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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.

The mitochondrion of malaria-causing Plasmodium spp. supports parasite energy requirements, pyrimidine and ubiquinone biosynthesis and [Fe-S] formation. As parasites transition from the host liver to asexual and sexual blood stages, metabolic shifts of ATP generation through glycolysis or mitochondrial oxidative phosphorylation are accompanied by change in mitochondrial number, branching complexity and development of cristae. The final step of synthesis of cardiolipin (CL), a critical phospholipid for mitochondrial biogenesis and function, is catalyzed by cardiolipin synthase (Cls). Plasmodium spp. carry an uncharacterized, putative bacterial-type Cls distinct from Cls of mammalian hosts. We probed enzyme activity of the phospholipase D-type recombinant Plasmodium falciparum Cls. Antibodies generated against Pf Cls localized it to the mitochondrion in asexual blood stages; additional Pf Cls signal was observed in the cytosolic periphery in late-gametocytes, accompanied by CL staining in the parasite plasma membrane. To investigate the impact of Cls on parasite life cycle progression, we generated its knockout in the rodent parasite P. berghei . Pb Cls KO parasites had significantly impaired asexual blood-stage proliferation associated with lower abundance of CL molecular species. They showed a marked reduction in mitochondrial membrane potential and basal oxygen consumption rate. While Pb Cls-deficient parasites completed development within the mosquito and generated sporozoites capable of hepatocyte invasion, they exhibited a severe defect in liver-stage maturation. Plasmodium Cls is thus a vital component of malaria parasite development with a critical role in maintaining mitochondrial function.

DNA damage, caused by various oncogenic stresses, can induce cell death or cellular senescence as an important tumor suppressor mechanism. Senescent cells display the features of a senescence-associated secretory phenotype (SASP), secreting inflammatory proteins into surrounding tissues, and contributing to various age-related pathologies. In addition to this inflammatory protein secretion, the release of extracellular vesicles (EVs) is also upregulated in senescent cells. However, the molecular mechanism underlying this phenomenon remains unclear. Here, we show that DNA damage activates the ceramide synthetic pathway, via the downregulation of sphingomyelin synthase 2 (SMS2) and the upregulation of neutral sphingomyelinase 2 (nSMase2), leading to an increase in senescence-associated EV (SA-EV) biogenesis. The EV biogenesis pathway, together with the autophagy-mediated degradation pathway, functions to block apoptosis by removing cytoplasmic DNA fragments derived from chromosomal DNA or bacterial infections. Our data suggest that this SA-EV pathway may play a prominent role in cellular homeostasis, particularly in senescent cells. In summary, DNA damage provokes SA-EV release by activating the ceramide pathway to protect cells from excessive inflammatory responses.

Mitochondria are essential and dynamic organelles in eukaryotes. Cardiolipin (CL) is a key phospholipid in mitochondrial membranes, playing important roles in maintaining the functional integrity and dynamics of mitochondria in animals and yeasts. However, CL's role in plants is just beginning to be elucidated. In this study, we used Arabidopsis thaliana to examine the subcellular distribution of CL and CARDIOLIPIN SYNTHASE (CLS) and analyzed loss-of-function els mutants for defects in mitochondrial morphogenesis and stress response. We show that CL localizes to mitochondria and is enriched at specific domains, and CLS targets to the inner membrane of mitochondria with its terminus in the intermembrane space. Furthermore, els mutants exhibit significantly impaired growth as well as altered structural integrity and morphogenesis of mitochondria. In contrast to animals and yeasts, in which CL's effect on mitochondrial fusion is more profound, Arabidopsis CL plays a dominant role in mitochondrial fission and exerts this function, at least in part, through stabilizing the protein complex of the major mitochondrial fission factor, DYNAMIN-RELATED PROTEIN3. CL also plays a role in plant responses to heat and extended darkness, stresses that induce programmed cell death. Our study has uncovered conserved and plant-specific aspects of CL biology in mitochondrial dynamics and the organism response to environmental stresses.

Phosphorus is a macronutrient taken up by cells as inorganic phosphate (Pi). How cells sense cellular Pi levels is poorly characterized. Here, we report that SPX domains—which are found in eukaryotic phosphate transporters, signaling proteins, and inorganic polyphosphate polymerases—provide a basic binding surface for inositol polyphosphate signaling molecules (InsPs), the concentrations of which change in response to Pi availability. Substitutions of critical binding surface residues impair InsP binding in vitro, inorganic polyphosphate synthesis in yeast, and Pi transport in Arabidopsis. In plants, InsPs trigger the association of SPX proteins with transcription factors to regulate Pi starvation responses. We propose that InsPs communicate cytosolic Pi levels to SPX domains and enable them to interact with a multitude of proteins to regulate Pi uptake, transport, and storage in fungi, plants, and animals.

