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Angewandte Chemie (International Ed. in... Aug 2022A short total synthesis of tunicamycin V (1), a non-selective phosphotransferase inhibitor, is achieved via a Büchner-Curtius-Schlotterbeck type reaction....
A short total synthesis of tunicamycin V (1), a non-selective phosphotransferase inhibitor, is achieved via a Büchner-Curtius-Schlotterbeck type reaction. Tunicamycin V can be synthesized in 15 chemical steps from D-galactal with 21 % overall yield. The established synthetic scheme is operationally very simple and flexible to introduce building blocks of interest. The inhibitory activity of one of the designed analogues 28 against human dolichyl-phosphate N-acetylglucosaminephosphotransferase 1 (DPAGT1) is 12.5 times greater than 1. While tunicamycins are cytotoxic molecules with a low selectivity, the novel analogue 28 displays selective cytostatic activity against breast cancer cell lines including a triple-negative breast cancer.
Topics: Antineoplastic Agents; Cytostatic Agents; Humans; Tunicamycin
PubMed: 35594368
DOI: 10.1002/anie.202203225 -
Investigative Ophthalmology & Visual... Nov 2023Endoplasmic reticulum (ER) and mitochondrial stress are independently associated with corneal endothelial cell (CEnC) loss in many corneal diseases, including Fuchs'...
PURPOSE
Endoplasmic reticulum (ER) and mitochondrial stress are independently associated with corneal endothelial cell (CEnC) loss in many corneal diseases, including Fuchs' endothelial corneal dystrophy (FECD). However, the role of ER stress in mitochondrial dysfunction contributing to CEnC apoptosis is unknown. The purpose of this study is to explore the crosstalk between ER and mitochondrial stress in CEnC.
METHODS
Human corneal endothelial cell line (HCEnC-21T) and human corneal endothelial tissues were treated with ER stressor tunicamycin. ER stress-reducing chemical 4-phenyl butyric acid (4-PBA) was used in HCEnC-21T after tunicamycin. Fuchs' corneal endothelial cell line (F35T) was used to determine differential activation of ER stress with respect to HCEnC-21T at the baseline. ER stress, mitochondrial-mediated intrinsic apoptotic, mitochondrial fission, and fusion proteins were determined using immunoblotting and immunohistochemistry. Mitochondrial bioenergetics were assessed by mitochondrial membrane potential (MMP) loss and ATP production at 48 hours after tunicamycin. Mitochondria dynamics (shape, area, perimeter) were also analyzed at 24 hours using transmission electron microscopy.
RESULTS
Treatment of HCEnC-21T cell line with tunicamycin activated three ER stress pathways (PERK-eIF2α-CHOP, IRE1α-XBP1, and ATF6), reduced cell viability, upregulated mitochondrial-mediated intrinsic apoptotic molecules (cleaved caspase 9, caspase 3, PARP, Bax, cytochrome C), downregulated anti-apoptotic Bcl-2 protein, initiated mitochondrial dysfunction by loss of MMP and lowering of ATP production, and caused mitochondrial swelling and fragmentation with increased expression of mitochondrial fission proteins (Fis1 and p-Drp1). Fuchs' CEnC (F35T) cell line also showed activation of the ER stress-related proteins (p-eIF2α, GRP78, CHOP, XBP1) compared to HCEnC-21T at the baseline. The 4-PBA ameliorated cell loss and reduced cleaved caspase 3 and 9, thereby rescuing tunicamycin-induced cell death but not mitochondrial bioenergetics in HCEnC-21T cell line.
CONCLUSIONS
Tunicamycin-induced ER stress disrupts mitochondrial bioenegetics, dynamics and contributes to the loss of CEnC viability. This novel study highlights the importance of ER-mitochondria crosstalk and its contribution to CEnCs apoptosis, seen in many corneal diseases, including FECD.
Topics: Humans; Caspase 3; Endoribonucleases; Tunicamycin; Protein Serine-Threonine Kinases; Apoptosis; Endoplasmic Reticulum Stress; Corneal Diseases; Fuchs' Endothelial Dystrophy; Butyric Acid; Energy Metabolism; Endothelial Cells; Adenosine Triphosphate
PubMed: 37962528
DOI: 10.1167/iovs.64.14.18 -
Oxidative Medicine and Cellular... 2022Primary open-angle glaucoma (POAG) is the most common form of glaucoma, for which elevated intraocular pressure (IOP) is a major risk factor. IOP is mainly regulated by...
