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Infection and Immunity Jul 1987A parasitic protozoan, Leishmania mexicana amazonensis, was previously made resistant to tunicamycin (J.A. Kink and K.-P. Chang, Proc. Natl. Acad. Sci. USA 84:1253-1257,...
A parasitic protozoan, Leishmania mexicana amazonensis, was previously made resistant to tunicamycin (J.A. Kink and K.-P. Chang, Proc. Natl. Acad. Sci. USA 84:1253-1257, 1987). In the present study, six different tunicamycin-resistant variants were biologically and biochemically compared with their parental wild type to further delineate the mechanism of tunicamycin resistance and that of their virulence observed. In contrast to their parental wild type, all tunicamycin-resistant variants were found to grow and differentiate in tunicamycin-containing medium. The 50% lethal doses of tunicamycin for variants resistant to 10 or 80 micrograms of tunicamycin per ml were 20- and 100-fold higher, respectively, than that of the wild type. Specific activity of the microsomal N-acetylglucosamine-1-phosphate transferase was 4- to 12-fold higher in the tunicamycin-resistant cells than in their parental wild type and tunicamycin-sensitive revertants. The level of the enzyme activity is proportional to the degree of drug resistance. Inhibition kinetics studies showed that the enzyme from all groups was equally sensitive to the drug, with a 50% effective concentration of 1 to 1.3 micrograms of tunicamycin per ml. Thus, tunicamycin resistance of the variants is caused primarily by an increased level of their enzyme without alteration of its structure. Protein glycosylation determined by the incorporation of 2-D-[3H]mannose was about twofold higher in the tunicamycin-resistant variants than in their parental wild type. The increased glycosyltransferase activity in the latter apparently renders their protein glycosylation insensitive to the inhibition by tunicamycin. A major membrane glycoprotein of 63 kilodaltons (gp63) on the leishmania surface was found to be about threefold higher in the tunicamycin-resistant variants than in the wild type, as determined by immunoprecipitation with a monoclonal antibody specific for this antigen. Tunicamycin treatment of the wild type and tunicamycin-resistant variants caused changes in the electrophoretic mobility of this molecule, indicating a higher degree of its glycosylation in the latter cells. The tunicamycin-resistant variants parasitized macrophages in vitro more effectively than did the wild type, accounting for their virulence seen in mice. Thus, a high level of the glycosyltransferase enables the tunicamycin-resistant cells not only to overcome the inhibitory effect of tunicamycin on protein glycosylation but also to express their virulence, possibly by regulating N glycosylation of leishmanial proteins critical for leishmanias to establish intracellular parasitism.
Topics: Animals; Cell Differentiation; Drug Resistance; Glycoproteins; Glycosylation; Leishmania mexicana; Leishmaniasis; Mice; Microsomes; Phosphotransferases; Protein Biosynthesis; Protein Processing, Post-Translational; Transferases (Other Substituted Phosphate Groups); Tunicamycin
PubMed: 3036710
DOI: 10.1128/iai.55.7.1692-1700.1987 -
Microbial Cell Factories Oct 2022Tunicamycin inhibits the first step of protein N-glycosylation modification. However, the physiological, transcriptomic, and N-glycomic effects of tunicamycin on...
Endoplasmic reticulum-quality control pathway and endoplasmic reticulum-associated degradation mechanism regulate the N-glycoproteins and N-glycan structures in the diatom Phaeodactylum tricornutum.
