Selective release of plasma-membrane enzymes from rat hepatocytes by a phosphatidylinositol-specific phospholipase C.
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ABSTRACT:
SUBMITTER: Shukla SD
PROVIDER: S-EPMC1162522 | biostudies-other | 1980 Apr
REPOSITORIES: biostudies-other
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Project description:Amphitropic proteins, such as the virulence factor phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis , often depend on lipid-specific recognition of target membranes. However, the recognition mechanisms for zwitterionic lipids, such as phosphatidylcholine, which is enriched in the outer leaflet of eukaryotic cells, are not well understood. A 500 ns long molecular dynamics simulation of PI-PLC at the surface of a lipid bilayer revealed a strikingly high number of interactions between tyrosines at the interfacial binding site and lipid choline groups with structures characteristic of cation-? interactions. Membrane affinities of PI-PLC tyrosine variants mostly tracked the simulation results, falling into two classes: (i) those with minor losses in affinity, Kd(mutant)/Kd(wild-type) ? 5 and (ii) those where the apparent Kd was 50-200 times higher than wild-type. Estimating ??G for these Tyr/PC interactions from the apparent Kd values reveals that the free energy associated with class I is ~1 kcal/mol, comparable to the value predicted by the Wimley-White hydrophobicity scale. In contrast, removal of class II tyrosines has a higher energy cost: ~2.5 kcal/mol toward pure PC vesicles. These higher energies correlate well with the occupancy of the cation-? adducts throughout the MD simulation. Together, these results strongly indicate that PI-PLC interacts with PC headgroups via cation-? interactions with tyrosine residues and suggest that cation-? interactions at the interface may be a mechanism for specific lipid recognition by amphitropic and membrane proteins.
| S-EPMC3797534 | biostudies-literature
Project description:We previously screened genes that were transcriptionally activated during the early stage of wound response in tobacco plants (Nicotiana tabacum), and isolated a particular clone, which encoded a membrane-located protein, designated as NtC7. Upon overexpression in tobacco plants, NtC7 conferred a marked tolerance to osmotic stress, suggesting it to be involved in maintenance of osmotic adjustments. In this study, we searched for proteins which interact with NtC7 by the yeast two-hybrid screening, and isolated a clone encoding phosphoinositide-specific phospholipase C, designated as NtPI-PLC. Physical interaction between NtC7 and C2 domain of NtPI-PLC was confirmed by the pull-down assay. Expression of fused protein to green-fluorescence protein in onion epidermal cell layers indicated both proteins to predominantly localize to the plasma membrane. Their interaction in planta was shown by the bimolecular fluorescence complementation, which exhibited a clear fluorescence of reconstituted yellow fluorescence protein. Transcripts of NtC7 and NtPI-PLC were markedly increased 30 to 60 min after wounding. PI-PLC is one of key enzymes in metabolism of inositol phospholipids, which function in signal transduction and also in response to stresses including osmotic changes. It was shown to localize to plasma-membrane and, to a lesser extent, to cytosol. However, molecular mechanism of membrane localization has remained to be determined, because of the apparent lack of domains for membrane association. The present results suggest that one of such mechanisms is tethering NtPI-PLC to the plasma membrane through interaction with NtC7, which possesses a transmembrane domain at the C-terminus.
| S-EPMC2634064 | biostudies-literature
Project description:A major glycoprotein of rat hepatoma plasma membranes was selectively released as a soluble form by incubating the membrane with phosphatidylinositol-specific phospholipase C. The soluble form corresponding to the glycoprotein was also prepared by butan-1-ol extraction of microsomal membranes at pH 5.5, whereas extraction at pH 8.5 yielded an electrophoretically different form with a hydrophobic nature. The soluble glycoprotein extracted at pH 5.5 was purified by sequential chromatography on concanavalin A-Sepharose, Sephacryl S-300 and anti-(alkaline phosphatase) IgG-Sepharose, the last step being used to remove a contaminating alkaline phosphatase. The glycoprotein thus purified was a single protein with Mr 130,000 in SDS/polyacrylamide-gel electrophoresis, although it behaved as a dimer in gel filtration on Sephacryl S-300. The glycoprotein was analysed for amino acid and carbohydrate composition. The composition of the carbohydrate moiety, which amounted to 64% by weight, suggested that the glycoprotein contained much larger numbers of N-linked oligosaccharide chains than those with O-linkage. It was confirmed that the purified glycoprotein was immunologically identical not only with that released by the phospholipase C but also with the hydrophobic form extracted with butan-1-ol at pH 8.5. The results indicate that the glycoprotein of rat hepatoma plasma membranes, which has an unusually high content of carbohydrate, is another membrane protein released by phosphatidylinositol-specific phospholipase C, as documented for alkaline phosphatase, acetylcholinesterase and Thy-1 antigen.
