Inhibition of Golgi stress alleviates sepsis-induced cardiomyopathy by reducing inflammation and apoptosis.

BACKGROUND: Sepsis is often accompanied by multiple organ dysfunction, in which the incidence of cardiac injury is about 60%, and is closely related to high mortality. Recent studies have shown that Golgi stress is involved in liver injury, kidney injury, and lung injury in sepsis. However, whether it is one of the key mechanisms of sepsis-induced cardiomyopathy (SIC) is still unclear. The aim of this study is to investigate whether Golgi stress mediates SIC and the specific mechanism. METHODS: Sepsis model of male C57BL/6J mice was established by cecal ligation and puncture. To observe the effect of Golgi stress on SIC, mice were injected with Golgi stimulant (Brefeldin A) or Golgi inhibitor (Glutathione), respectively. The 7-day survival rate of mice were recorded, and myocardial injury indicators including cardiac function, myocardial enzymes, myocardial pathological tissue score, myocardial inflammatory factors, and apoptosis were detected. The morphology of Golgi was observed by immunofluorescence, and the Golgi stress indices including GM-130, GOLPH3 and Goligin97 were detected by WB and qPCR. RESULTS: After CLP, the cardiac function of mice was impaired and the levels of myocardial enzymes were significantly increased. Golgi stress was accompanied by increased myocardial inflammation and apoptosis. Moreover, the expressions of morphological proteins GM-130 and Golgin97 were decreased, and the expression of stress protein GOLPH3 was increased. In addition, Brefeldin A increased 7-day mortality and the above indicators in mice. The use of glutathione improves all of the above indicators. CONCLUSION: Golgi stress mediates SIC, and the inhibition of Golgi stress can improve SIC by inhibiting apoptosis and inflammation.

Electrostatic Impact of Brefeldin A on Thiocyanate Probes Surrounding the Interface of Arf1-BFA-ARNO4M, a Protein-Drug-Protein Complex.

Protein-protein interactions regulate many cellular processes, making them ideal drug candidates. Design of such drugs, however, is hindered by a lack of understanding of the factors that contribute to the interaction specificity. Specific protein-protein complexes possess both structural and electrostatic complementarity, and while structural complementarity of protein complexes has been extensively investigated, fundamental understanding of the complicated networks of electrostatic interactions at these interfaces is lacking, thus hindering the rational design of orthosterically binding small molecules. To better understand the electrostatic interactions at protein interfaces and how a small molecule could contribute to and fit within that environment, we used a model protein-drug-protein system, Arf1-BFA-ARNO4M, to investigate how small molecule brefeldin A (BFA) perturbs the Arf1-ARNO4M interface. By using nitrile probe labeled Arf1 sites and measuring vibrational Stark effects as well as temperature dependent infrared shifts, we measured changes in the electric field and hydrogen bonding at this interface upon BFA binding. At all five probe locations of Arf1, we found that the vibrational shifts resulting from BFA binding corroborate trends found in Poisson-Boltzmann calculations of surface potentials of Arf1-ARNO4M and Arf1-BFA-ARNO4M, where BFA contributes negative electrostatic potential to the protein interface. The data also corroborate previous hypotheses about the mechanism of interfacial binding and confirm that alternating patches of hydrophobic and polar interactions lead to BFA binding specificity. These findings demonstrate the impact of BFA on this protein-protein interface and have implications for the design of other interfacial drug candidates.

The development of resistance to an inhibitor of a cellular protein reveals a critical interaction between the enterovirus protein 2C and a small GTPase Arf1.

The cellular protein GBF1, an activator of Arf GTPases (ArfGEF: Arf guanine nucleotide exchange factor), is recruited to the replication organelles of enteroviruses through interaction with the viral protein 3A, and its ArfGEF activity is required for viral replication, however how GBF1-dependent Arf activation supports the infection remains enigmatic. Here, we investigated the development of resistance of poliovirus, a prototype enterovirus, to increasing concentrations of brefeldin A (BFA), an inhibitor of GBF1. High level of resistance required a gradual accumulation of multiple mutations in the viral protein 2C. The 2C mutations conferred BFA resistance even in the context of a 3A mutant previously shown to be defective in the recruitment of GBF1 to replication organelles, and in cells depleted of GBF1, suggesting a GBF1-independent replication mechanism. Still, activated Arfs accumulated on the replication organelles of this mutant even in the presence of BFA, its replication was inhibited by a pan-ArfGEF inhibitor LM11, and the BFA-resistant phenotype was compromised in Arf1-knockout cells. Importantly, the mutations strongly increased the interaction of 2C with the activated form of Arf1. Analysis of other enteroviruses revealed a particularly strong interaction of 2C of human rhinovirus 1A with activated Arf1. Accordingly, the replication of this virus was significantly less sensitive to BFA than that of poliovirus. Thus, our data demonstrate that enterovirus 2Cs may behave like Arf1 effector proteins and that GBF1 but not Arf activation can be dispensable for enterovirus replication. These findings have important implications for the development of host-targeted anti-viral therapeutics.