Tunicamycin inhibits the first step of protein N-glycosylation modification. However, the physiological, transcriptomic, and N-glycomic effects of tunicamycin on important marine diatom Phaeodactylum tricornutum are still unknown. In this study, comprehensive approaches were used to study the effects of tunicamycin stress. The results showed that cell growth and photosynthesis were significantly inhibited in P. tricornutum under the tunicamycin stress. The soluble protein content was significantly decreased, while the soluble sugar and neutral lipid were dramatically increased to orchestrate the balance of carbon and nitrogen metabolisms. The stress of 0.3 μg ml tunicamycin resulted in the differential expression of ERQC and ERAD related genes. The upregulation of genes involved in ERQC pathway, the activation of anti-oxidases and the differential expression of genes related with ERAD mechanism might be important for maintaining homeostasis in cell. The identification of N-glycans, especially complex-type N-glycan structures enriched the N-glycan database of diatom P. tricornutum and provided important information for studying the function of N-glycosylation modification on proteins. As a whole, our study proposed working models of ERQC and ERAD will provide a solid foundation for further in-depth study of the related mechanism and the diatom expression system.
Topics: Endoplasmic Reticulum-Associated Degradation; Diatoms; Tunicamycin; Endoplasmic Reticulum; Glycoproteins; Polysaccharides; Carbon; Sugars; Nitrogen; Lipids; Quality Control
PubMed: 36266689
DOI: 10.1186/s12934-022-01941-y -
ACS Chemical Biology Nov 2020The alarming growth of antibiotic resistance that is currently ongoing is a serious threat to human health. One of the most promising novel antibiotic targets is MraY...
The alarming growth of antibiotic resistance that is currently ongoing is a serious threat to human health. One of the most promising novel antibiotic targets is MraY (phospho-MurNAc-pentapeptide-transferase), an essential enzyme in bacterial cell wall synthesis. Through recent advances in biochemical research, there is now structural information available for MraY, and for its human homologue GPT (GlcNAc-1-P-transferase), that opens up exciting possibilities for structure-based drug design. The antibiotic compound tunicamycin is a natural product inhibitor of MraY that is also toxic to eukaryotes through its binding to GPT. In this work, we have used tunicamycin and modified versions of tunicamycin as tool compounds to explore the active site of MraY and to gain further insight into what determines inhibitor potency. We have investigated tunicamycin variants where the following motifs have been modified: the length and branching of the tunicamycin fatty acyl chain, the saturation of the fatty acyl chain, the 6″-hydroxyl group of the GlcNAc ring, and the ring structure of the uracil motif. The compounds are analyzed in terms of how potently they bind to MraY, inhibit the activity of the enzyme, and affect the protein thermal stability. Finally, we rationalize these results in the context of the protein structures of MraY and GPT.
Topics: Anti-Bacterial Agents; Bacterial Infections; Bacterial Proteins; Catalytic Domain; Clostridium; Clostridium Infections; Guanosine Triphosphate; Humans; Molecular Docking Simulation; Transferases; Transferases (Other Substituted Phosphate Groups); Tunicamycin
PubMed: 33164499
DOI: 10.1021/acschembio.0c00423 -
Evaluation of Cell Models to Study Monocyte Functions in PMM2 Congenital Disorders of Glycosylation.Frontiers in Immunology 2022Congenital disorders of glycosylation (CDG) are inherited metabolic diseases characterized by mutations in enzymes involved in different steps of protein glycosylation,...