Primary open-angle glaucoma (POAG) is the most common form of glaucoma, for which elevated intraocular pressure (IOP) is a major risk factor. IOP is mainly regulated by dynamic balance of aqueous humor (AH) production and outflow via the conventional trabecular meshwork/Schlemm's canal (TM/SC) pathway. Dysfunctions of TM cells due to endoplasmic reticulum (ER) stress have been demonstrated to increase the resistance of AH outflow, resulting in IOP elevation. Epigallocatechin-3-gallate (EGCG), the most abundant polyphenolic component in green tea, has been shown to alleviate ER stress in several diseases while its potential roles in alleviating ER stress in TM cells have not been determined. In this study, we investigate the mitigation of tunicamycin-induced ER stress in TM cells by EGCG. MTT assay was used to measure the cell viability of human TM (HTM) cells and primary porcine TM (PTM) cells. ER stress levels in both HTM cells and primary PTM cells were detected by quantitative real-time PCR. The primary PTM cells isolated from porcine TM tissues were characterized by immunostaining. We found that 40 M and 80 M EGCG pretreatment substantially promoted HTM cell survival under 3 M tunicamycin-induced ER stress. Pretreatment of 40 M EGCG markedly reduced the expression of ER stress markers , , and evoked by 3 M tunicamycin in HTM cells. Furthermore, 40 M EGCG pretreatment significantly decreased the expressions of , , and at the mRNA level induced by 3 M tunicamycin and improved cell viability in primary PTM cells. Our results show that EGCG is capable of protecting TM cells from ER stress. EGCG provides a promising therapeutic option for POAG treatment.
Topics: Humans; Swine; Animals; Trabecular Meshwork; Endoplasmic Reticulum Stress; Tunicamycin; Glaucoma, Open-Angle
PubMed: 36406768
DOI: 10.1155/2022/7435754 -
Nature Structural & Molecular Biology Mar 2018N-linked glycosylation is a predominant post-translational modification of protein in eukaryotes, and its dysregulation is the etiology of several human disorders. The...
N-linked glycosylation is a predominant post-translational modification of protein in eukaryotes, and its dysregulation is the etiology of several human disorders. The enzyme UDP-N-acetylglucosamine:dolichyl-phosphate N-acetylglucosaminephosphotransferase (GlcNAc-1-P-transferase or GPT) catalyzes the first and committed step of N-linked glycosylation in the endoplasmic reticulum membrane, and it is the target of the natural product tunicamycin. Tunicamycin has potent antibacterial activity, inhibiting the bacterial cell wall synthesis enzyme MraY, but its usefulness as an antibiotic is limited by off-target inhibition of human GPT. Our understanding of how tunicamycin inhibits N-linked glycosylation and efforts to selectively target MraY are hampered by a lack of structural information. Here we present crystal structures of human GPT in complex with tunicamycin. Structural and functional analyses reveal the difference between GPT and MraY in their mechanisms of inhibition by tunicamycin. We demonstrate that this difference could be exploited to design MraY-specific inhibitors as potential antibiotics.
Topics: Anti-Bacterial Agents; Bacterial Proteins; Enzyme Inhibitors; Glycosylation; Humans; Magnesium; Models, Molecular; Protein Binding; Protein Multimerization; Substrate Specificity; Transferases; Transferases (Other Substituted Phosphate Groups); Tunicamycin
PubMed: 29459785
DOI: 10.1038/s41594-018-0031-y -
International Journal of Molecular... Aug 2017Disorders of hepatic energy metabolism, which can be regulated by endoplasmic reticulum (ER) stress, lead to metabolic diseases such as hepatic steatosis and...
Disorders of hepatic energy metabolism, which can be regulated by endoplasmic reticulum (ER) stress, lead to metabolic diseases such as hepatic steatosis and hypoglycemia. Tunicamycin, a pharmacological ER stress inducer, is used to develop an anti-cancer drug. However, the effects of tunicamycin on hepatic energy metabolism have not been well elucidated. Mice were intraperitoneally injected with tunicamycin or vehicle. Twenty-four hours later, hepatic triglyceride and glycogen content and serum lipids profiles were analyzed, as well as the expression of lipogenic and gluconeogenic genes. Tunicamycin significantly induced hepatic a yellowish color and ER stress, as well as increasing serum levels of aspartate transaminase and alanine transaminase. Besides, tunicamycin remarkably increased hepatic triglyceride content and suppressed the expression of apolipoprotein B100. In addition, tunicamycin-treated mice had lower serum levels of triglyceride, apolipoprotein B, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol. Gene expression of peroxisome proliferator-activated receptor α was decreased by tunicamycin, but the protein level was increased. Furthermore, blood glucose level and hepatic glycogen content were decreased in tunicamycin-treated mice. Protein kinase B signaling was attenuated in the tunicamycin-treated liver, but the expression and activities of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase were unchanged. Tunicamycin alters hepatic energy homeostasis by increasing triglyceride accumulation and decreasing glycogen content.
