaub, Kjetil Ask, James C Paton, Adrienne W Paton, Richard C Austin, Joan C Krepinsky.

  The 78 kDa glucose-regulated protein (GRP78) is a well-established endoplasmic reticulum (ER)-resident chaperone that maintains protein homeostasis and regulates the unfolded protein response. Under conditions of ER stress, GRP78 is also expressed at the cell surface and implicated in tumorigenesis, immunity, and cellular signaling events. The role of cell surface-associated GRP78 (csGRP78) in the pathogenesis of diabetic nephropathy has not yet been defined. Here, we explored the role of csGRP78 in regulating high glucose (HG)-induced profibrotic AKT Ser/Thr kinase (AKT) signaling and upregulation of extracellular matrix (ECM) proteins. Using primary kidney mesangial cells (MCs), we show that HG treatment, but not the osmotic control mannitol, induces csGRP78 expression through an ER stress-dependent mechanism. We found that csGRP78, known to be located on the outer membrane leaflet, interacts with the transmembrane protein integrin β1 and activates focal adhesion kinase (FAK) and downstream phosphoinositide 3-kinase (PI3K)/AKT signaling. Localization of GRP78 at the cell surface and its interaction with integrin β1 were also required for ECM protein synthesis in response to HG. Surprisingly, both the N and C termini of csGRP78 were necessary for this profibrotic response. Increased localization of GRP78 at the plasma membrane was also found in the glomerular mesangial area of type 1 diabetic mice in two different models (streptozotocin-induced and Akita). In freshly isolated glomeruli from Akita mice, csGRP78 co-localized with the mesangial cell-surface marker α8-integrin. In conclusion, our work reveals a role for csGRP78 in HG-induced profibrotic responses in MC, informing a potential approach to treating diabetic nephropathy.

Keywords:  78 kDa glucose-regulated protein (GRP78); cell stress; chaperone; diabetic nephropathy; extracellular matrix; fibrosis; integrin; kidney disease

DOI:  https://doi.org/10.1074/jbc.RA118.006939
J Cell Physiol. 2019 Mar 25.

Salubrinal, a novel inhibitor of eIF-2α dephosphorylation, promotes erythropoiesis at early stage targeted by ufmylation pathway.

Fanghui Chen, Chaofeng Xing, Wei Zhang, Jun Li, Tianxiang Hu, Lian Li, Honglin Li, Yafei Cai.

  Ufmylation was proved to play a crucial role in hematopoietic stem cell (HSC) survival and erythroid differentiation, ufmylation deficiency induces acute anemia and lethality of embryos and adults in mouse models. To screen some compounds to rescue phenotypes induced by gene deletion, in this study, we used DDRGK1F/F ; CreERT2 conditional knockout mice, DDRGK1F/F ; CreERT2 bone marrow (BM) and fetal liver cells (FL), Uba5, and DDRGK1 knockdown human CD34 cell in vivo and in vitro, we found salubrinal, a novel inhibitor of eIF-2α dephosphorylation, promoted erythropoiesis at early stage, and partly rescued the acute anemia induce by DDRGK1 deficiency through upregulation of ufmylation and erythroid transcription factors. In phenylhydrazine (PHZ)-induced hemolytic anemia mice, interestingly, salubrinal could significantly improve hemocrit and red blood cell (RBC) indices of the mice treated with PHZ via upregulation of ufmylation. Its novel function was verified to attenuate unfolded protein response (UPR) and cell death programs, and to keep endoplasmic reticulum (ER) homeostasis in HSCs. Taken together results, it suggested that salubrinal may be a promising antianemic agent targeted by ufmylation.

Keywords:  DDRGK1; erythropoiesis; salubrinal; ufmylation

DOI:  https://doi.org/10.1002/jcp.28493
Sci Rep. 2019 Mar 26. 9(1): 5199

Pancreatic stone protein/regenerating protein is a potential biomarker for endoplasmic reticulum stress in beta cells.

Stephen Stone, Damien Abreu, Jana Mahadevan, Rie Asada, Kelly Kries, Rolf Graf, Bess A Marshall, Tamara Hershey, Fumihiko Urano.