Congenital disorders of glycosylation (CDG) are inherited metabolic diseases characterized by mutations in enzymes involved in different steps of protein glycosylation, leading to aberrant synthesis, attachment or processing of glycans. Recently, immunological dysfunctions in several CDG types have been increasingly documented. Despite these observations, detailed studies on immune cell dysfunction in PMM2-CDG and other CDG types are still scarce. Studying PMM2-CDG patient immune cells is challenging due to limited availability of patient material, which is a result of the low incidence of the disease and the often young age of the subjects. Dedicated immune cell models, mimicking PMM2-CDG, could circumvent many of these problems and facilitate research into the mechanisms of immune dysfunction. Here we provide initial observations about the immunophenotype and the phagocytic function of primary PMM2-CDG monocytes. Furthermore, we assessed the suitability of two different glycosylation-impaired human monocyte models: tunicamycin-treated THP-1 monocytes and PMM2 knockdown THP-1 monocytes induced by shRNAs. We found no significant differences in primary monocyte subpopulations of PMM2-CDG patients as compared to healthy individuals but we did observe anomalous surface glycosylation patterns in PMM2-CDG patient monocytes as determined using fluorescent lectin binding. We also looked at the capacity of monocytes to bind and internalize fungal particles and found a slightly increased uptake of by PMM2-CDG monocytes as compared to healthy monocytes. Tunicamycin-treated THP-1 monocytes showed a highly decreased uptake of fungal particles, accompanied by a strong decrease in glycosylation levels and a high induction of ER stress. In contrast and despite a drastic reduction of the PMM2 enzyme activity, PMM2 knockdown THP-1 monocytes showed no changes in global surface glycosylation levels, levels of fungal particle uptake similar to control monocytes, and no ER stress induction. Collectively, these initial observations suggest that the absence of ER stress in PMM2 knockdown THP-1 cells make this model superior over tunicamycin-treated THP-1 cells and more comparable to primary PMM2-CDG monocytes. Further development and exploitation of CDG monocyte models will be essential for future in-depth studies to ultimately unravel the mechanisms of immune dysfunction in CDG.
Topics: Congenital Disorders of Glycosylation; Glycosylation; Humans; Monocytes; Phosphotransferases (Phosphomutases); Tunicamycin
PubMed: 35603178
DOI: 10.3389/fimmu.2022.869031 -
Methods in Molecular Biology (Clifton,... 2018The unfolded protein response (UPR) is a highly regulated signaling pathway that is largely conserved across eukaryotes. It is essential for cell homeostasis under...
The unfolded protein response (UPR) is a highly regulated signaling pathway that is largely conserved across eukaryotes. It is essential for cell homeostasis under environmental and physiological conditions that perturb the protein folding in the endoplasmic reticulum (ER). Arabidopsis is one of the outstanding multicellular model systems in which to investigate the UPR. Here, we described a protocol to induce the UPR in plants, specifically arabidopsis, and to estimate their ability to cope with ER stress through the quantification of physiological parameters.
Topics: Arabidopsis; Arabidopsis Proteins; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Tunicamycin; Unfolded Protein Response
PubMed: 29043682
DOI: 10.1007/978-1-4939-7389-7_18 -
Nan Fang Yi Ke Da Xue Xue Bao = Journal... Jun 2022To explore the mechanism by which inositol-requiring enzyme-1 (IRE1) regulates autophagy function of chondrocytes through calcium homeostasis endoplasmic reticulum...
OBJECTIVE
To explore the mechanism by which inositol-requiring enzyme-1 (IRE1) regulates autophagy function of chondrocytes through calcium homeostasis endoplasmic reticulum protein (CHERP).
METHODS
Cultured human chondrocytes (C28/I2 cells) were treated with tunicamycin, 4μ8c, rapamycin, or both 4μ8c and rapamycin, and the expressions of endoplasmic reticulum (ER) stress- and autophagy-related proteins were detected with Western blotting. Primary chondrocytes from ERN1 knockout (ERN1 CKO) mice and wild-type mice were examined for ATG5 and ATG7 mRNA expressions, IRE1 and p-IRE1 protein expressions, and intracellular calcium ion content using qPCR, Western blotting and flow cytometry. The effect of bafilomycin A1 treatment on LC3 Ⅱ/LC3 Ⅰ ratio in the isolated chondrocytes was assessed with Western blotting. Changes in autophagic flux of the chondrocytes in response to rapamycin treatment were detected using autophagy dual fluorescent virus. The changes in autophagy level in C28/I2 cells overexpressing CHERP and IRE1 were detected using immunofluorescence assay.