Topics: Animals; Blood Glucose; Endoplasmic Reticulum Stress; Energy Metabolism; Gene Expression Regulation; Glycogen; Homeostasis; Lipogenesis; Lipoproteins; Liver; Male; Mice, Inbred C57BL; Triglycerides; Tunicamycin
PubMed: 28777337
DOI: 10.3390/ijms18081710 -
Cell Death & Disease Aug 2023Unfolded protein response (UPR) maintains the endoplasmic reticulum (ER) homeostasis, survival, and physiological function of mammalian cells. However, how cells adapt...
Unfolded protein response (UPR) maintains the endoplasmic reticulum (ER) homeostasis, survival, and physiological function of mammalian cells. However, how cells adapt to ER stress under physiological or disease settings remains largely unclear. Here by a genome-wide CRISPR screen, we identified that RBBP8, an endonuclease involved in DNA damage repair, is required for ATF4 activation under ER stress in vitro. RNA-seq analysis suggested that RBBP8 deletion led to impaired cell cycle progression, retarded proliferation, attenuated ATF4 activation, and reduced global protein synthesis under ER stress. Mouse tissue analysis revealed that RBBP8 was highly expressed in the liver, and its expression is responsive to ER stress by tunicamycin intraperitoneal injection. Hepatocytes with RBBP8 inhibition by adenovirus-mediated shRNA were resistant to tunicamycin (Tm)-induced liver damage, cell death, and ER stress response. To study the pathological role of RBBP8 in regulating ATF4 activity, we illustrated that both RBBP8 and ATF4 were highly expressed in liver cancer tissues compared with healthy controls and highly expressed in Ki67-positive proliferating cells within the tumors. Interestingly, overexpression of RBBP8 in vitro promoted ATF4 activation under ER stress, and RBBP8 expression showed a positive correlation with ATF4 expression in liver cancer tissues by co-immunostaining. Our findings provide new insights into the mechanism of how cells adapt to ER stress through the crosstalk between the nucleus and ER and how tumor cells survive under chemotherapy or other anticancer treatments, which suggests potential therapeutic strategies against liver disease by targeting DNA damage repair, UPR or protein synthesis.
Topics: Animals; Mice; Clustered Regularly Interspaced Short Palindromic Repeats; Tunicamycin; Unfolded Protein Response; Liver Neoplasms; Mammals
PubMed: 37591836
DOI: 10.1038/s41419-023-06046-x -
Shock (Augusta, Ga.) Apr 2017The endoplasmic reticulum (ER) is a critical organelle that synthesizes secretory proteins and serves as the main calcium storage site of the cell. The accumulation of...
The endoplasmic reticulum (ER) is a critical organelle that synthesizes secretory proteins and serves as the main calcium storage site of the cell. The accumulation of unfolded proteins at the ER results in ER stress. Although the association between ER stress and the pathogenesis of many metabolic conditions have been well characterized using both in vivo and in vitro models, no standardized model concerning ER stress exists. Here, we report a standardized model of ER stress using two well-characterized ER stress-inducing agents, thapsigargin and tunicamycin. Our aim in this current study was 2-fold: to characterize and establish which agent is optimal for in vitro use to model acute ER stress and to evaluate which agent is optimal for in vivo use. To study the first aim we used two well-established metabolic cell lines; human hepatocellular carcinoma (HepG2s) and differentiated mouse adipocytes (3T3-L1). In the second aim we utilized C57BL/6J mice that were randomized into three treatment groups of sham, thapsigargin, and tunicamycin. Our in vitro results showed that tunicamycin worked as a rapid and efficacious inducer of ER stress in adipocytes consistently, whereas thapsigargin and tunicamycin were equally effective in inducing ER stress in hepatocytes. In regards to our in vivo results, we saw that tunicamycin was superior in not only inducing ER stress but also recapturing the metabolic alterations associated with ER stress. Thus, our findings will help guide and inform researchers as to which ER stress agent is appropriate with regards to their model.
Topics: 3T3-L1 Cells; Adipocytes; Animals; Endoplasmic Reticulum Stress; Hep G2 Cells; Hepatocytes; Humans; Immunoblotting; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Polymerase Chain Reaction; Thapsigargin; Tunicamycin
PubMed: 27755507
DOI: 10.1097/SHK.0000000000000759 -
Virology Nov 2021Ibaraki virus (IBAV) causes Ibaraki disease. We have previously shown that IBAV NS3 protein is highly glycosylated and that tunicamycin, an inhibitor of N-linked...
