Monitoring Protein Import into the Endoplasmic Reticulum in Living Cells with Proximity Labeling

The proper trafficking of eukaryotic proteins is essential to cellular function. Genetic, environmental, and other stresses can induce protein mistargeting, and in turn threaten cellular protein homeostasis. Current methods for measuring protein mistargeting are difficult to translate to living cells, and thus the role of cellular signaling networks in stress-dependent protein mistargeting processes, such as ER pre-emptive quality control (ER pQC), are difficult to parse. Herein, we use genetically encoded peroxidases to characterize protein import into the endoplasmic reticulum (ER). We show that the ERHRP/cytAPEX pair provides good selectivity and sensitivity for identifying protein mistargeting, using the known ER pQC substrate transthyretin (TTR). Although ERHRP labeling induces formation of detergent-resistant TTR aggregates, this is minimized by using low ERHRP expression, without loss of labeling efficiency. cytAPEX labeling recovers TTR that is mistargeted as a consequence of Sec61 inhibition or ER stress-induced ER pQC. Furthermore, we demonstrate that stress-free activation of the ER stress-associated transcription factor ATF6 recapitulates the TTR import deficiency of ER pQC. Hence, proximity labeling is an effective strategy for characterizing factors that influence ER protein import in living cells.

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α-Synuclein Decreases the Abundance of Proteasome Subunits and Alters Ubiquitin Conjugates in Yeast

Cells ◽

10.3390/cells10092229 ◽

2021 ◽

Vol 10 (9) ◽

pp. 2229

Author(s):

Blagovesta Popova ◽

Dajana Galka ◽

Nicola Häffner ◽

Dan Wang ◽

Kerstin Schmitt ◽

...

Keyword(s):

Dopaminergic Neurons ◽

Cellular Protein ◽

Protein Homeostasis ◽

Wild Type ◽

Yeast Growth ◽

Independent Manner ◽

Proteasome Subunits ◽

Dependent Protein ◽

The Brain

Parkinson’s disease (PD) is the most prevalent movement disorder characterized with loss of dopaminergic neurons in the brain. One of the pathological hallmarks of the disease is accumulation of aggregated α-synuclein (αSyn) in cytoplasmic Lewy body inclusions that indicates significant dysfunction of protein homeostasis in PD. Accumulation is accompanied with highly elevated S129 phosphorylation, suggesting that this posttranslational modification is linked to pathogenicity and altered αSyn inclusion dynamics. To address the role of S129 phosphorylation on protein dynamics further we investigated the wild type and S129A variants using yeast and a tandem fluorescent timer protein reporter approach to monitor protein turnover and stability. Overexpression of both variants leads to inhibited yeast growth. Soluble S129A is more stable and additional Y133F substitution permits αSyn degradation in a phosphorylation-independent manner. Quantitative cellular proteomics revealed significant αSyn-dependent disturbances of the cellular protein homeostasis, which are increased upon S129 phosphorylation. Disturbances are characterized by decreased abundance of the ubiquitin-dependent protein degradation machinery. Biotin proximity labelling revealed that αSyn interacts with the Rpt2 base subunit. Proteasome subunit depletion by reducing the expression of the corresponding genes enhances αSyn toxicity. Our studies demonstrate that turnover of αSyn and depletion of the proteasome pool correlate in a complex relationship between altered proteasome composition and increased αSyn toxicity.

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Engineering ER-stress dependent non-conventional mRNA splicing

eLife ◽

10.7554/elife.35388 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 10

Author(s):

Weihan Li ◽

Voytek Okreglak ◽

Jirka Peschek ◽

Philipp Kimmig ◽

Meghan Zubradt ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Endoplasmic Reticulum ◽

Protein Folding ◽

Er Stress ◽

Schizosaccharomyces Pombe ◽

Transcriptional Response ◽

Structural Features ◽

Mrna Splicing ◽

Protein Homeostasis ◽

Stress Dependent

The endoplasmic reticulum (ER) protein folding capacity is balanced with the protein folding burden to prevent accumulation of un- or misfolded proteins. The ER membrane-resident kinase/RNase Ire1 maintains ER protein homeostasis through two fundamentally distinct processes. First, Ire1 can initiate a transcriptional response through a non-conventional mRNA splicing reaction to increase the ER folding capacity. Second, Ire1 can decrease the ER folding burden through selective mRNA decay. In Saccharomyces cerevisiae and Schizosaccharomyces pombe, the two Ire1 functions have been evolutionarily separated. Here, we show that the respective Ire1 orthologs have become specialized for their functional outputs by divergence of their RNase specificities. In addition, RNA structural features separate the splicing substrates from the decay substrates. Using these insights, we engineered an S. pombe Ire1 cleavage substrate into a splicing substrate, which confers S. pombe with both Ire1 functional outputs.

