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Cell Stress & Chaperones Sep 2022Pulmonary arterial hypertension (PAH) is driven by vascular remodelling due to inflammation and cellular stress, including endoplasmic reticulum stress (ER stress). The...
Pulmonary arterial hypertension (PAH) is driven by vascular remodelling due to inflammation and cellular stress, including endoplasmic reticulum stress (ER stress). The main ER-stress chaperone, glucose-regulated protein 78 kDa (GRP78), is known to have protective effects in inflammatory diseases through extracellular signalling. The aim of this study is to investigate its significance in PAH. Human pulmonary arterial smooth muscle cells (PASMC) were stimulated with compounds that induce ER stress, after which the secretion of GRP78 into the cell medium was analysed by western blot. We found that when ER stress was induced in PASMC, there was also a time-dependent secretion of GRP78. Next, naïve PASMC were treated with conditioned medium (CM) from the ER-stressed donor PASMC. Incubation with CM from ER-stressed PASMC reduced the viability, oxidative stress, and expression of inflammatory and ER-stress markers in target cells. These effects were abrogated when the donor cells were co-treated with Brefeldin A to inhibit active secretion of GRP78. Direct treatment of PASMC with recombinant GRP78 modulated the expression of key inflammatory markers. Additionally, we measured GRP78 plasma levels in 19 PAH patients (Nice Group I) and correlated the levels to risk stratification according to ESC guidelines. Here, elevated plasma levels of GRP78 were associated with a favourable risk stratification. In conclusion, GRP78 is secreted by PASMC under ER stress and exhibits protective effects from the hallmarks of PAH in vitro. Circulating GRP78 may serve as biomarker for risk adjudication of patients with PAH. Proposed mechanism of ER-stress-induced GRP78 secretion by PASMC. Extracellular GRP78 can be measured as a circulating biomarker and is correlated with favourable clinical characteristics. Conditioned medium from ER-stressed PASMC reduces extensive viability, ROS formation, inflammation, and ER stress in target cells. These effects can be abolished by blocking protein secretion in donor cells by using Brefeldin A.
Topics: Brefeldin A; Cell Proliferation; Cells, Cultured; Culture Media, Conditioned; Glucose; Humans; Hypertension, Pulmonary; Inflammation; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Pulmonary Artery; Reactive Oxygen Species; Vascular Remodeling
PubMed: 36029373
DOI: 10.1007/s12192-022-01292-y -
Marine Drugs Jun 2022Mangrove-associated fungi are rich sources of novel and bioactive compounds. A total of 102 fungal strains were isolated from the medicinal mangrove collected from the...
Mangrove-associated fungi are rich sources of novel and bioactive compounds. A total of 102 fungal strains were isolated from the medicinal mangrove collected from the South China Sea. Eighty-four independent culturable isolates were identified using a combination of morphological characteristics and internal transcribed spacer (ITS) sequence analyses, of which thirty-seven strains were selected for phylogenetic analysis. The identified fungi belonged to 22 genera within seven taxonomic orders of one phyla, of which four genera , , , and were first isolated from mangroves. The cytotoxic activity of organic extracts from 55 identified fungi was evaluated against human lung cancer cell lines (A-549), human cervical carcinoma cell lines (HeLa), human hepatoma cells (HepG2), and human acute lymphoblastic leukemia cell lines (Jurkat). The crude extracts of 31 fungi (56.4%) displayed strong cytotoxicity at the concentration of 50 μg/mL. Furthermore, the fungus sp. (HS-N-27) still showed strong cytotoxic activity at the concentration of 25 µg/mL. Integrating cytotoxic activity-guided strategy and fingerprint analysis, a well-known natural Golgi-disruptor and Arf-GEFs inhibitor, brefeldin A, was isolated from the target active strain HS-N-27. It displayed potential activity against A549, HeLa and HepG2 cell lines with the IC values of 101.2, 171.9 and 239.1 nM, respectively. Therefore, combining activity-guided strategy with fingerprint analysis as a discovery tool will be implemented as a systematic strategy for quick discovery of active compounds.
Topics: Acanthaceae; Antineoplastic Agents; Ascomycota; Brefeldin A; Fungi; Gene Library; Humans; Phylogeny
PubMed: 35877725
DOI: 10.3390/md20070432 -
Proceedings of the National Academy of... Jun 2013ADP-ribosylation is a posttranslational modification that modulates the functions of many target proteins. We previously showed that the fungal toxin brefeldin A (BFA)...