Sphingomyelin synthase is responsible for the production of sphingomyelin (SGM), the second most abundant phospholipid in mammalian plasma, from ceramide, a major sphingolipid. Knowledge of the effects of cigarette smoke on SGM production is limited. In the present study, we examined the effect of chronic cigarette smoke on sphingomyelin synthase (SGMS) activity and evaluated how the deficiency of Sgms2, one of the two isoforms of mammalian SGMS, impacts pulmonary function. Sgms2-knockout and wild-type control mice were exposed to cigarette smoke for 6 months, and pulmonary function testing was performed. SGMS2-dependent signaling was investigated in these mice and in human monocyte-derived macrophages of nonsmokers and human bronchial epithelial (HBE) cells isolated from healthy nonsmokers and subjects with chronic obstructive pulmonary disease (COPD). Chronic cigarette smoke reduces SGMS activity and Sgms2 gene expression in mouse lungs. Sgms2-deficient mice exhibited enhanced airway and tissue resistance after chronic cigarette smoke exposure, but had similar degrees of emphysema, compared with smoke-exposed wild-type mice. Sgms2−/− mice had greater AKT phosphorylation, peribronchial collagen deposition, and protease activity in their lungs after smoke inhalation. Similarly, we identified reduced SGMS2 expression and enhanced phosphorylation of AKT and protease production in HBE cells isolated from subjects with COPD. Selective inhibition of AKT activity or overexpression of SGMS2 reduced the production of several matrix metalloproteinases in HBE cells and monocyte-derived macrophages. Our study demonstrates that smoke-regulated Sgms2 gene expression influences key COPD features in mice, including airway resistance, AKT signaling, and protease production.

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.

CD1d-restricted natural killer T (NKT) cells include two major subgroups. The most widely studied are Vα14Jα18 ⁺ invariant NKT (iNKT) cells that recognize the prototypical α-galactosylceramide antigen, whereas the other major group uses diverse T-cell receptor (TCR) α-and β-chains, does not recognize α-galactosylceramide, and is referred to as diverse NKT (dNKT) cells. dNKT cells play important roles during infection and autoimmunity, but the antigens they recognize remain poorly understood. Here, we identified phosphatidylglycerol (PG), diphosphatidylglycerol (DPG, or cardiolipin), and phosphatidylinositol from Mycobacterium tuberculosis or Corynebacterium glutamicum as microbial antigens that stimulated various dNKT, but not iNKT, hybridomas. dNKT hybridomas showed distinct reactivities for diverse antigens. Stimulation of dNKT hybridomas by microbial PG was independent of Toll-like receptor-mediated signaling by antigen-presenting cells and required lipid uptake and/or processing. Furthermore, microbial PG bound to CD1d molecules and plate-bound PG/CD1d complexes stimulated dNKT hybridomas, indicating direct recognition by the dNKT cell TCR. Interestingly, despite structural differences in acyl chain composition between microbial and mammalian PG and DPG, lipids from both sources stimulated dNKT hybridomas, suggesting that presentation of microbial lipids and enhanced availability of stimulatory self-lipids may both contribute to dNKT cell activation during infection.

The major membrane phospholipid classes, described thus far, include phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), and phosphatidylinositol (PtdIns). Here, we demonstrate the natural occurrence and genetic origin of an exclusive and rather abundant lipid, phosphatidylthreonine (PtdThr), in a common eukaryotic model parasite, Toxoplasma gondii. The parasite expresses a novel enzyme PtdThr synthase (TgPTS) to produce this lipid in its endoplasmic reticulum. Genetic disruption of TgPTS abrogates de novo synthesis of PtdThr and impairs the lytic cycle and virulence of T. gondii. The observed phenotype is caused by a reduced gliding motility, which blights the parasite egress and ensuing host cell invasion. Notably, the PTS mutant can prevent acute as well as yet-incurable chronic toxoplasmosis in a mouse model, which endorses its potential clinical utility as a metabolically attenuated vaccine. Together, the work also illustrates the functional speciation of two evolutionarily related membrane phospholipids, i.e., PtdThr and PtdSer.