Identification of propranolol and derivatives that are chemical inhibitors of phosphatidate phosphatase as potential broad-spectrum fungicides.

Plant diseases cause enormous economic losses in agriculture and threaten global food security, and application of agrochemicals is an important method of crop disease control. Exploration of disease-resistance mechanisms and synthesis of highly bioactive agrochemicals are thus important research objectives. Here, we show that propranolol, a phosphatidate phosphatase (Pah) inhibitor, effectively suppresses fungal growth, sporulation, sexual reproduction, and infection of diverse plants. The MoPah1 enzyme activity of the rice blast fungus Magnaporthe oryzae is inhibited by propranolol. Alterations in lipid metabolism are associated with inhibited hyphal growth and appressorium formation caused by propranolol in M. oryzae. Propranolol inhibits a broad spectrum of 12 plant pathogens, effectively inhibiting infection of barley, wheat, maize, tomato, and pear. To improve antifungal capacity, we synthesized a series of propranolol derivatives, one of which shows a 16-fold increase in antifungal ability and binds directly to MoPah1. Propranolol and its derivatives can also reduce the severity of rice blast and Fusarium head blight of wheat in the field. Taken together, our results demonstrate that propranolol suppresses fungal development and infection through mechanisms involved in lipid metabolism. Propranolol and its derivatives may therefore be promising candidates for fungicide development.

Lusianthridin ameliorates high fat diet-induced metabolic dysfunction-associated fatty liver disease via activation of FXR signaling pathway.

Metabolic dysfunction-associated fatty liver disease (MAFLD) is a common chronic liver disease, but there are few specific medications for it. Lusianthridin, a major phenanthrene component that originates from Dendrobium Sonia, has various in vitro biological functions. In this study, we aimed to evaluate the therapeutic effects of lusianthridin on high-fat diet (HFD)-induced MAFLD as well as to examine the mechanism of its effects. We fed male mice high-fat-diet for 12 weeks to induce MAFLD and then continued to feed them, either with or without lusianthridin, for another six weeks. We found that lusianthridin decreased serum triacylglycerol, hepatic triacylglycerol, and serum low density lipoprotein cholesterol. It also reduced hepatic lipid accumulation based on the results of morphology analysis. Besides, it improved hepatic inflammation as well, including a decrease in serum alanine aminotransferase and a reduction in macrophage and neutrophil infiltration. Mechanistically, surface plasmon resonance, cell thermal shift assay and dual-luciferase report system results suggested that lusianthridin combined with farnesoid X receptor (FXR) ligand binding region and activated its transcriptional activity. Lusianthridin also decreased de no lipogenesis though inhibiting Srebp1c and downstream Scd-1, Lpin1 and Dgat2 expression in a FXR-dependent manner in oleic acid treated L02 cells. Correspondingly, lusianthridin inhibited Srebp1c and downstream lipogenesis in MAFLD liver tissues of mice at both of genetic and protein levels. Finally, the protective effects of lusianthridin on hepatic steaotosis were abolished in Fxr-/- mice. Taken together, our results suggested that lusianthridin attenuated high-fat-diet induced MAFLD via activation the FXR signaling pathway.

Quantitative proteomics analysis based on tandem mass tag labeling coupled with labeling coupled with liquid chromatography-tandem mass spectrometry discovers the effect of silibinin on non-alcoholic fatty liver disease in mice.

In recent years, the beneficial effects of silibinin (SIL) on nonalcoholic fatty liver disease (NAFLD) have attracted widespread attention. We tried to study the intervention effect of SIL on NAFLD, and explore the potential mechanisms and targets of SIL on NAFLD improvement. Thirty-three male C57BL6/J mice were divided into three groups, and, respectively, fed a normal diet (ND), a high-fat diet (HFD) or a HFD given SIL treatment (HFD+SIL). Biochemical indexes and histopathological changes of mice in each group were detected. In addition, quantitative proteomics analysis based on tandem mass tag (TMT) labeling coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and bioinformatics analysis was performed on protein changes in the livers. SIL could reduce the weight of mice, reduce liver lipid deposition, and improve glucose metabolism. Through comparison among the three experimental groups, a total of 30 overlapping proteins were found. These identified proteins were closely linked to liver lipid metabolism and energy homeostasis. Moreover, some drug targets were found, namely perilipin-2, phosphatidate phosphatase LPIN1, farnesyl pyrophosphate synthase, and glutathione S-transferase A1. In conclusions, high-fat diet increases the expressions of proteins implicated in lipid synthesis and transport in the liver, which can result in disorders of liver lipid metabolism. SIL can decrease liver lipid deposition and increase insulin sensitivity by regulating the expressions of these proteins. It not only improves the disorder of lipid metabolism in vivo, but also improves the disorder of glucose metabolism.

A novel mode of action for a microbial-derived immunotoxin: the cytolethal distending toxin subunit B exhibits phosphatidylinositol 3,4,5-triphosphate phosphatase activity.

The Actinobacillus actinomycetemcomitans cytolethal distending toxin (Cdt) is a potent immunotoxin that induces G(2) arrest in human lymphocytes. We now show that the CdtB subunit exhibits phosphatidylinositol (PI)-3,4,5-triphosphate phosphatase activity. Breakdown product analysis indicates that CdtB hydrolyzes PI-3,4,5-P(3) to PI-3,4-P(2) and therefore functions in a manner similar to phosphatidylinositol 5-phosphatases. Conserved amino acids critical to catalysis in this family of enzymes were mutated in the cdtB gene. The mutant proteins exhibit reduced phosphatase activity along with decreased ability to induce G(2) arrest. Consistent with this activity, Cdt induces time-dependent reduction of PI-3,4,5-P(3) in Jurkat cells. Lymphoid cells with defects in SHIP1 and/or ptase and tensin homolog deleted on chromosome 10 (PTEN) (such as Jurkat, CEM, Molt) and, concomitantly, elevated PI-3,4,5-P(3) levels were more sensitive to the toxin than HUT78 cells which contain functional levels of both enzymes and low levels of PI-3,4,5-P(3). Finally, reduction of Jurkat cell PI-3,4,5-P(3) synthesis using the PI3K inhibitors, wortmannin and LY290004, protects cells from toxin-induced cell cycle arrest. Collectively, these studies show that the CdtB not only exhibits PI-3,4,5-P(3) phosphatase activity, but also that toxicity in lymphocytes is related to this activity.