ADP-ribosylation is a posttranslational modification that modulates the functions of many target proteins. We previously showed that the fungal toxin brefeldin A (BFA) induces the ADP-ribosylation of C-terminal-binding protein-1 short-form/BFA-ADP-ribosylation substrate (CtBP1-S/BARS), a bifunctional protein with roles in the nucleus as a transcription factor and in the cytosol as a regulator of membrane fission during intracellular trafficking and mitotic partitioning of the Golgi complex. Here, we report that ADP-ribosylation of CtBP1-S/BARS by BFA occurs via a nonconventional mechanism that comprises two steps: (i) synthesis of a BFA-ADP-ribose conjugate by the ADP-ribosyl cyclase CD38 and (ii) covalent binding of the BFA-ADP-ribose conjugate into the CtBP1-S/BARS NAD(+)-binding pocket. This results in the locking of CtBP1-S/BARS in a dimeric conformation, which prevents its binding to interactors known to be involved in membrane fission and, hence, in the inhibition of the fission machinery involved in mitotic Golgi partitioning. As this inhibition may lead to arrest of the cell cycle in G2, these findings provide a strategy for the design of pharmacological blockers of cell cycle in tumor cells that express high levels of CD38.
Topics: ADP-ribosyl Cyclase; ADP-ribosyl Cyclase 1; Adenosine Diphosphate Ribose; Alcohol Oxidoreductases; Animals; Binding Sites; Binding, Competitive; Blotting, Western; Brefeldin A; Cytosol; DNA-Binding Proteins; HeLa Cells; Humans; Membrane Glycoproteins; Models, Molecular; NAD; Protein Binding; Protein Processing, Post-Translational; Protein Structure, Tertiary; Rats
PubMed: 23716697
DOI: 10.1073/pnas.1222413110 -
Scientific Reports Oct 2013cAMP is a universal second messenger. Its signalling is mediated by protein kinase A, Epac and certain types of ion channels in mammalians. cAMP signalling is involved...
cAMP is a universal second messenger. Its signalling is mediated by protein kinase A, Epac and certain types of ion channels in mammalians. cAMP signalling is involved in many physiological processes ranging from vision to the control of insulin secretion, pacemaker activity and gene transcription and therefore selective pharmacological interference is of medical interest. Whereas selective inhibitors of PKA and selective activators of Epac are well established, no inhibitors of Epac were available until recently. Here the action of four of the novel Epac inhibitors was analysed by biophysical means. ESI-05 is confirmed as a selective inhibitor of Epac2. No direct action of Brefeldin A on Epac could be demonstrated. ESI-09 and HJC0197 were found to act as chemicals with general protein denaturing properties and do not act on Epac selectively.
Topics: Animals; Benzene Derivatives; Brefeldin A; Cyclic AMP; Dose-Response Relationship, Drug; Guanine Nucleotide Exchange Factors; Humans; Hydrazones; Isoxazoles; Mice; Protein Denaturation; Sulfones; Thermodynamics; Transition Temperature
PubMed: 24149987
DOI: 10.1038/srep03032 -
The Journal of Biological Chemistry Apr 1999Activation of ADP-ribosylation factors (ARFs), approximately 20-kDa guanine nucleotide-binding proteins that play an important role in intracellular vesicular...
Activation of ADP-ribosylation factors (ARFs), approximately 20-kDa guanine nucleotide-binding proteins that play an important role in intracellular vesicular trafficking, depends on guanine nucleotide-exchange proteins (GEPs), which accelerate replacement of bound GDP with GTP. Two major families of ARF GEPs are known: approximately 200-kDa molecules that are inhibited by brefeldin A (BFA), a fungal metabolite that blocks protein secretion and causes apparent disintegration of Golgi structure, and approximately 50-kDa GEPs that are insensitive to BFA. We describe here two human brain cDNAs that encode BFA-inhibited GEPs. One is a approximately 209-kDa protein 99.5% identical in deduced amino acid sequence (1, 849 residues) to a BFA-inhibited ARF GEP (p200) from bovine brain. The other smaller protein, which is approximately 74% identical (1, 785 amino acids), represents a previously unknown gene. We propose that the former, p200, be named BIG1 for (brefeldin A-inhibited GEP1) and the second, which encodes a approximately 202-kDa protein, BIG2. A protein containing sequences found in BIG2 had been purified earlier from bovine brain. Human tissues contained a 7.5-kilobase BIG1 mRNA and a 9.4-kilobase BIG2 transcript. The BIG1 and BIG2 genes were localized, respectively, to chromosomes 8 and 20. BIG2, synthesized as a His6 fusion protein in Sf9 cells, accelerated guanosine 5'-3-O-(thio)triphosphate binding by recombinant ARF1, ARF5, and ARF6. It activated native ARF (mixture of ARF1 and ARF3) more effectively than it did any of the nonmyristoylated recombinant ARFs. BIG2 activity was inhibited by BFA in a concentration-dependent manner but not by B17, a structural analog without effects on Golgi function. Although several clones for approximately 50-kDa BFA-insensitive ARF GEPs are known, these new clones for the approximately 200-kDa BIG1 and BIG2 should facilitate characterization of this rather different family of proteins as well as the elucidation of mechanisms of regulation of BFA-sensitive ARF function in Golgi transport.