Cell surface associated protein mucin 15 (MUC15) is elevated in preeclampsia.

BACKGROUND: Mucins are a family of proteins that protect the epithelium. A particular type of mucin, MUC15 is highly expressed in the placenta. This study aimed to characterise MUC15 in preeclampsia and investigate its role in placental stem cell biology. METHODS: MUC15 mRNA and protein were measured in placentas from patients with early onset (<34 weeks' gestation) preeclampsia. Circulating serum MUC15 was measured via ELISA. MUC15 was localised in the placenta using in situ hybridisation. MUC15 mRNA expression was measured across differentiation of human trophoblast stem cells (hTSCs) to syncytiotrophoblast and extravillous trophoblasts. MUC15 was measured after syncytialised hTSCs were cultured in hypoxic (1% O2) and proinflammatory (TNF α, IL-6) conditions. MUC15 secretion was assessed when syncytialised hTSCs were treated with brefeldin A (impairs protein trafficking) and batimastat (inhibits matrix metalloproteinases). RESULTS: MUC15 protein was significantly increased in the placenta (P = 0.0003, n = 32 vs n = 20 controls) and serum (P = 0.016, n = 32 vs n = 22 controls) of patients with preeclampsia, whilst MUC15 mRNA remained unchanged (n = 61 vs n = 18 controls). MUC15 mRNA (P = 0.005) and protein secretion (P = 0.006) increased following differentiation to syncytiotrophoblast cells. In situ hybridisation confirmed MUC15 localised to the syncytiotrophoblast cell within the placenta. Neither hypoxic or inflammatory conditions changed MUC15 mRNA expression or secretion. Brefeldin A treated hTSCs did not alter MUC15 secretion, whilst batimastat reduced MUC15 secretion (P = 0.044). CONCLUSIONS: MUC15 is increased in early onset preeclampsia and is cleaved by matrix metalloproteinases. Increased MUC15 may reflect a protective mechanism associated with placental dysfunction. Further research will aid in confirming this.

Design and Synthesis of Brefeldin A-Isothiocyanate Derivatives with Selectivity and Their Potential for Cervical Cancer Therapy.

Brefeldin A has a wide range of anticancer activity against a variety of tumor cells. Its poor pharmacokinetic properties and significant toxicity seriously hinder its further development. In this manuscript, 25 brefeldin A-isothiocyanate derivatives were designed and synthesized. Most derivatives showed good selectivity between HeLa cells and L-02 cells. In particular, 6 exhibited potent antiproliferative activity against HeLa cells (IC50 = 1.84 µM) with no obvious cytotoxic activity to L-02 (IC50 > 80 µM). Further cellular mechanism tests indicated that 6 induced HeLa cell cycle arrest at G1 phase. Cell nucleus fragmentation and decreased mitochondrial membrane potential suggested 6 could induce apoptosis in HeLa cells through the mitochondrial-dependent pathway.

5-(4-Hydroxyphenyl)-3H-1,2-dithiole-3-thione derivatives of brefeldin A: Design, synthesis and cytotoxicity in MDA-MB-231 human breast cancer cells.

27 novel 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione derivatives of brefeldin A were designed and synthesized to make them more conducive to the cancer treatment. The antiproliferative activity of all the target compounds was tested against six human cancer cell lines and one human normal cell line. Compound 10d exhibited nearly the most potent cytotoxicity with IC50 values of 0.58, 0.69, 1.82, 0.85, 0.75, 0.33 and 1.75 µM against A549, DU-145, A375, HeLa, HepG2, MDA-MB-231 and L-02 cell lines. Moreover, 10d inhibited metastasis and induced apoptosis of MDA-MB-231 cells in a dose-dependent manner. The potent anticancer effects of 10d were prompted based on the aforementioned results, the therapeutic potential of 10d for breast cancer was worth further exploration.

Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells.

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane system. This includes the secretory transport of newly synthesized proteins to the cell surface or the outside of the cell, the endocytic transport of extracellular cargoes or plasma membrane components into the cell, and the recycling or shuttling transport of cargoes between the subcellular organelles, etc. Membrane trafficking events are crucial to the development, growth, and environmental adaptation of all eukaryotic cells and, thus, are under stringent regulation. Cell-surface receptor kinases, which perceive ligand signals from the extracellular space, undergo both secretory and endocytic transport. Commonly used approaches to study the membrane trafficking events using a plasma membrane-localized leucine-rich-repeat receptor kinase, ERL1, are described here. The approaches include plant material preparation, pharmacological treatment, and confocal imaging setup. To monitor the spatiotemporal regulation of ERL1, this study describes the co-localization analysis between ERL1 and a multi-vesicular body marker protein, RFP-Ara7, the time series analysis of these two proteins, and the z-stack analysis of ERL1-YFP treated with the membrane trafficking inhibitors brefeldin A and wortmannin.

Endosidin 5 disruption of the Golgi apparatus and extracellular matrix secretion in the unicellular charophyte Penium margaritaceum.

BACKGROUND AND AIMS: Endosidins are a group of low-molecular-weight compounds, first identified by 'chemical biology' screening assays, that have been used to target specific components of the endomembrane system. In this study, we employed multiple microscopy-based screening techniques to elucidate the effects of endosidin 5 (ES5) on the Golgi apparatus and the secretion of extracellular matrix (ECM) components in Penium margaritaceum. These effects were compared with those caused by treatments with brefeldin A and concanamycin A. Penium margaritaceum's extensive Golgi apparatus and endomembrane system make it an outstanding model organism for screening changes to the endomembrane system. Here we detail changes to the Golgi apparatus and secretion of ECM material caused by ES5. METHODS: Changes to extracellular polymeric substance (EPS) secretion and cell wall expansion were screened using fluorescence microscopy. Confocal laser scanning microscopy and transmission electron microscopy were used to assess changes to the Golgi apparatus, the cell wall and the vesicular network. Electron tomography was also performed to detail the changes to the Golgi apparatus. KEY RESULTS: While other endosidins were able to impact EPS secretion and cell wall expansion, only ES5 completely inhibited EPS secretion and cell wall expansion over 24 h. Short treatments of ES5 resulted in displacement of the Golgi bodies from their typical linear alignment. The number of cisternae decreased per Golgi stack and trans face cisternae in-curled to form distinct elongate circular profiles. Longer treatment resulted in a transformation of the Golgi body to an irregular aggregate of cisternae. These alterations could be reversed by removal of ES5 and returning cells to culture. CONCLUSIONS: ES5 alters secretion of ECM material in Penium by affecting the Golgi apparatus and does so in a markedly different way from other endomembrane inhibitors such as brefeldin A and concanamycin A.

Elucidation of OSW-1-Induced Stress Responses in Neuro2a Cells.

OSW-1, a steroidal saponin isolated from the bulbs of Ornithogalum saundersiae, is a promising compound for an anticancer drug; however, its cytotoxic mechanisms have not been fully elucidated. Therefore, we analyzed the stress responses triggered by OSW-1 in the mouse neuroblastoma cell line Neuro2a by comparing it with brefeldin A (BFA), a Golgi apparatus-disrupting reagent. Among the Golgi stress sensors TFE3/TFEB and CREB3, OSW-1 induced dephosphorylation of TFE3/TFEB but not cleavage of CREB3, and induction of the ER stress-inducible genes GADD153 and GADD34 was slight. On the other hand, the induction of LC3-II, an autophagy marker, was more pronounced than the BFA stimulation. To elucidate OSW-1-induced gene expression, we performed a comprehensive gene analysis using a microarray method and observed changes in numerous genes involved in lipid metabolism, such as cholesterol, and in the regulation of the ER-Golgi apparatus. Abnormalities in ER-Golgi transport were also evident in the examination of secretory activity using NanoLuc-tag genes. Finally, we established Neuro2a cells lacking oxysterol-binding protein (OSBP), which were severely reduced by OSW-1, but found OSBP deficiency had little effect on OSW-1-induced cell death and the LC3-II/LC3-I ratio in Neuro2a cells. Future work to elucidate the relationship between OSW-1-induced atypical Golgi stress responses and autophagy induction may lead to the development of new anticancer agents.

