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The N-acetylglucosamine-1-phosphate transferase (WecA)is a potential target for developing anti-tuberculosis drugs, due to its critical role in the synthesis of mycobacterial cell wall. The enzymatic study of WecA and the discovery of WecA inhibitors are therefore justified. However, WecA is a membrane protein with 11 transmembrane domains, making it difficult to be obtained, and even more difficult to perform activity studies. In order to gain sufficient WecA protein for activity investigation, the ( ) Lemo21(DE3) strain was utilised in this study. The expression level of WecA was precisely regulated by T7 lysozyme. Purified WecA was obtained by affinity chromatography and identified by mass spectrometry. The kinetic properties of WecA were determined based on the detection of the product UMP. In addition, tunicamycin proved to be a competitive inhibitor. These results will lay theoretical foundations for the elucidation of WecA catalytic mechanism and the development of WecA inhibitors.

High infiltration of M2-polarized macrophages in the primary tumor indicates unfavorable prognosis and poor overall survival in the patients with triple-negative breast cancer (TNBC). Thus, reversing M2-polarized tumor-associated macrophages in the tumors has been considered as a potential therapeutic strategy for TNBC. Sphingomyelin synthase 2 (SMS2) is the key enzyme for sphingomyelin production, which plays an important role in plasma membrane integrity and function. In this study we investigated whether SMS2 inhibitor or SMS2 gene knockout could reduce macrophages M2 polarization and tumor progression in a mouse model of TNBC. We showed that SMS2 mRNA expression was linked to immunosuppressive tumor microenvironment and poor prognosis in TNBC patients. The knockout of SMS2 or application of 15w (a specific SMS2 inhibitor) markedly decreased the generation of M2-type macrophages in vitro, and reduced the tumor weight and lung metastatic niche formation in a 4T1-TNBC mouse model. We further demonstrated that the in vivo antitumor efficacy of 15w was accompanied by a multifaceted remodeling of tumor immune environment reflecting not only the suppression of M2-type macrophages but also diminished levels of regulatory T cells and myeloid-derived suppressor cells leading to a dramatically improved infiltration of antitumor CD8 + T lymphocytes. Collectively, our results reveal a novel and important role of SMS2 in the protumorigenic function and may offer a new strategy for macrophage-targeted anticancer therapy.

The mechanism of action of 2-hydroxyoleic acid (2OHOA), a potent antitumor compound, has not yet been fully elucidated. Here, we show that human cancer cells have markedly lower levels of sphingomyelin (SM) than nontumor (MRC-5) cells. In this context, 2OHOA treatment strongly augments SM mass (4.6-fold), restoring the levels found in MRC-5 cells, while a loss of phosphatidylethanolamine and phosphatidylcholine is observed (57 and 30%, respectively). The increased SM mass was due to a rapid and highly specific activation of SM synthases (SMS). This effect appeared to be specific against cancer cells as it did not affect nontumor MRC-5 cells. Therefore, low SM levels are associated with the tumorigenic transformation that produces cancer cells. SM accumulation occurred at the plasma membrane and caused an increase in membrane global order and lipid raft packing in model membranes. These modifications would account for the observed alteration by 2OHOA in the localization of proteins involved in cell apoptosis (Fas receptor) or differentiation (Ras). Importantly, SMS inhibition by D609 diminished 2OHOA effect on cell cycle. Therefore, we propose that the regulation of SMS activity in tumor cells is a critical upstream event in 2OHOA antitumor mechanism, which also explains its specificity for cancer cells, its potency, and the lack of undesired side effects. Finally, the specific activation of SMS explains the ability of this compound to trigger cell cycle arrest, cell differentiation, and autophagy or apoptosis in cancer cells.

The cell envelope of Mycobacterium tuberculosis, the causative agent of tuberculosis in humans, contains lipids with unusual structures. These lipids play a key role in both virulence and resistance to the various hostile environments encountered by the bacteria during infection. They are synthesized by complex enzymatic systems, including type-I polyketide synthases and type-I and -II fatty acid synthases, which require a post-translational modification to become active. This modification consists of the covalent attachment of the 4'-phosphopantetheine moiety of Coenzyme A catalyzed by phosphopantetheinyl transferases (PPTases). PptT, one of the two PPTases produced by mycobacteria, is involved in post-translational modification of various type-I polyketide synthases required for the formation of both mycolic acids and lipid virulence factors in mycobacteria. Here we identify PptT as a new target for anti-tuberculosis drugs; we address all the critical issues of target validation to demonstrate that PptT can be used to search for new drugs. We confirm that PptT is essential for the growth of M. bovis BCG in vitro and show that it is required for persistence of M. bovis BCG in both infected macrophages and immunodeficient mice. We generated a conditional expression mutant of M. tuberculosis, in which the expression of the pptT gene is tightly regulated by tetracycline derivatives. We used this construct to demonstrate that PptT is required for the replication and survival of the tubercle bacillus during the acute and chronic phases of infection in mice. Finally, we developed a robust and miniaturized assay based on scintillation proximity assay technology to search for inhibitors of PPTases, and especially of PptT, by high-throughput screening. Our various findings indicate that PptT meets the key criteria for being a therapeutic target for the treatment of mycobacterial infections.

