Intron retention in PI-PLC γ1 mRNA as a key mechanism affecting MMP expression in human primary fibroblast-like synovial cells.

The intron retention (IR) is a phenomenon utilized by cells to allow diverse fates at the same mRNA, leading to a different pattern of synthesis of the same protein. In this study, we analyzed the modulation of phosphoinositide-specific phospholipase C (PI-PLC) enzymes by Harpagophytum procumbens extract (HPE) in synoviocytes from joins of osteoarthritis (OA) patients. In some samples, the PI-PLC γ1 isoform mature mRNA showed the IR and, in these synoviocytes, the HPE treatment increased the phenomenon. Moreover, we highlighted that as a consequence of IR, a lower amount of PI-PLC γ1 was produced. The decrease of PI-PLC γ1 was associated with the decrease of metalloprotease-3 (MMP-3), and MMP-13, and ADAMTS-5 after HPE treatment. The altered expression of MMPs is a hallmark of the onset and progression of OA, thus substances able to decrease their expression are very desirable. The interesting outcomes of this study are that 35% of analyzed synovial tissues showed the IR phenomenon in the PI-PLC γ1 mRNA and that the HPE treatment increased this phenomenon. For the first time, we found that the decrease of PI-PLC γ1 protein in synoviocytes interferes with MMP production, thus affecting the pathways involved in the MMP expression. This finding was validated by the silencing of PI-PLC γ1 in synoviocytes where the IR phenomenon was not present. Our results shed new light on the biochemical mechanisms involved in the degrading enzyme production in the joint of OA patients, suggesting a new therapeutic target and highlighting the importance of personalized medicine.

Phosphatidylinositol-phospholipase C3 negatively regulates the hypersensitive response via complex signaling with MAP kinase, phytohormones, and reactive oxygen species in Nicotiana benthamiana.

Phospholipid signaling plays important roles in plant immune responses. Here, we focused on two phospholipase C3 (PLC3) orthologs in the Nicotiana benthamiana genome, NbPLC3-1 and NbPLC3-2. We generated NbPLC3-1 and NbPLC3-2-double-silenced plants (NbPLC3s-silenced plants). In NbPLC3s-silenced plants challenged with Ralstonia solanacearum 8107, induction of hypersensitive response (HR)-related cell death and bacterial population reduction was accelerated, and the expression level of Nbhin1, a HR marker gene, was enhanced. Furthermore, the expression levels of genes involved in salicylic acid and jasmonic acid signaling drastically increased, reactive oxygen species production was accelerated, and NbMEK2-induced HR-related cell death was also enhanced. Accelerated HR-related cell death was also observed by bacterial pathogens Pseudomonas cichorii, P. syringae, bacterial AvrA, oomycete INF1, and TMGMV-CP with L1 in NbPLC3s-silenced plants. Although HR-related cell death was accelerated, the bacterial population was not reduced in double NbPLC3s and NbCoi1-suppressed plants nor in NbPLC3s-silenced NahG plants. HR-related cell death acceleration and bacterial population reduction resulting from NbPLC3s-silencing were compromised by the concomitant suppression of either NbPLC3s and NbrbohB (respiratory oxidase homolog B) or NbPLC3s and NbMEK2 (mitogen activated protein kinase kinase 2). Thus, NbPLC3s may negatively regulate both HR-related cell death and disease resistance through MAP kinase- and reactive oxygen species-dependent signaling. Disease resistance was also regulated by NbPLC3s through jasmonic acid- and salicylic acid-dependent pathways.

Phosphatidylinositol-phospholipase C4 suppresses the hypersensitive response of Nicotiana benthamiana.

Phospholipid signaling plays an important role in plant immune responses. Here, we isolated two phospholipase C4 (PLC4) orthologs in the Nicotiana benthamiana genome, designated as N. benthamiana PLC4-1 and PLC4-2 (NbPLC4-1 and NbPLC4-2). We created NbPLC4-1- and NbPLC4-2- silenced plants. Induction of the hypersensitive response (HR), including HR cell death and bacterial population reduction, was accelerated in both NbPLC4-1- and NbPLC4-2-silenced plants challenged with N. benthamiana-incompatible Ralstonia solanacearum 8107. The NbPLC4-1- and NbPLC4-2-silenced plants also showed enhanced expression of Nbhin1, a HR marker gene. Expressions of genes for salicylic acid (SA) and jasmonic acid (JA) signaling were drastically increased in NbPLC4-1- and NbPLC4-2-silenced plants by R. solanacearum inoculation. In addition, NbPLC4-1 and NbPLC4-2 silencing triggered reactive oxygen species (ROS) hyper-production. These results suggest that NbPLC4s are closely associated with JA, SA, and ROS signaling and act as negative regulators of the HR in N. benthamiana.