Exposure to brefeldin A induces unusual expression of hybrid- and complex-type free N-glycans in HepG2 cells.

This study determined the effect of brefeldin A (BFA) on the free N-glycomic profile of HepG2 cells to better understand the effect of blocking intracellular vesicle formation and transport of proteins from the endoplasmic reticulum to the Golgi apparatus. A series of exoglycosidase- and endoglycosidase-assisted analyses clarified the complex nature of altered glycomic profiles. A key feature of BFA-mediated alterations in Gn2-type glycans was the expression of unusual hybrid-, monoantennary- and complex-type free N-glycans (FNGs). BFA-mediated alterations in Gn1-type glycans were characterized by the expression of unusual hybrid- and monoantennary-FNGs, without significant expression of complex-type FNGs. A time course analysis revealed that sialylated hybrid- and complex-type Gn2-type FNGs were generated later than asialo-Gn2-type FNGs, and the expression profiles of Gn2-type FNGs and N-glycans were found to be similar, suggesting that the metabolic flux of FNGs is the same as that of protein-bound N-glycans. Subcellular glycomic analysis revealed that almost all FNGs were detected in the cytoplasmic extracts. Our data suggest that hybrid-, monoantennary- and complex-type Gn2-type FNGs were cleaved from glycoproteins in the cytosol by cytosolic PNGase, and subsequently digested by cytosolic endo-ß-N-acetylglucosaminidase (ENGase) to generate Gn1-type FNGs. The substrate specificity of ENGase explains the limited expression of complex Gn1 type FNGs.

Absorption and Transport Mechanism of Red Meat-Derived N-glycolylneuraminic Acid and Its Damage to Intestinal Barrier Function through the NF-κB Signaling Pathway.

N-glycolylneuraminic acid (Neu5Gc) is a specific factor in red meat that induces intestinal disease. Our aim was to investigate the effect of Neu5Gc on the intestinal barrier as well as its mechanism of endocytosis and exocytosis. Ten specific inhibitors were used to explore the mechanism of Neu5Gc endocytosis and exocytosis by Caco-2 cells. Amiloride hydrochloride and cytochalasin D had the strongest inhibitory effect on the endocytosis of Neu5Gc. Sodium azide, dynasore, chlorpromazine hydrochloride, and nystatin also inhibited Neu5Gc endocytosis. Dynasore exhibited a stronger inhibitory effect than that of chlorpromazine hydrochloride or nystatin alone. Exocytosis inhibitors, including nocodazole, brefeldin A, monensin, and bafilomycin A, inhibited the transmembrane transport of Neu5Gc. Monensin promoted the exocytosis of Neu5Gc from Caco-2 cells. In another experiment, we observed no significant inhibitory effects of monensin and brefeldin A. Dietary concentrations of Neu5Gc induced prominent damage to intestinal tight junction proteins zonula occludens-1 (ZO-1), occludin, and claudin-1 and promoted the phosphorylation of IκB-α and P65 to activate the canonical Nuclear Factor kappa-B (NF-κB) pathway. Neu5Gc increased the RNA levels of pro-inflammatory factors IL-1ß, IL-6, and TNF-α and inhibited those of anti-inflammatory factors TGF-ß and IL-10. BAY, an NF-κB signaling pathway inhibitor, attenuated these changes. Reductions in the levels of ZO-1, occludin, and claudin-1 were recovered in response to BAY. Our data reveal the endocytosis and exocytosis mechanism of Neu5Gc and prove that Neu5Gc can activate the canonical NF-κB signaling pathway, regulate the transcription of inflammatory factors, thereby damaging intestinal barrier function.

Effect of Common ER Stress-Inducing Drugs on the Growth and Lipid Phenotypes of Chlamydomonas and Arabidopsis.