There are no approved therapies for Ebola virus infection. Here, to find potential therapeutic targets, we perform a screen for genes essential for Ebola virus (EBOV) infection. We identify GNPTAB , which encodes the α and β subunits of N-acetylglucosamine-1-phosphate transferase. We show that EBOV infection of a GNPTAB knockout cell line is impaired, and that this is reversed by reconstituting GNPTAB expression. Fibroblasts from patients with mucolipidosis II, a disorder associated with mutations in GNPTAB , are refractory to EBOV, whereas cells from their healthy parents support infection. Impaired infection correlates with loss of the expression of cathepsin B, known to be essential for EBOV entry. GNPTAB activity is dependent upon proteolytic cleavage by the SKI-1/S1P protease. Inhibiting this protease with the small-molecule PF-429242 blocks EBOV entry and infection. Disruption of GNPTAB function may represent a strategy for a host-targeted therapy for EBOV. Genetic screens are important tools to identify host factors associated with viral infections. Here, Flint et al. perform a genome-wide CRISPR screen using infectious Ebola virus (EBOV) and show that the host transferase GNPTAB is required for EBOV infection and a potential target for antiviral therapies

Antifungal drugs acting via new mechanisms of action are urgently needed to combat the increasing numbers of severe fungal infections caused by pathogens such as Candida albicans. The phosphopantetheinyl transferase of Aspergillus fumigatus, encoded by the essential gene pptB, has previously been identified as a potential antifungal target. This study investigated the function of its orthologue in C. albicans, PPT2/C1_09480W by placing one allele under the control of the regulatable MET3 promoter, and deleting the remaining allele. The phenotypes of this conditional null mutant showed that, as in A. fumigatus, the gene PPT2 is essential for growth in C. albicans, thus fulfilling one aspect of an efficient antifungal target. The catalytic activity of Ppt2 as a phosphopantetheinyl transferase and the acyl carrier protein Acp1 as a substrate were demonstrated in a fluorescence transfer assay, using recombinant Ppt2 and Acp1 produced and purified from E.coli. A fluorescence polarisation assay amenable to high-throughput screening was also developed. Therefore we have identified Ppt2 as a broad-spectrum novel antifungal target and developed tools to identify inhibitors as potentially new antifungal compounds.

We have been investigating the role that phosphatidylethanolamine (PE) and phosphatidylcholine (PC) content plays in modulating the solubility of the Parkinson's disease protein alpha-synuclein (α-syn) using Saccharomyces cerevisiae and Caenorhabditis elegans. One enzyme that synthesizes PE is the conserved enzyme phosphatidylserine decarboxylase (Psd1/yeast; PSD-1/worms), which is lodged in the inner mitochondrial membrane. We previously found that decreasing the level of PE due to knockdown of Psd1/psd-1 affects the homeostasis of α-syn in vivo. In S. cerevisiae, the co-occurrence of low PE and α-syn in psd1Δ cells triggers mitochondrial defects, stress in the endoplasmic reticulum, misprocessing of glycosylphosphatidylinositol-anchored proteins, and a 3-fold increase in the level of α-syn. The goal of this study was to identify drugs that rescue this phenotype. We screened the Prestwick library of 1121 Food and Drug Administration-approved drugs using psd1Δ + α-syn cells and identified cyclosporin A, meclofenoxate hydrochloride, and sulfaphenazole as putative protective compounds. The protective activity of these drugs was corroborated using C. elegans in which α-syn is expressed specifically in the dopaminergic neurons, with psd-1 depleted by RNAi. Worm populations were examined for dopaminergic neuron survival following psd-1 knockdown. Exposure to cyclosporine, meclofenoxate, and sulfaphenazole significantly enhanced survival at day 7 in α-syn-expressing worm populations whereby 50-55% of the populations displayed normal neurons, compared to only 10-15% of untreated animals. We also found that all three drugs rescued worms expressing α-syn in dopaminergic neurons that were deficient in the phospholipid cardiolipin following cardiolipin synthase (crls-1) depletion by RNAi. We discuss how these drugs might block α-syn pathology in dopaminergic neurons.