Differential changes in phospholipase D and phosphatidate phosphohydrolase activities in ischemia-reperfusion of rat heart.

Phospholipase D (PLD2) produces phosphatidic acid (PA), which is converted to 1,2 diacylglycerol (DAG) by phosphatidate phosphohydrolase (PAP2). Since PA and DAG regulate Ca(2+) movements, we examined PLD2 and PAP2 in the sarcolemma (SL) and sarcoplasmic reticular (SR) membranes from hearts subjected to ischemia and reperfusion (I-R). Although SL and SR PLD2 activities were unaltered after 30 min ischemia, 5 min reperfusion resulted in a 36% increase in SL PLD2 activity, whereas 30 min reperfusion resulted in a 30% decrease in SL PLD2 activity, as compared to the control value. SR PLD2 activity was decreased (39%) after 5 min reperfusion, but returned to control levels after 30 min reperfusion. Ischemia for 60 min resulted in depressed SL and SR PLD2 activities, characterized with reduced V(max) and increased K(m) values, which were not reversed during reperfusion. Although the SL PAP2 activity was decreased (31%) during ischemia and at 30 min reperfusion (28%), the SR PAP2 activity was unchanged after 30 min ischemia, but was decreased after 5 min reperfusion (25%) and almost completely recovered after 30 min reperfusion. A 60 min period of ischemia followed by reperfusion caused an irreversible depression of SL and SR PAP2 activities. Our results indicate that I-R induced cardiac dysfunction is associated with subcellular changes in PLD2 and PAP2 activities.

Evidence for the involvement of protein kinase C in acidic pH-induced contraction in spontaneously hypertensive rat aorta.

This study was performed to test the hypothesis that activation of protein kinase C (PKC) is a mechanism underlying the acidic pH-induced contraction (APIC) in spontaneously hypertensive rat (SHR) aorta. Changing pH of the bathing solution from 7.4 to 6.5 induced a marked contraction of SHR aorta. PKC inhibitors, GF109203X and calphostin C markedly inhibited the APIC selectively, without having a marked effect on the KCl-induced contraction. Inhibitors of mitogen-activated protein kinase kinase, U0126 and PD98059 mildly but significantly attenuated the APIC. However, at the similar concentrations both U0126 and PD98059 inhibited the KCl-induced contraction in a manner similar to that observed in APIC. D-609, an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC) markedly inhibited the APIC and the extent of inhibition by this compound was similar to that shown by PKC inhibitors. Whereas, U-73122 and propranolol, inhibitors of phosphatidylinositol-specific PLC and phosphatidate phosphohydrolase, respectively, had no affect on the APIC. A tyrosine kinase inhibitor, tyrphostin 23 and GF109203X inhibited the APIC in an additive manner, and together they abolished the contractile response. From all these results, it is suggested that a significant component of the contraction observed in response to acidosis in SHR aorta is dependent upon the activation of PKC that seems to be the downstream event of the activation of PC-PLC. Furthermore, PKC- and tyrosine kinase-dependent pathways underlying the APIC are independent of each other.

Progesterone triggers the rapid activation of phospholipase D in the amphibian oocyte plasma membrane when initiating the G2/M transition.

Previous reports indicate that, in the Rana pipiens oocyte, progesterone triggers a rapid rise in 1,2-diacylglycerol (DAG) derived from phosphatidylcholine (PC) in the plasma membranes. This DAG transient, which appears and is terminated within 60-90 s, is derived both from a phospholipase which we assumed to be phospholipase C and from sphingomyelin (SM) synthase. We now find that progesterone stimulates PC and DAG turnover primarily via the phospholipase D (PLD) and phosphatidic acid phosphohydrolase (PAP) pathways as well as via the SM-ceramide pathway. Rana oocytes were prelabeled with [3H]choline chloride under conditions in which about 70% is incorporated into PC of the plasma membrane of the intact oocyte or with [3H]lysoplatelet activating factor (1-O-octadecyl-sn-glycero-3-phosphocholine, lysoPAF) which is selectively incorporated into plasma membrane PC. Progesterone induced the release of [3H]choline from intact oocytes into the medium within 60-90 s. This choline release was dose-dependent and was not inhibited by a putative PC-specific phospholipase C inhibitor, D609. Progesterone also induced a transient rise in [3H]lysoPAF-derived [3H]DAG within 1-2 min followed by a rise in [3H]PA. In the presence of 20 mM ethanol, progesterone stimulated formation of [3H]lysoPAF-derived phosphatidylethanol, indicating progesterone activation of PC-specific PLD and concomitant formation of PA. A DGK inhibitor (D102) reduced the level of [3H]PA, produced a sustained rise in [3H]DAG and was a weak inducer of meiosis in oocytes not exposed to progesterone. A PA phosphohydrolase inhibitor (propranolol) elevated [3H]PA and completely inhibited the progesterone-induced rise in DAG. Progesterone thus acts at oocyte plasma membrane receptors to release PC-derived DAG via both SM synthase and PC-PLD. The duration of the DAG signal is regulated by the coordinate action of DGK and PAP.