Endoplasmic reticulum (ER) stress is caused by the stress-induced accumulation of unfolded proteins in the ER. Several compounds are used to induce the unfolded protein response (UPR) in animals, with different modes of action, but which ER stress-inducing drugs induce ER stress in microalgae or land plants is unclear. In this study, we examined the effects of seven chemicals that were reported to induce ER stress in animals on the growth, UPR gene expression and fatty acid profiles of Chlamydomonas reinhardtii (Chlamydomonas) and Arabidopsis thaliana (Arabidopsis): 2-deoxyglucose, dithiothreitol (DTT), tunicamycin (TM), thapsigargin, brefeldin A (BFA), monensin (MON) and eeyarestatin I. In both model photosynthetic organisms, DTT, TM, BFA and MON treatment induced ER stress, as indicated by the induction of spliced bZIP1 and bZIP60, respectively. In Chlamydomonas, DTT, TM and BFA treatment induced the production of transcripts related to lipid biosynthesis, but MON treatment did not. In Arabidopsis, DTT, TM, BFA and MON inhibited seed germination and seedling growth with the activation of bZIP60. These findings lay the foundation for using four types of ER stress-inducing drugs in photosynthetic organisms, and they help uncover the mode of action of each compound.

Microscopy and Immunocytochemistry-Based Methods to Study Cell Wall Biosynthetic Enzymes in the Golgi.

The Golgi apparatus has essential roles in all eukaryotic cells, and its importance in plants is further exemplified by a critical role in building a cellulosic cell wall. The Golgi apparatus houses numerous cell wall-synthesizing or cell wall-modifying enzymes to generate the complex cell wall structure. However, several putative cell wall biosynthetic candidates await characterization, which requires verification of the subcellular localization and enzymatic products. Here, we describe detailed methods to analyze the localization of proteins that are transiently produced in tobacco leaves or stably produced in transgenic plants, by confocal microscopy using fluorescent-tagged proteins along with known Golgi markers or the trafficking inhibitor brefeldin A. We also detail a procedure to analyze the enzymatic products through antibody-based immunoblotting after cell wall enrichment.

IL17F Expression as an Early Sign of Oxidative Stress-Induced Cytotoxicity/Apoptosis.

Interleukin 17F (IL17F) has been found to be involved in various inflammatory pathologies and has recently become a target for therapeutic purposes. In contrast to IL17F secreted by immune cells, the focus of this study is to describe the triggers of IL17F release in non-immune cells with a particular focus on IL17F-induced fibrosis. IL17F induction was examined in human lung epithelial (BEAS-2B) and myeloid cell lines as well as in peripheral blood mononuclear cells after in vitro exposure to aqueous cigarette smoke extract (CSE), inorganic mercury, cadmium or the apoptosis inducer brefeldin A. Fibrosis was examined in vitro, evaluating the transition of human primary dermal fibroblasts to myofibroblasts. We observed that all stressors were able to induce IL17F gene expression regardless of cell type. Interestingly, its induction was associated with cytotoxic/apoptotic signs. Inhibiting oxidative stress by N-acetylcysteine abrogated CSE-induced cytotoxic and IL17F-inducing effects. The induction of IL17F was accompanied by IL17F protein expression. The transition of fibroblasts into myofibroblasts was not influenced by either recombinant IL17F or supernatants of CSE-exposed BEAS-2B. In addition to IL17F secretion by specialized or activated immune cells, we underscored the cell type-independent induction of IL17F by mechanisms of inhibitable oxidative stress-induced cytotoxicity. However, IL17F was not involved in dermal fibrosis under the conditions used in this study.

Phospholipase Dδ and phosphatidic acid mediate heat-induced nuclear localization of glyceraldehyde-3-phosphate dehydrogenase in Arabidopsis.

Cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC) is a glycolytic enzyme, but undergoes stress-induced nuclear translocation for moonlighting. We previously reported that in response to heat stress, GAPC accumulated in the nucleus to modulate transcription and thermotolerance. Here we show a cellular and molecular mechanism that mediates heat-induced nuclear translocation of cytosolic GAPC in Arabidopsis thaliana. Heat-induced GAPC nuclear accumulation and plant heat tolerance were reduced in Arabidopsis phospholipase D (PLD) knockout mutants of pldδ and pldα1pldδ, but not of pldα1. These changes were restored to wild type by genetic complementation with active PLDδ, but not with catalytically inactive PLDδ. GAPC overexpression enhanced the seedling thermotolerance and the expression of heat-inducible genes, but this effect was abolished in the pldδ background. Heat stress elevated the levels of the PLD product phosphatidic acid (PA) in the nucleus in wild type, but not in pldδ plants. Lipid labeling demonstrated the heat-induced nuclear co-localization of PA and GAPC, which was impaired by zinc, which inhibited the PA-GAPC interaction, and by the membrane trafficking inhibitor brefeldin A (BFA). The GAPC nuclear accumulation and seedling thermotolerance were also decreased by treatment with zinc or BFA. Our data suggest that PLDδ and PA are critical for the heat-induced nuclear translocation of GAPC. We propose that PLDδ-produced PA mediates the process via lipid-protein interaction and that the lipid mediation acts as a cellular conduit linking stress perturbations at cell membranes to nuclear functions in plants coping with heat stress.