Ceramide can be converted into sphingomyelin by sphingomyelin synthases (SMS) 1 and 2. In this study, we show that in human leukemia Jurkat cells, which express mainly SMS1, Fas ligand (FasL) treatment inhibited SMS activity in a dose- and time-dependent manner before nuclear fragmentation. The SMS inhibition elicited by FasL (1) was abrogated by benzyloxycarbonyl valyl-alanyl-aspartyl-( O -methyl)-fluoromethylketone (zVAD-fmk), a broad-spectrum caspase inhibitor; (2) did not occur in caspase-8-deficient cells and (3) was not affected in caspase-9-deficient cells. Western blot experiments showed SMS1 cleavage in a caspase-dependent manner upon FasL treatment. In a cell-free system, caspase-2, -7, -8 and -9, but not caspase-3 and -10, cleaved SMS1. In HeLa cells, SMS1 was Golgi localized and relocated throughout the cytoplasm in cells exhibiting an early apoptotic phenotype on FasL treatment. zVAD-fmk prevented FasL-induced SMS1 relocation. Thus, FasL-mediated SMS1 inhibition and relocation depend on caspase activation and likely represent proximal events in Fas signaling. FasL-induced ceramide production and cell death were enhanced in cells stably expressing an siRNA against SMS1. Conversely, in cells stably overexpressing SMS1, FasL neither increased ceramide generation nor efficiently induced cell death. Altogether, our data show that SMS1 is a novel caspase target that is functionally involved in the regulation of FasL-induced apoptosis.

Sphingomylin participates in sperm function in animals, and also regulates the Akt and ERK signaling pathways, both of which are associated with the asthenospermia. Sphingomyelin synthase 2 (SMS2) is involved in the biosynthesis of sphingomylin. To determine the relationship between SMS2 and human sperm function, we analyzed the distribution of SMS2 in human sperm and testes, and SMS2 expression in patients with asthenospermia and normozoospermia; human sperm were treated with anti-SMS2, and the sperm motility, penetration ability into methylcellulose, capacitation and acrosome reaction, and sperm [Ca2+]i imaging were evaluated, while the Akt and ERK pathway and cleaved caspase 3 were also analyzed. Results showed that SMS2 was localized in the testis and human sperm, and the protein levels of normozoospermia were higher than asthenospermia. Inhibition of SMS2 activity significantly decreased sperm motility and penetration ability into methylcellulose, but had no influence on capacitation and acrosome reaction, or on intracellular [Ca2+]i compared to IgG-treated control groups. Moreover, the phosphorylation level of Akt was decreased, whereas the phosphorylation of ERK and cleaved-caspase 3 levels were significantly increased. Taken together, SMS2 can affect sperm motility and penetration ability into methylcellulose, and participate in apoptosis associated with the Akt and ERK signaling pathways.

Sphingomyelin (SM), a major sphingolipid in the lipid raft microdomains of the cell membrane, is synthesized by plasma membrane–bound sphingomyelin synthase 2 (SMS2). SMS2 is required for the maintenance of plasma membrane microdomain fluidity and receptor-mediated responses to inflammation in macrophages. However, the exact mechanism of SMS2 activation in endothelial barrier disruption and lung injury is not fully understood. To define the role of SMS activation in lung injury, we hypothesized that the inhibition of SM synthesis may provide protection against acute lung injury (ALI) by preserving endothelial barrier function. Using SMS2-silencing RNA (siRNA) treatment in human pulmonary endothelial cells (HPAECs) and tricyclodecan-9-yl-xanthogenate (D609), a competitive inhibitor of SMS, and phosphatidylcholine-specific phospholipase C in a murine model of bacterial LPS injury, we studied the role of sphingomyelin synthesis in ALI. Results show that pretreating mice with D609 significantly attenuated LPS-induced lung injury, as measured by a significant decrease in wet to dry ratio, bronchoalveolar lavage fluid cell and protein counts, and myeloperoxidase activity in lung tissue. Similarly, LPS-induced endothelial barrier disruption was significantly reduced in HPAECs pretreated with D609 or SMS2 siRNA, as demonstrated by an increase in paracellular integrity on an FITC-dextran assay, by the inhibition of LPS-induced stress fibers, and by the formation of cortical actin rings and lamellipodia at the periphery. These results indicate that D609 attenuates LPS-mediated endothelial barrier dysfunction and lung injury in mice through inhibition of SMS, suggesting a novel and essential role of SMS inhibition in modulating endothelial barrier integrity via actin cytoskeletal activation, with a potential therapeutic role in ALI.