Ibaraki virus (IBAV) causes Ibaraki disease. We have previously shown that IBAV NS3 protein is highly glycosylated and that tunicamycin, an inhibitor of N-linked glycosylation, suppressed NS3 glycosylation and viral propagation. Since tunicamycin is known to cause endoplasmic reticulum (ER) stress, we explored the effects of ER stress and NS3 glycosylation on IBAV infection using tunicamycin and thapsigargin. These reagents both induced ER stress and NS3 glycosylation inhibition in a concentration-dependent manner, and as in our previous report, high concentrations of tunicamycin and thapsigargin suppressed IBAV propagation. However, lower concentrations of these reagents produced limited differences in IBAV propagation, despite their ability to suppress NS3 glycosylation and induce ER stress. These findings suggest that a considerable degree of NS3 glycosylation inhibition and ER stress induction does not suppress IBAV propagation. Conversely, lower concentrations of thapsigargin enhanced IBAV propagation, suggesting that moderate ER stress could benefit IBAV.
Topics: Animals; Cricetinae; Endoplasmic Reticulum Stress; Gene Expression Regulation, Viral; Glycosylation; Orbivirus; Thapsigargin; Tunicamycin; Viral Nonstructural Proteins; Virus Replication
PubMed: 34418796
DOI: 10.1016/j.virol.2021.08.003 -
Cells Nov 2022Neuropeptide Y (NPY), a sympathetic neurotransmitter, is involved in various physiological functions, and its dysregulation is implicated in several neurodegenerative...
Neuropeptide Y (NPY), a sympathetic neurotransmitter, is involved in various physiological functions, and its dysregulation is implicated in several neurodegenerative diseases. Glutamate excitotoxicity, endoplasmic reticulum (ER) stress, and oxidative stress are the common mechanisms associated with numerous neurodegenerative illnesses. The present study aimed to elucidate the protective effects of NPY against glutamate toxicity and tunicamycin-induced ER stress in the human neuroblastoma SH-SY5Y cell line. We exposed the SH-SY5Y cells to glutamate and tunicamycin for two different time points and analyzed the protective effects of NPY at different concentrations. The protective effects of NPY treatments were assessed by cell viability assay, and the signalling pathway changes were evaluated by biochemical techniques such as Western blotting and immunofluorescence assays. Our results showed that treatment of SH-SY5Y cells with NPY significantly increased the viability of the cells in both glutamate toxicity and ER stress conditions. NPY treatments significantly attenuated the glutamate-induced pro-apoptotic activation of ERK1/2 and JNK/BAD pathways. The protective effects of NPY were further evident against tunicamycin-induced ER stress. NPY treatments significantly suppressed the ER stress activation by downregulating BiP, phospho-eIF2α, and CHOP expression. In addition, NPY alleviated the Akt/FoxO3a pathway in acute oxidative conditions caused by glutamate and tunicamycin in SH-SY5Y cells. Our results demonstrated that NPY is neuroprotective against glutamate-induced cell toxicity and tunicamycin-induced ER stress through anti-apoptotic actions.
Topics: Humans; Neuroprotective Agents; Neuropeptide Y; Neuroblastoma; Glutamic Acid; Tunicamycin; Cell Line, Tumor
PubMed: 36429093
DOI: 10.3390/cells11223665 -
Drug Discovery Today Jul 2018The rapid growth of antibiotic-resistant bacterial infections is of major concern for human health. Therefore, it is of great importance to characterize novel targets... (Review)
Review
The rapid growth of antibiotic-resistant bacterial infections is of major concern for human health. Therefore, it is of great importance to characterize novel targets for the development of antibacterial drugs. One promising protein target is MraY (UDP-N-acetylmuramyl-pentapeptide: undecaprenyl phosphate N-acetylmuramyl-pentapeptide-1-phosphate transferase or MurNAc-1-P-transferase), which is essential for bacterial cell wall synthesis. Here, we summarize recent breakthroughs in structural studies of bacterial MraYs and the closely related human GPT (UDP-N-acetylglucosamine: dolichyl phosphate N-acetylglucosamine-1-phosphate transferase or GlcNAc-1-P-transferase). We present a detailed comparison of interaction modes with the natural product inhibitors tunicamycin and muraymycin D2. Finally, we speculate on possible routes to design an antibacterial agent in the form of a potent and selective inhibitor against MraY.
Topics: Animals; Anti-Bacterial Agents; Bacteria; Bacterial Proteins; Drug Design; Drug Resistance, Bacterial; Enzyme Inhibitors; Humans; Models, Molecular; Nucleosides; Peptides; Peptidoglycan; Protein Conformation; Structure-Activity Relationship; Transferases; Transferases (Other Substituted Phosphate Groups); Tunicamycin
PubMed: 29778697
DOI: 10.1016/j.drudis.2018.05.020