Sphingomyelin synthase-related protein generates diacylglycerol via the hydrolysis of glycerophospholipids in the absence of ceramide.

Diacylglycerol (DG) is a well-established lipid second messenger. Sphingomyelin synthase (SMS)-related protein (SMSr) produces DG and ceramide phosphoethanolamine (CPE) by the transfer of phosphoethanolamine from phosphatidylethanolamine (PE) to ceramide. We previously reported that human SMSr overexpressed in COS-7 cells significantly increased DG levels, particularly saturated and/or monounsaturated fatty acid-containing DG molecular species, and provided DG to DG kinase (DGK) δ, which regulates various pathophysiological events, including epidermal growth factor-dependent cell proliferation, type 2 diabetes, and obsessive-compulsive disorder. However, mammalian SMSr puzzlingly produces only trace amounts of CPE/DG. To clarify this discrepancy, we highly purified SMSr and examined its activities other than CPE synthase. Intriguingly, purified SMSr showed a DG-generating activity via hydrolysis of PE, phosphatidic acid (PA), phosphatidylinositol (PI), and phosphatidylcholine (PC) in the absence of ceramide. DG generation through the PA phosphatase (PAP) activity of SMSr was approximately 300-fold higher than that with PE and ceramide. SMSr hydrolyzed PI ten times stronger than PI(4,5)bisphosphate (PI(4,5)P2). The PAP and PC-phospholipase C (PLC) activities of SMSr were inhibited by propranolol, a PAP inhibitor, and by D609, an SMS/PC-PLC inhibitor. Moreover, SMSr showed substrate selectivity for saturated and/or monounsaturated fatty acid-containing PA molecular species, but not arachidonic-acid-containing PA, which is exclusively generated in the PI(4,5)P2 cycle. We confirmed that SMSr expressed in COS-7 cells showed PAP and PI-PLC activities. Taken together, our study indicated that SMSr possesses previously unrecognized enzyme activities, PAP and PI/PE/PC-PLC, and constitutes a novel DG/PA signaling pathway together with DGKδ, which is independent of the PI(4,5)P2 cycle.

Phosphatidylinositol-phospholipase C2 regulates pattern-triggered immunity in Nicotiana benthamiana.

Phospholipid signaling plays an important role in plant immune responses against phytopathogenic bacteria in Nicotiana benthamiana. Here, we isolated two phospholipase C2 (PLC2) orthologs in the N. benthamiana genome, designated as PLC2-1 and 2-2. Both NbPLC2-1 and NbPLC2-2 were expressed in most tissues and were induced by infiltration with bacteria and flg22. NbPLC2-1 and NbPLC2-2 (NbPLC2s) double-silenced plants showed a moderately reduced growth phenotype. The induction of the hypersensitive response was not affected, but bacterial growth and the appearance of bacterial wilt were accelerated in NbPLC2s-silenced plants when they were challenged with a virulent strain of Ralstonia solanacearum that was compatible with N. benthamiana. NbPLC2s-silenced plants showed reduced expression levels of NbPR-4, a marker gene for jasmonic acid signaling, and decreased jasmonic acid and jasmonoyl-L-isoleucine contents after inoculation with R. solanacearum. The induction of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) marker genes was reduced in NbPLC2s-silenced plants after infiltration with R. solanacearum or Pseudomonas fluorescens. Accordingly, the resistance induced by flg22 was compromised in NbPLC2s-silenced plants. In addition, the expression of flg22-induced PTI marker genes, the oxidative burst, stomatal closure, and callose deposition were all reduced in the silenced plants. Thus, NbPLC2s might have important roles in pre- and post-invasive defenses, namely in the induction of PTI.

Lipidic Aminoglycoside Derivatives: A New Class of Immunomodulators Inducing a Potent Innate Immune Stimulation.