RESULTS
Tunicamycin treatment significantly up-regulated ER stress-related proteins and LC3 Ⅱ/LC3 Ⅰ ratio and down-regulated the expression of p62 in C28/I2 cells ( < 0.05). Rapamycin obviously up-regulated LC3 Ⅱ/LC3 Ⅰ ratio ( < 0.001) in C28/I2 cells, but this effect was significantly attenuated by co-treatment with 4μ8c ( < 0.05). Compared with the cells from the wild-type mice, the primary chondrocytes from ERN1 knockout mice showed significantly down-regulated mRNA levels of ERN1 ( < 0.01), ATG5 ( < 0.001) and ATG7 ( < 0.001), lowered or even lost expressions of IRE1 and p-IRE1 proteins (P < 0.01), and increased expression of CHERP ( < 0.05) and intracellular calcium ion content ( < 0.001). Bafilomycin A1 treatment obviously increased LC3 Ⅱ/ LC3 Ⅰ ratio in the chondrocytes from both wild-type and ERN1 knockout mice ( < 0.01 or 0.05), but the increment was more obvious in the wild-type chondrocytes ( < 0.05). Treatment with autophagy dual-fluorescence virus resulted in a significantly greater fluorescence intensity of LC3-GFP in rapamycin-treated ERN1 CKO chondrocytes than in wild-type chondrocytes ( < 0.05). In C28/I2 cells, overexpression of CHERP obviously decreased the fluorescence intensity of LC3, and overexpression of IRE1 enhanced the fluorescence intensity and partially rescued the fluorescence reduction of LC3 caused by CHERP.
CONCLUSION
IRE1 deficiency impairs autophagy in chondrocytes by upregulating CHERP and increasing intracellular calcium ion content.
Topics: Animals; Autophagy; Calcium; Chondrocytes; Endoplasmic Reticulum; Endoribonucleases; Homeostasis; Inositol; Mice; Mice, Knockout; Protein Serine-Threonine Kinases; RNA, Messenger; Sirolimus; Tunicamycin
PubMed: 35790428
DOI: 10.12122/j.issn.1673-4254.2022.06.01 -
Antimicrobial Agents and Chemotherapy Aug 2018The tunicamycin biosynthetic gene cluster of consists of 14 genes ( to ) with a high degree of apparent translational coupling. Transcriptional analysis revealed that...
The tunicamycin biosynthetic gene cluster of consists of 14 genes ( to ) with a high degree of apparent translational coupling. Transcriptional analysis revealed that all of these genes are likely to be transcribed as a single operon from two promoters, p1 and p2. In-frame deletion analysis revealed that just six of these genes () are essential for tunicamycin production in the heterologous host , while five () with likely counterparts in primary metabolism are not necessary, but presumably ensure efficient production of the antibiotic at the onset of tunicamycin biosynthesis. Three genes are implicated in immunity, namely, and , which encode a two-component ABC transporter presumably required for export of the antibiotic, and , which encodes a putative -adenosylmethionine (SAM)-dependent methyltransferase. Expression of or in conferred resistance to exogenous tunicamycin. The results presented here provide new insights into tunicamycin biosynthesis and immunity.
Topics: ATP-Binding Cassette Transporters; Anti-Bacterial Agents; Base Sequence; Gene Deletion; Gene Expression Regulation, Bacterial; Genes, Bacterial; Genetic Complementation Test; Methyltransferases; Multigene Family; Operon; Promoter Regions, Genetic; Streptomyces; Streptomyces coelicolor; Tunicamycin
PubMed: 29844049
DOI: 10.1128/AAC.00130-18 -
Proteomics. Clinical Applications Sep 2022Parkinson's disease (PD) is the second most prevalent neurodegenerative disease. It is generally diagnosed clinically after the irreversible loss of dopaminergic neurons...
PURPOSE
Parkinson's disease (PD) is the second most prevalent neurodegenerative disease. It is generally diagnosed clinically after the irreversible loss of dopaminergic neurons and no general biomarkers currently exist. To gain insight into the underlying cellular causes of PD we aimed to quantify the proteomic differences between healthy control and PD patient cells.
EXPERIMENTAL DESIGN
Sequential Window Acquisition of all THeoretical Mass Spectra was performed on primary cells from healthy controls and PD patients.