Endoplasmic reticulum (ER) stress in beta cells is an important pathogenic component of both type 1 and type 2 diabetes mellitus, as well as genetic forms of diabetes, especially Wolfram syndrome. However, there are currently no convenient ways to assess ER stress in beta cells, raising the need for circulating ER stress markers indicative of beta cell health. Here we show that pancreatic stone protein/regenerating protein (PSP/reg) is a potential biomarker for ER stressed beta cells. PSP/reg levels are elevated in cell culture and mouse models of Wolfram syndrome, a prototype of ER stress-induced diabetes. Moreover, PSP/reg expression is induced by the canonical chemical inducers of ER stress, tunicamycin and thapsigargin. Circulating PSP/reg levels are also increased in some patients with Wolfram syndrome. Our results therefore reveal PSP/reg as a potential biomarker for beta cells under chronic ER stress, as is the case in Wolfram syndrome.

DOI: https://doi.org/10.1038/s41598-019-41604-4
Methods Enzymol. 2019 ;pii: S0076-6879(19)30002-3. [Epub ahead of print]619 1-26

Assays for protein retrotranslocation in ERAD.

Sonya Neal, Sascha H Duttke, Randolph Y Hampton.

  Elimination of misfolded proteins by endoplasmic reticulum (ER)-associated protein degradation (ERAD) ensures that proteins proceeding through the secretory pathway are correctly folded and processed, which is critical to minimize ER stress. All ERAD pathways include a protein translocation process termed retrotranslocation, in which ubiquitinated misfolded substrates are extracted from the ER and degraded by the cytosolic 26S proteasome. Despite being integral to ERAD, the retrotranslocation process has been largely obscure. Recently, an explosion of discoveries has provided key mechanistic insights into this novel route of protein transport. These advances were facilitated by the development of in vitro and in vivo assays that utilize components from the yeast Saccharomyces cerevisiae. The assays permit detailed study of the distinct steps in ERAD-linked retrotranslocation, including ubiquitination of selected ERAD substrates, substrate removal from the ER, maintenance of cytosolic substrate solubility in the cytosol, and substrate degradation. Here we provide detailed protocols for these assays that pertain to work on retrotranslocation of integral membrane proteins (ERAD-M substrates), with the expectation that these approaches can be adapted for many related biochemical processes.

Keywords:  Cdc48; Dfm1; Doa10; ER; ERAD; Hmg2; Hrd1; Retrochaperone; Retrotranslocation

DOI:  https://doi.org/10.1016/bs.mie.2019.01.002
Am J Respir Cell Mol Biol. 2019 Mar 25.

CircRNA-012091/PPP1R13B-Mediated Lung Fibrotic Response in Silicosis via ER Stress and Autophagy.

Yusi Cheng, Wei Luo, Zhuang Li, Mumin Cao, Zixin Zhu, Chen Han, Xiaoniu Dai, Wei Zhang, Jing Wang, Honghong Yao, Jie Chao.

  Silicosis is a progressive fibrotic disease of lung tissue caused by long-term inhalation of SiO2. However, relatively few studies on the direct effects of SiO2 on lung fibroblasts have been performed. PPP1R13B is a major member of the apoptosis-stimulating proteins of the p53 family (ASPPs), but its role in pulmonary fibrosis is unclear. To elucidate the role of PPP1R13B in the pathological process of silicosis, this study explored the molecular mechanisms related to PPP1R13B and the functional effects of proliferation and migration of fibroblasts. Through lentivirus transfection, western blotting and fluorescent in situ hybridization experiments, we found that SiO2 downregulated ciR-012091 expression in lung fibroblasts and induced upregulation of downstream PPP1R13B. Transfection of L929 cells with PPP1R13B CRISPR NIC plasmid inhibited the upregulation of endoplasmic reticulum stress (ERS) and autophagy-related protein expression in lung fibroblasts treated with SiO2 and induced decreases in cell proliferation, migration, and viability. Transfection of L929 cells with the PPP1R13B CRISPR ACT plasmid induced increases in cell proliferation, migration, and viability. In addition, the ERS inhibitor salubrinal and the autophagy inhibitor 3-MA inhibited the increased migration of L929 cells transfected with the PPP1R13B CRISPR ACT plasmid. These results suggest that PPP1R13B regulated by ciR-012091 promotes the proliferation and migration of lung fibroblasts through ERS and autophagy and plays a crucial role in the development of pulmonary fibrosis in silicosis.

Keywords:  ER Stress; PPP1R13B; autophagy; circRNA; silicosis

DOI:  https://doi.org/10.1165/rcmb.2019-0017OC