Topics: ADP-Ribosylation Factor 1; ADP-Ribosylation Factors; Amino Acid Sequence; Animals; Base Sequence; Brefeldin A; Cattle; Cloning, Molecular; DNA, Complementary; GTP-Binding Proteins; Guanine Nucleotide Exchange Factors; Humans; Molecular Sequence Data; Recombinant Proteins; Sequence Homology, Amino Acid
PubMed: 10212200
DOI: 10.1074/jbc.274.18.12308 -
Biochimica Et Biophysica Acta Sep 2009The COG complex is a cytosolic heteromeric Golgi complex constituted of 8 subunits (Cog1 to Cog8) and involved in retrograde vesicular Golgi trafficking. The involvement... (Review)
Review
The COG complex is a cytosolic heteromeric Golgi complex constituted of 8 subunits (Cog1 to Cog8) and involved in retrograde vesicular Golgi trafficking. The involvement of this complex in glycosylation and more specifically in Golgi glycosyltransferases localization has been highlighted with the discovery of COG subunit deficiencies leading to CDG (Congenital Disorders of Glycosylation), a group of inherited disorders of glycosylation. To date, many COG deficient CDG patients have been discovered and this article reviews the birth and rise of this group of defects. The architecture of the COG complex and its cellular functions in Golgi trafficking and Golgi glycosylation are discussed.
Topics: Abnormalities, Multiple; Brefeldin A; Congenital Disorders of Glycosylation; Glycosylation; Golgi Apparatus; Humans; Infant; Protein Transport
PubMed: 19028570
DOI: 10.1016/j.bbadis.2008.10.020 -
International Journal of Molecular... Sep 2022Fibrosis is a common final pathway of chronic kidney disease, which is a major incurable disease. Although fibrosis has an irreversible pathophysiology, the molecular...
Fibrosis is a common final pathway of chronic kidney disease, which is a major incurable disease. Although fibrosis has an irreversible pathophysiology, the molecular and cellular mechanisms responsible remain unclear and no specific treatment is available to halt the progress of renal fibrosis. Thus, an improved understanding of the cellular mechanism involved and a novel therapeutic approach are urgently required for end-stage renal disease (ESRD). We investigated the role played by interleukin-10 (IL-10, a potent anti-inflammatory cytokine) in kidney fibrosis and the mechanisms involved using mice and TCMK-1 cells (mouse kidney tubular epithelial cell line). Endoplasmic reticulum stress (ERS), apoptosis, and fibrosis in mice were more severe than in mice after unilateral ureteral obstruction (UUO). The 4-Phenylbutyrate (an ERS inhibitor) treatment induced dramatic reductions in ERS, apoptosis, and fibrosis-associated factors in the renal tissues of mice, compared to wild-type controls after UUO. On the other hand, in cultured TCMK-1 cells, the ERS inducers (tunicamycin, thapsigargin, or brefeldin A) enhanced the expressions of proapoptotic and profibrotic factors, though these effects were mitigated by IL-10. These results were supported by the observation that IL-10 siRNA transfection aggravated tunicamycin-induced CHOP and a-SMA expressions in TCMK-1 cells. We conclude that the anti-fibrotic effects of IL-10 were attributable to the inhibition of ERS-mediated apoptosis and believe that the results of this study improve the understanding of the cellular mechanism responsible for fibrosis and aid in the development of novel therapeutic approaches.
Topics: Animals; Apoptosis; Brefeldin A; Disease Models, Animal; Endoplasmic Reticulum Stress; Fibrosis; Interleukin-10; Kidney; Kidney Diseases; Mice; RNA, Small Interfering; Renal Insufficiency, Chronic; Thapsigargin; Tunicamycin; Ureteral Obstruction
PubMed: 36142626
DOI: 10.3390/ijms231810702 -
Bioorganic & Medicinal Chemistry Letters Jun 2017Docetaxel is a commonly used chemotherapeutic drug for patients with late stage prostate cancer. However, serious side effect and drug resistance limit its clinical...