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Endoplasmic Reticulum Stress, a Target for Drug Design and Drug Resistance in Parasitosis

Frontiers in Microbiology ◽

10.3389/fmicb.2021.670874 ◽

2021 ◽

Vol 12 ◽

Author(s):

Mei Peng ◽

Fang Chen ◽

Zhongdao Wu ◽

Jia Shen

Keyword(s):

Endoplasmic Reticulum ◽

Drug Design ◽

Endoplasmic Reticulum Stress ◽

Er Stress ◽

Parasitic Infection ◽

Artemisinin Resistance ◽

Public Health Problem ◽

Cellular Protein ◽

Host Cells ◽

Protein Homeostasis

Endoplasmic reticulum stress (ER stress) can be induced when cellular protein homeostasis is damaged, and cells can activate the unfolded protein response (UPR) to restore protein homeostasis or induce cell death to facilitate the survival of the whole system. Globally, parasites are a constant threat to human health and are therefore considered a serious public health problem. Parasitic infection can cause ER stress in host cells, and parasites also possess part or all of the UPR under ER stress conditions. In this review, we aim to clarify the role of ER stress pathways and related molecules in parasites for their survival and development, the pathogenesis of parasitosis in hosts, and the artemisinin resistance of Plasmodium, which provides some potential drug design targets to inhibit survival of parasites, relieves pathological damage of parasitosis, and solves the problem of artemisinin resistance.

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c-Jun Inhibits Thapsigargin-Induced ER Stress Through Up-Regulation of DSCR1/Adapt78

Experimental Biology and Medicine ◽

10.3181/0803-rm-84 ◽

2008 ◽

Vol 233 (10) ◽

pp. 1289-1300 ◽

Cited By ~ 25

Author(s):

Peng Zhao ◽

Xiaoyan Xiao ◽

Agnes S. Kim ◽

M. Fatima Leite ◽

Jinxia Xu ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Cell Death ◽

Er Stress ◽

Wild Type Mouse ◽

Mouse Fibroblast ◽

Expression Levels ◽

Mouse Fibroblasts ◽

The Unfolded Protein Response ◽

Calcineurin Activity

The endoplasmic reticulum (ER) is exquisitely sensitive to changes in its internal environment. Various conditions, collectively termed “ER stress”, can perturb ER function, leading to the activation of a complex response known as the unfolded protein response (UPR). Although c-Jun N-terminal kinase (JNK) activation is nearly always associated with cell death by various stimuli, the functional role of JNK in ER stress-induced cell death remains unclear. JNK regulates gene expression through the phosphorylation and activation of transcription factors, such as c-Jun. Here, we investigated the role of c-Jun in the regulation of ER stress-related genes. c-Jun expression levels determined the response of mouse fibroblasts to ER stress induced by thapsigargin (TG, an inhibitor of sarco/endoplasmic reticulum Ca2+ ATPase). c-jun−/− mouse fibroblast cells were more sensitive to TG-induced cell death compared to wild-type mouse fibroblasts, while reconstitution of c-Jun expression in c-jun−/− cells (c-Jun Re) enhanced resistance to TG-induced cell death. The expression levels of ER chaperones Grp78 and Gadd153 induced by TG were lower in c-Jun Re than in c-jun−/− cells. Moreover, TG treatment significantly increased calcineurin activity in c-jun−/− cells, but not in c-Jun Re cells. In c-Jun Re cells, TG induced the expression of Adapt78, also known as the Down syndrome critical region 1 (DSCR1), which is known to block calcineurin activity. Taken together, our findings suggest that c-Jun, a transcription factor downstream of the JNK signaling pathway, up-regulates Adapt78 expression in response to TG-induced ER stress and contributes to protection against TG-induced cell death.