RESULTS
In total, 1948 proteins were quantified and 228 proteins were significantly differentially expressed in PD patient cells. In PD patient cells, we identified seven significantly increased proteins involved in the unfolded protein response (UPR) and focused on cells with high and low amounts of PDIA6 and HYOU1. We discovered that PD patients with high amounts of PDIA6 and HYOU1 proteins were more sensitive to endoplasmic reticulum stress, in particular to tunicamycin. Data is available via ProteomeXchange with identifier PXD030723.
CONCLUSIONS AND CLINICAL RELEVANCE
This data from primary patient cells has uncovered a critical role of the UPR in patients with PD and may provide insight to the underlying cellular dysfunctions in these patients.
Topics: Biomarkers; Humans; Neurodegenerative Diseases; Parkinson Disease; Proteomics; Tunicamycin
PubMed: 35579911
DOI: 10.1002/prca.202200015 -
Biomolecules Jan 2021Renal injury observed in several pathologies has been associated with lipid accumulation in the kidney. While it has been suggested that the accumulation of renal lipids...
Renal injury observed in several pathologies has been associated with lipid accumulation in the kidney. While it has been suggested that the accumulation of renal lipids depends on free fatty acids released from adipose tissue, it is not known whether in situ renal lipogenesis due to endoplasmic reticulum (ER) stress contributes to kidney injury. The aim of the present study was to elucidate the role of pharmacological ER stress in renal structure and function and its effect on renal lipid metabolism of C57BL/6 mice. ER stress increased serum creatinine and induced kidney structural abnormalities. Tunicamycin-administered mice developed hyperinsulinemia, augmented lipolysis and increased circulating leptin and adiponectin. Renal unfolded protein response (UPR) gene expression markers, the lipogenic transcription factor SREBP1 and the phosphorylation of eIF2α increased 8 h after tunicamycin administration. At 24 h, an increase in BiP protein content was accompanied by a reduction in p-eIF2α and increased SREBP-1 and FASn protein content, in addition to a significant increase in triglyceride content and a reduction in AMPK. Thus, ER stress induces in situ lipid synthesis, leading to renal lipid accumulation and functional alterations. Future pharmacological and/or dietary strategies must target renal ER stress to prevent kidney damage and the progression of metabolic diseases.
Topics: Animals; Body Weight; Endocrine System; Endoplasmic Reticulum Stress; Kidney; Lipogenesis; Male; Metabolic Networks and Pathways; Mice, Inbred C57BL; Organ Size; Tunicamycin; Unfolded Protein Response; Mice
PubMed: 33430288
DOI: 10.3390/biom11010073 -
Bioscience, Biotechnology, and... 2013We have developed a new series of R4L1 Gateway binary vectors (R4L1pGWB), which carry the bialaphos resistance gene (bar) or the UDP-N-acetylglucosamine:dolichol... (Review)
Review
Development of gateway binary vectors R4L1pGWB possessing the bialaphos resistance gene (bar) and the tunicamycin resistance gene as markers for promoter analysis in plants.
We have developed a new series of R4L1 Gateway binary vectors (R4L1pGWB), which carry the bialaphos resistance gene (bar) or the UDP-N-acetylglucosamine:dolichol phosphate N-acetylglucosamine-1-P transferase (GPT) gene as selection markers that confer BASTA® and tunicamycin resistance on plants respectively. R4L1pGWBs have an attR4-attL1-reporter and can accept an attL4-promoter-attR1 entry clone for easy construction of an attB4-promoter-attB1-reporter clone. The new R4L1pGWBs facilitate promoter:reporter analysis in pre-existing transgenic plants that are resistant to kanamycin or hygromycin.
Topics: Biomarkers; Cinnamates; Drug Resistance, Microbial; Genetic Vectors; Hygromycin B; Kanamycin; Organophosphorus Compounds; Plants, Genetically Modified; Promoter Regions, Genetic; Tunicamycin; Uridine Diphosphate Sugars
PubMed: 23924715
DOI: 10.1271/bbb.130405