Interleukin-10 Protects against Ureteral Obstruction-Induced Kidney Fibrosis by Suppressing Endoplasmic Reticulum Stress and Apoptosis.

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 IL-10-/- mice and TCMK-1 cells (mouse kidney tubular epithelial cell line). Endoplasmic reticulum stress (ERS), apoptosis, and fibrosis in IL-10-/- mice were more severe than in IL-10+/+ 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 IL-10-/- 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.

Design and Characterization of a Natural Arf-GEFs Inhibitor Prodrug CHNQD-01255 with Potent Anti-Hepatocellular Carcinoma Efficacy In Vivo.

Brefeldin A (BFA), a well-known natural Arf-GEFs inhibitor, is effective against hepatocellular carcinoma (HCC), while the poor solubility, serious toxicity, and short half-life limit its potential. Herein, distinct corresponding prodrugs of BFA, including esters 1-15, carbonates 16-24 and 30-32, and carbamates 25-29, were synthesized and evaluated. CHNQD-01255 (16) with improved aqueous solubility (15-20 mg/mL) demonstrated favorable pharmacokinetic profiles. It behaved as expected by undergoing rapid conversion to BFA in vivo, and achieved sufficient high plasma exposure, prolonged half-life, as well as the improved bioavailability of BFA (F = 18.96%). Meanwhile, CHNQD-01255 significantly suppressed tumor growth (TGI = 61.0%) at a dose of 45 mg/kg (p.o.) in the xenograft model. Notably, the improved safety profile of CHNQD-01255 (MTD > 750 mg/kg, p.o.) was confirmed to be superior to that of BFA (MTD < 506 mg/kg). Overall, CHNQD-01255 may serve as a safe and effective new anti-HCC prodrug.

The Golgi-localized sphingosine-1-phosphate phosphatase is indispensable for Leishmania major.

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.

The unconventional prefoldin RPB5 interactor mediates the gravitropic response by modulating cytoskeleton organization and auxin transport in Arabidopsis.

Gravity-induced root curvature involves the asymmetric distribution of the phytohormone auxin. This response depends on the concerted activities of the auxin transporters such as PIN-FORMED (PIN) proteins for auxin efflux and AUXIN RESISTANT 1 (AUX1) for auxin influx. However, how the auxin gradient is established remains elusive. Here we identified a new mutant with a short root, strong auxin distribution in the lateral root cap and an impaired gravitropic response. The causal gene encoded an Arabidopsis homolog of the human unconventional prefoldin RPB5 interactor (URI). AtURI interacted with prefoldin 2 (PFD2) and PFD6, two ß-type PFD members that modulate actin and tubulin patterning in roots. The auxin reporter DR5rev :GFP showed that asymmetric auxin redistribution after gravistimulation is disordered in aturi-1 root tips. Treatment with the endomembrane protein trafficking inhibitor brefeldin A indicated that recycling of the auxin transporter PIN2 is disrupted in aturi-1 roots as well as in pfd mutants. We propose that AtURI cooperates with PFDs to recycle PIN2 and modulate auxin distribution.

Brefeldin A from the Deep-Sea-Derived Fungus Fusarium sp. Targets on RIPK3 to Inhibit TNFα-Induced Necroptosis.

From the deep-sea-derived Fusarium sp. ZEN-48, four known compounds were obtained. Their structures were established by extensive analyses of the NMR, HR-ESI-MS, and the X-ray crystallographic data as brefeldin A (BFA, 1), brevianamide F (2), N-acetyltryptamine (3), and (+)-diaporthin (4). Although BFA was extensively investigated for its potent bioactivities, its role on TNFα-induced necroptosis was incompletely understood. In this study, BFA showed significant inhibition on TNFα-induced necroptosis by disrupting the necrosome formation and suppressing the phosphorylation of RIPK3 and MLKL (IC50 =0.5 µM). While, it had no effect on TNFα-induced NF-κB/MAPKs activation and apoptosis. The finding raised significant implications of BFA for necroptosis-related inflammatory disease therapy and new drug development from marine fungi.