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RHBDL4 protects against endoplasmic reticulum stress by regulating the morphology and distribution of ER sheets

10.1101/2021.06.15.448480 ◽

2021 ◽

Author(s):

Viorica Liebe Lastun ◽

Matthew Freeman

Keyword(s):

Cell Cycle ◽

Endoplasmic Reticulum ◽

Endoplasmic Reticulum Stress ◽

Er Stress ◽

Liver Steatosis ◽

Physiological Role ◽

Cell Types ◽

Rhomboid Protease ◽

Rapid Changes

In metazoans, the architecture of the endoplasmic reticulum (ER) differs between cell types, and undergoes major changes through the cell cycle and according to physiological needs. Although much is known about how the different ER morphologies are generated and maintained, especially the ER tubules, how context dependent changes in ER shape and distribution are regulated and the factors involved are less characterized. Here, we show that RHBDL4, an ER-resident rhomboid protease, modulates the shape and distribution of the ER, especially under conditions that require rapid changes in the ER sheet distribution, including ER stress. RHBDL4 interacts with CLIMP-63, a protein involved in ER sheet stabilisation, and with the cytoskeleton. Mice lacking RHBDL4 are sensitive to ER stress and develop liver steatosis, a phenotype associated with unresolved ER stress. Our data introduce a new physiological role of RHBDL4 and also imply that this function does not require its enzymatic activity.

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Licochalcone A-Induced Human Bladder Cancer T24 Cells Apoptosis Triggered by Mitochondria Dysfunction and Endoplasmic Reticulum Stress

BioMed Research International ◽

10.1155/2013/474272 ◽

2013 ◽

Vol 2013 ◽

pp. 1-9 ◽

Cited By ~ 20

Author(s):

Xuan Yuan ◽

Defang Li ◽

Hong Zhao ◽

Jiangtao Jiang ◽

Penglong Wang ◽

...

Keyword(s):

Bladder Cancer ◽

Endoplasmic Reticulum ◽

Er Stress ◽

Parp Cleavage ◽

Ros Generation ◽

Human Bladder Cancer ◽

Human Bladder ◽

Licochalcone A ◽

T24 Cells ◽

Stress Dependent

Licochalcone A (LCA), a licorice chalconoid, is considered to be a bioactive agent with chemopreventive potential. This study investigated the mechanisms involved in LCA-induced apoptosis in human bladder cancer T24 cells. LCA significantly inhibited cells proliferation, increased reactive oxygen species (ROS) levels, and caused T24 cells apoptosis. Moreover, LCA induced mitochondrial dysfunction, caspase-3 activation, and poly-ADP-ribose polymerase (PARP) cleavage, which displayed features of mitochondria-dependent apoptotic signals. Besides, exposure of T24 cells to LCA triggered endoplasmic reticulum (ER) stress; as indicated by the enhancement in 78 kDa glucose-regulated protein (GRP 78), growth arrest and DNA damage-inducible gene 153/C/EBP homology protein (GADD153/CHOP) expression, ER stress-dependent apoptosis is caused by the activation of ER-specific caspase-12. All the findings from our study suggest that LCA initiates mitochondrial ROS generation and induces oxidative stress that consequently causes T24 cell apoptosis via the mitochondria-dependent and the ER stress-triggered signaling pathways.

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Abstract 357: Hepatic ERK2 Suppresses Endoplasmic Reticulum Stress, Leading to Protection from Oxidative Stress and Endothelial Dysfunction

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.33.suppl_1.a357 ◽

2013 ◽

Vol 33 (suppl_1) ◽

Author(s):

Takehiko Kujiraoka ◽

Yasushi Satoh ◽

Makoto Ayaori ◽

Yasunaga Shiraishi ◽

Yuko Arai-Nakaya ◽

...

Keyword(s):