Development of simple and fully characterized immunomodulatory molecules is an active area of research to enhance current immunotherapies. Monophosphoryl lipid A (MPL), a nontoxic lipidic derivative from bacteria, is the first and currently only adjuvant approved in humans. However, its capacity to induce a potent response against weak immunogenic tumoral-associated antigens remains limited. Herein, a new generation of lipidic immunomodulators to conduct a structure-activity relationship study to determine the minimal structural elements conferring immunomodulatory properties is introduced. Two lead molecules characterized by a short succinyl linker between two oleyl chains and a polar headgroup consisting of either naturally occurring tobramycin (DOST) or kanamycin (DOSK) are identified. These two lipoaminoglycosides self-assemble in very small vesicles. In a wide variety of cells including 3D human cell culture, DOST and DOSK induce the upregulation of proinflammatory cytokines and interferon-inducible proteins in a dose and time-dependent manner via a caveolae-dependent proinflammatory mechanism and phosphatidylinositol phospholipase C activation. Furthermore, after intratumoral administration, these lipoaminoglycosides induce an efficient immune response leading to significant antitumor activity in a mouse breast cancer model. Altogether, these findings indicate that DOST and DOSK are two groundbreaking synthetic lipid immunostimulators that can be used as adjuvants to enhance current immunotherapeutic treatments.

The ßγ-crystallin domain of Lysinibacillus sphaericus phosphatidylinositol phospholipase C plays a central role in protein stability.

ßγ-crystallin has emerged as a superfamily of structurally homologous proteins with representatives across all domains of life. A major portion of this superfamily is constituted by microbial members. This superfamily has also been recognized as a novel group of Ca2+-binding proteins with a large diversity and variable properties in Ca2+ binding and stability. We have recently described a new phosphatidylinositol phospholipase C from Lysinibacillus sphaericus (LS-PIPLC) which was shown to efficiently remove phosphatidylinositol from crude vegetable oil. Here, the role of the C-terminal ßγ-crystallin domain of LS-PIPLC was analyzed in the context of the whole protein. A truncated protein in which the C-terminal ßγ-crystallin domain was deleted (LS-PIPLCΔCRY) is catalytically as efficient as the full-length protein (LS-PIPLC). However, the thermal and chemical stability of LS-PIPLCΔCRY are highly affected, demonstrating a stabilizing role for this domain. It is also shown that the presence of Ca2+ increases the thermal and chemical stability of the protein both in aqueous media and in oil, making LS-PIPLC an excellent candidate for use in industrial soybean oil degumming.

Development of a highly efficient oil degumming process using a novel phosphatidylinositol-specific phospholipase C enzyme.

Enzymatic degumming using phospholipase C (PLC) enzymes may be used in environmentally friendly processes with improved oil recovery yields. In this work, phosphatidylinositol-specific phospholipase C (PIPLC) candidates obtained from an in silico analysis were evaluated for oil degumming. A PIPLC from Lysinibacillus sphaericus was shown to efficiently remove phosphatidylinositol from crude oil, and when combined with a second phosphatidylcholine and phosphatidylethanolamine-specific phospholipase C, the three major phospholipids were completely hydrolyzed, providing an extra yield of oil greater than 2.1%, compared to standard methods. A remarkably efficient fed-batch Escherichia coli fermentation process producing ∼14 g/L of the recombinant PIPLC enzyme was developed, which may facilitate the adoption of this cost-effective oil-refining process.

Plant phospholipase C family: Regulation and functional role in lipid signaling.

Phospholipase C (PLC), a major membrane phospholipid hydrolyzing enzyme generates signaling messengers such as diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) in animals, and their phosphorylated forms such as phosphatidic acid (PA) and inositol hexakisphosphate (IP6) are thought to regulate various cellular processes in plants. Based on substrate specificity, plant PLC family is sub-divided into phosphatidylinositol-PLC (PI-PLC) and phosphatidylcholine-PLC (PC-PLC) groups. The activity of plant PLCs is regulated by various factors and the major ones include, Ca(2+) concentration, phospholipid substrate, post-translational modifications and interacting proteins. Most of the PLC members have been localized at the plasma membrane, suited for their function of membrane lipid hydrolysis. Several PLC members have been implicated in various cellular processes and signaling networks, triggered in response to a number of environmental cues and developmental events in different plant species, which makes them potential candidates for genetically engineering the crop plants for stress tolerance and enhancing the crop productivity. In this review article, we are focusing mainly on the plant PLC signaling and regulation, potential cellular and physiological role in different abiotic and biotic stresses, nutrient deficiency, growth and development.

Trypanosoma rangeli: an alkaline ecto-phosphatase activity is involved with survival and growth of the parasite.