Docetaxel is a commonly used chemotherapeutic drug for patients with late stage prostate cancer. However, serious side effect and drug resistance limit its clinical success. Brefeldin A is a 16-membered macrolide antibiotic from mangrove-derived Fungus Aspergillus sp. (9Hu), which exhibited potent cytotoxicity against human cancer cells. In the present study, we determined the effect of brefeldin A on docetaxel-induced growth inhibition and apoptosis in human prostate cancer PC-3 cells. Brefeldin A in combination with docetaxel inhibited the growth of PC-3 cells in monolayer and in three dimensional cultures. The combination also potently stimulated apoptosis in PC-3 cells as determined by propidium iodide staining and morphological assessment. Mechanistic studies showed that growth inhibition and apoptosis in PC-3 cells treated with brefeldin A and docetaxel were associated with decrease in the level of Bcl-2. The present study indicates that combined brefeldin A with docetaxel may represent a novel approach for improving the efficacy of docetaxel, and Bcl-2 may serve as a target for brefeldin A to enhance the effects of docetaxel chemotherapy.
Topics: Apoptosis; Brefeldin A; Cell Line, Tumor; Cell Proliferation; Docetaxel; Humans; Male; Prostatic Neoplasms; Taxoids
PubMed: 28462831
DOI: 10.1016/j.bmcl.2017.04.047 -
Developmental Biology Sep 2014Myoblast fusion (a critical process by which muscles grow) occurs in a multi-step fashion that requires actin and membrane remodeling; but important questions remain...
Myoblast fusion (a critical process by which muscles grow) occurs in a multi-step fashion that requires actin and membrane remodeling; but important questions remain regarding the spatial/temporal regulation of and interrelationship between these processes. We recently reported that the Rho-GAP, GRAF1, was particularly abundant in muscles undergoing fusion to form multinucleated fibers and that enforced expression of GRAF1 in cultured myoblasts induced robust fusion by a process that required GAP-dependent actin remodeling and BAR domain-dependent membrane sculpting. Herein we developed a novel line of GRAF1-deficient mice to explore a role for this protein in the formation/maturation of myotubes in vivo. Post-natal muscles from GRAF1-depleted mice exhibited a significant and persistent reduction in cross-sectional area, impaired regenerative capacity and a significant decrease in force production indicative of lack of efficient myoblast fusion. A significant fusion defect was recapitulated in isolated myoblasts depleted of GRAF1 or its closely related family member GRAF2. Mechanistically, we show that GRAF1 and 2 facilitate myoblast fusion, at least in part, by promoting vesicle-mediated translocation of fusogenic ferlin proteins to the plasma membrane.
Topics: Animals; Biological Transport; Brefeldin A; Cell Fusion; Cell Membrane; Cells, Cultured; GTPase-Activating Proteins; Golgi Apparatus; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle Development; Muscle Fibers, Skeletal; Muscle Proteins; Myoblasts; Protein Synthesis Inhibitors; RNA Interference; RNA, Small Interfering; Vesicular Transport Proteins
PubMed: 25019370
DOI: 10.1016/j.ydbio.2014.06.025 -
Scientific Reports Sep 2022Sphingosine-1-phosphate phosphatase (SPP) catalyzes the dephosphorylation of sphingosine-1-phosphate (S1P) into sphingosine, the reverse reaction of sphingosine kinase....
Sphingosine-1-phosphate phosphatase (SPP) catalyzes the dephosphorylation of sphingosine-1-phosphate (S1P) into sphingosine, the reverse reaction of sphingosine kinase. In mammals, S1P acts as a potent bioactive molecule regulating cell proliferation, migration, and immunity. In Leishmania, S1P production is crucial for the synthesis of ethanolamine and choline phospholipids, and cell survival under stress conditions. To better understand the roles of S1P, we characterized a SPP ortholog in Leishmania major which displays activity towards S1P but not structurally related lipids such as ceramide-1-phosphate or lysophosphatidic acid. While this enzyme is found in the endoplasmic reticulum in mammalian cells, L. major SPP is localized at the Golgi apparatus. Importantly, chromosomal SPP alleles cannot be deleted from L. major even with the addition of a complementing episome, suggesting that endogenously expressed SPP is essential. Finally, SPP overexpression in L. major leads to a slower growth rate and heightened sensitivity to brefeldin A and sodium orthovanadate. Together, these results suggest that the equilibrium between S1P and sphingosine is vital for the function of Golgi apparatus in Leishmania.
Topics: Animals; Brefeldin A; Ceramides; Choline; Ethanolamines; Golgi Apparatus; Leishmania major; Lysophospholipids; Mammals; Membrane Proteins; Phosphates; Phosphoric Monoester Hydrolases; Sodium; Sphingosine; Vanadates
PubMed: 36163400
DOI: 10.1038/s41598-022-20249-w