Oxidative Stress ◽

Insulin Resistance ◽

Endoplasmic Reticulum ◽

Fatty Acid ◽

Endothelial Dysfunction ◽

Er Stress ◽

Vascular Complications ◽

Metabolic Remodeling ◽

And Oxidative Stress

Background Insulin signaling comprises 2 major cascades, the IRS/PI3K/Akt and Ras/Raf/MEK/ERK pathways. Many studies on the tissue-specific effects of the former pathway had been conducted, however, the role of the latter cascade in tissue-specific insulin resistance had not been investigated. High glucose/fatty acid toxicity, inflammation and oxidative stress, all of which are associated with insulin resistance, can activate ERK. Liver plays a central role of metabolism and hepatosteatosis (HST) is associated with vascular diseases. The aim of this study is to elucidate the role of hepatic ERK2 in HST, metabolic remodeling and endothelial dysfunction. Methods Serum biomarkers of vascular complications in human were compared between subjects with and without HST diagnosed by echography for regular medical checkup. Next, we created liver-specific ERK2 knockout mice (LE2KO) and fed them with a high-fat/high-sucrose diet (HFHSD) for 20 weeks. The histological analysis, the expression of hepatic sarco/endoplasmic reticulum (ER) Ca 2+ -ATPase 2 (SERCA2) and glucose-tolerance/insulin-sensitivity (GT/IS) were tested. Vascular superoxide production and endothelial function were evaluated with dihydroethidium staining and isometric tension measurement of aorta. Results The presence of HST significantly increased HOMA-IR, an indicator of insulin resistance or atherosclerotic index in human. HFHSD-fed LE2KO revealed a marked exacerbation in HST and metabolic remodeling represented by the impairment of GT/IS, elevated serum free fatty acid and hyperhomocysteinemia without changes in body weight, blood pressure and serum cholesterol/triglyceride levels. In the HFHSD-fed LE2KO, mRNA and protein expressions of hepatic SERCA2 were significantly decreased, which resulted in hepatic ER stress. Induction of vascular superoxide production and remarkable endothelial dysfunction were also observed in them. Conclusions Hepatic ERK2 revealed the suppression of hepatic ER stress and HST in vivo , which resulted in protection from vascular oxidative stress and endothelial dysfunction. HST with hepatic ER stress can be a prominent risk of vascular complications by metabolic remodeling and oxidative stress in obese-related diseases.

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Abstract 590: Increased Endoplasmic Reticulum Stress in Atherosclerotic Plaques Associated With Acute Coronary Syndrome

Circulation ◽

10.1161/circ.116.suppl_16.ii_107-a ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Masafumi Myoishi ◽

Testuo Minamino ◽

Masafumi Kitakaze

Keyword(s):

Endoplasmic Reticulum ◽

Coronary Artery ◽

Er Stress ◽

Atherosclerotic Plaques ◽

Coronary Syndrome ◽

Er Chaperone ◽

Unstable Plaques ◽

Induced Apoptosis ◽

Stress Dependent ◽

Dependent Pathway

Background Endoplasmic reticulum (ER) responds to various stresses by up-regulation of ER chaperones, and prolonged ER stress eventually causes apoptosis. Although apoptosis is considered to be essential for the progression and rupture of atherosclerotic plaques, the influence of ER stress and apoptosis on rupture of unstable coronary plaques remains unclear. Methods and Results We obtained 152 coronary artery segments at autopsy and 40 atherectomy specimens from 71 and 40 patients, respectively . Smooth muscle cells (SMCs) and macrophages in the fibrous caps of thin cap atheroma and ruptured plaques, but not in the fibrous caps of thick cap atheroma and fibrous plaques, showed a marked increase in the expression of ER chaperone and numbers of apoptotic cells. ER chaperones also expressed higher in atherectomy specimens from patients with unstable angina pectoris than with stable angina. To explore the plausible molecular mechanism of activation of ER stress and the mechanistic link to apoptosis, we investigated plaque lipids such as oxysterols. Among oxysterols, expression of 7-ketocholesterol was increased in the fibrous caps of thin cap atheroma compared with thick cap atheroma. Treatment of either cultured coronary artery SMCs or THP-1 cells with 7-ketocholesterol induced upregulation of ER chaperones and apoptosis, while these changes were prevented by antioxidants. We also investigated possible signaling pathways for ER-initiated apoptosis and found that the CHOP (a transcription factor induced by ER stress)-dependent pathway was activated in unstable plaques. In addition, knockdown of CHOP expression by siRNA decreased ER stress-dependent death of cultured coronary artery SMCs and THP-1 cells. Conclusions Increased ER stress occurs in unstable plaques. Our findings suggest that ER stress-induced apoptosis of SMCs and macrophages may contribute to plaque vulnerability.