The aim of this work was to investigate whether an alkaline ecto-phosphatase activity is present in the surface of Trypanosoma rangeli. Intact short epimastigote forms were assayed for ecto-phosphatase activity to study kinetics and modulators using ß-glycerophosphate (ß-GP) and p-nitrophenyl phosphate (pNPP) as substrates. Its role in parasite development and differentiation was also studied. Competition assays using different proportions of ß-GP and pNPP evidenced the existence of independent and non-interacting alkaline and acid phosphatases. Hydrolysis of ß-GP increased progressively with pH, whereas the opposite was evident using pNPP. The alkaline enzyme was inhibited by levamisole in a non-competitive fashion. The Ca(2+) present in the reaction medium was enough for full activity. Pretreatment with PI-PLC decreased the alkaline but not the acid phosphatase evidence that the former is catalyzed by a GPI-anchored enzyme, with potential intracellular signaling ability. ß-GP supported the growth and differentiation of T. rangeli to the same extent as high orthophosphate (Pi). Levamisole at the IC50 spared significantly parasite growth when ß-GP was the sole source of Pi and stopped it in the absence of ß-GP, indicating that the alkaline enzyme can utilize phosphate monoesters present in serum. These results demonstrate the existence of an alkaline ecto-phosphatase in T. rangeli with selective requirements and sensitivity to inhibitors that participates in key metabolic processes in the parasite life cycle.

Identification of phospholipase C in Leptospira interrogans and the mechanism of macrophage apopto-sis induced by phospholipase C / 中华微生物学和免疫学杂志

Objective To investigate the function of phosphatidylinositol phospholipase C encoded by LB361 gene of L.interrogans ( L-PI-PLC) and its mechanism in inducing macrophage apoptosis .Meth-ods The PI-PLC domains in the sequence of LB 361 gene of L.interrogans serovar Lai strain were analyzed by bioinformatics method .Prokaryotic expression system was established to express the recombinant L -PI-PLC ( rL-PI -PLC).The enzymatic activity of rL-PI-PLC in hydrolyzing phosphatidylinositol -4,5-bisphos -phate (PIP2) substrate into inositol-1,4,5-trisphosphate (IP3) was determined by IP3 fluorescence polariza-tion assay.LB361gene expressions at mRNA and protein levels as well as the secretion of LB 361gene prod -ucts were detected by real-time fluorescent quantitative RT -PCR and Western blot assay after infection of hu-man THP-1 macrophages with L.interrogans serovar Lai strain.A LB361 gene-transfected THP -1cell line was generated for evaluation of the mechanism of LB 361 gene products in elevating intracellular free Ca 2+( [Ca 2+] i) concentration and inducing the apoptosis of transfected THP -1 cells with the use of laser confocal microscopy and flow cytometry.Re sul ts The rL-PI-PLC hydrolyzed PIP2 into IP3 with a Km of 199 μmol/L and a Kcat of 8.566×10-5 S-1 .The expressions of LB361gene at mRNA and protein levels were both signifi -cantly up-regulated after infection of THP-1 cells with L.interrogans serovar Lai strain .Moreover , the exter-nal secretion of L-PI-PLC was also found during infection .The concentrations of IP 3 and [ Ca2+] i in the LB361 gene-transfected THP-1 cells were significantly increased compared to those in the non-transfected THP-1 cells, resulting in a high [Ca2+]i-dependent apoptosis of partial THP-1 cells.Conclusion PI-PLC is encoded by the LB361 gene of L.interrogans, which could induce the apoptosis of macrophages through el-evating [ Ca2+] i concentration during infection of microphages with L.interrogans.

Islet cells modified with PD-L1-GPI molecule inhibit attack of reactive T cells / 中国病理生理杂志

AIM:To investigate the immune depressive effect on the reactive T cells and to explore the immunologic injury mechanism of beta cells of islet in type 1 diabetes mellitus(DM-1).METHODS:pAd5/PD-L1-GPI adenovirus vector with target gene was constructed and transfected into NIT cells which are known as a mouse insuloma cell line.The highly expressed membrane protein of PD-L1-GPI was confirmed by Western blotting.The peripheral blood non-adherence lymph leukocytes and target cells were cultured to detect lymph leukocyte proliferation and the T cell function.The level of IL-2,TNF-? and IFN-? were detected in the cell culture fluid.RESULTS:Compared with the control group,the NIT cells modified with PD-L1-GPI inhibited the sensitized lymph leukocyte proliferation effectively and down-regulated the level of some cytokine secretions such as IL-2,IFN-? and TNF-?(P