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Role of O-GlcNAcylation and endoplasmic reticulum stress on obesity and insulin resistance

Turkish Journal of Biochemistry ◽

10.1515/tjb-2018-0303 ◽

2019 ◽

Vol 44 (5) ◽

pp. 599-610 ◽

Cited By ~ 1

Author(s):

Benan Pelin Sermikli ◽

Gulizar Aydogdu ◽

Afsar Abbasi Taghidizaj ◽

Erkan Yilmaz

Keyword(s):

Insulin Resistance ◽

Endoplasmic Reticulum ◽

Western Blot ◽

Er Stress ◽

Insulin Receptor ◽

Serine Phosphorylation ◽

Wild Type ◽

Adipose Tissues ◽

Insulin Resistant

Abstract Background Obesity is a global public health problem. Obesity closely associated with various metabolic diseases such as; insulin resistance, hypertension, dyslipidemia and cardiovascular diseases. Endoplasmic reticulum (ER) stress is a critical factor for insulin resistance. O-linked N-acetyl-glucosamine (O-GlcNAc); is the post-translational modification which is has a vital role in biological processes; including cell signaling, in response to nutrients, stress and other extracellular stimuli. Materials and methods In this study, we aimed to investigate the role of O-GlcNAc modification in the context of obesity and obesity-associated insulin resistance in adipose tissue. For this purpose, first, the visceral and epididymal adipose tissues of obese and insulin resistant C57BL/6 Lepob/Lepob and wild-type mice were used to determine the O-GlcNAc modification pattern by western blot. Secondly, the external stimulation of O-GlcNAc modification in wild-type mice achieved by intraperitoneal 5 mg/kg/day glucosamine injection every 24 h for 5 days. The effect of increased O-GlcNAc modification on insulin resistance and ER stress investigated in adipose tissues of glucosamine challenged wild-type mice through regulation of the insulin signaling pathway and unfolded protein response (UPR) elements by western blot. In addition to that, the O-GlcNAc status of the insulin receptor substrate-1 (IRS1) investigated in epididymal and visceral adipose tissues of ob/ob, wild-type and glucosamine challenged mice by immunoprecipitation. Results We found that reduced O-GlcNAc levels in visceral and epididymal adipose tissues of obese and insulin-resistant ob/ob mice, although interestingly we observed that increased O-GlcNAc modification in glucosamine challenged wild-type mice resulted in insulin resistance and ER stress. Furthermore, we demonstrated that the IRS1 was modified with O-GlcNAc in visceral and epididymal adipose tissues in both ob/ob mice and glucosamine-injected mice, and was compatible with the serine phosphorylation of this modification. Conclusion Our results suggest that O-GlcNAcylation of proteins is a crucial factor for intracellular trafficking regulates insulin receptor signaling and UPR depending on the cellular state of insulin resistance.

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Chemotherapy Resistance Explained through Endoplasmic Reticulum Stress-Dependent Signaling

Cancers ◽

10.3390/cancers11030338 ◽

2019 ◽

Vol 11 (3) ◽

pp. 338 ◽

Cited By ~ 17

Author(s):

Entaz Bahar ◽

Ji-Ye Kim ◽

Hyonok Yoon

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Molecular Mechanism ◽

Molecular Mechanisms ◽

Cellular Proliferation ◽

Protein Quality ◽

Chemotherapy Resistance ◽

Persistent Problem ◽

Stress Dependent ◽

The Relationship

Cancers cells have the ability to develop chemotherapy resistance, which is a persistent problem during cancer treatment. Chemotherapy resistance develops through different molecular mechanisms, which lead to modification of the cancer cells signals needed for cellular proliferation or for stimulating an immune response. The endoplasmic reticulum (ER) is an important organelle involved in protein quality control, by promoting the correct folding of protein and ER-mediated degradation of unfolded or misfolded protein, namely, ER-associated degradation. Disturbances of the normal ER functions causes an accumulation of unfolded or misfolded proteins in the ER lumen, resulting in a condition called “ER stress (ERS).” ERS triggers the unfolded protein response (UPR)—also called the ERS response (ERSR)—to restore homeostasis or activate cell death. Although the ERSR is one emerging potential target for chemotherapeutics to treat cancer, it is also critical for chemotherapeutics resistance, as well. However, the detailed molecular mechanism of the relationship between the ERSR and tumor survival or drug resistance remains to be fully understood. In this review, we aim to describe the most vital molecular mechanism of the relationship between the ERSR and chemotherapy resistance. Moreover, the review also discusses the molecular mechanism of ER stress-mediated apoptosis on cancer treatments.

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