| S-EPMC1147524 | biostudies-other
Project description:Purified phosphatidylinositol-specific phospholipase C from Staphylococcus aureus released a substantial proportion of the total alkaline phosphatase activity from a wide range of tissues from several mammalian species. Co-purification of the phospholipase C and alkaline phosphatase-releasing activities and the inhibition of both these activities by iso-osmotic salt solutions suggested that the releasing effect was unlikely to be due to a contaminant.
| S-EPMC1183647 | biostudies-other
Project description:Staphylococcus aureus (S. aureus) commonly colonizes the human skin and nostrils. However, it is also associated with a wide variety of diseases. S. aureus is frequently isolated from the skin of patients with atopic dermatitis (AD), and is linked to increased disease severity. S. aureus impairs the skin barrier and triggers inflammation through the secretion of various virulence factors. S. aureus secretes phosphatidylinositol-specific phospholipase C (PI-PLC), which hydrolyses phosphatidylinositol and cleaves glycosylphosphatidylinositol-anchored proteins. However, the role of S. aureus PI-PLC in the pathogenesis of skin diseases, including AD, remains unclear. In this study, we sought to determine the role of S. aureus PI-PLC in the pathogenesis of skin diseases. PI-PLC was observed to enhance the invasion and persistence of S. aureus in keratinocytes. Besides, PI-PLC promoted the penetration of S. aureus through the epidermal barrier in a mouse model of AD and the human organotypic epidermal equivalent. Furthermore, the loss of PI-PLC attenuated epidermal hyperplasia and the infiltration of Gr-1+ cells and CD4+ cells induced by S. aureus infection in the mouse model of AD. Collectively, these results indicate that PI-PLC eases the entry of S. aureus into the dermis and aggravates acanthosis and immune cell infiltration in infected skin.
| S-EPMC7575579 | biostudies-literature
Project description:Calcium-dependent phosphatidylinositol-specific phospholipase C from Streptomyces antibioticus (saPLC1) catalyzes hydrolysis of phosphatidylinositol (PI) into inositol 1-phosphate by a unique mechanism involving formation of inositol 1,6-cyclic phosphate (1,6-IcP) as an intermediate. This work examines the rates and products of cleavage of phosphorothioate and phosphorodithioate analogues of PI in which sulfur was introduced into the phosphate moiety at a nonbridging position (pro-R or pro-S), a bridging position, or both. The replacement of the pro-S oxygen in the phosphoryl moiety of PI by sulfur results in a 3 x 10(7)-fold decrease of the catalytic rate constant, whereas alteration of the pro-R oxygen results in only a modest rate reduction. The addition of the second sulfur atom into the bridging position of the S(p) isomer of the phosphorothioate analogue causes a dramatic (2 x 10(5)-fold) increase of the rate of cleavage but has a negligible effect on the R(p) isomer. These differences are consistent with a change in the mechanism for the S(p) isomer of the phosphorodithioate analogue into a more dissociative type, where the leaving group carries a large amount of negative charge. In addition, hydrolysis of the diastereomers of the phosphorothioate analogues of 1,6-IcP, inositol cis-1,6-IcPs and inositol trans-1,6-IcPs, affords two distinct products, inositol 1-phosphorothioate and inositol 6-phosphorothioate, respectively. Formation of inositol 6-phosphorothioate is explained by the binding of trans-1,6-IcPs in the active site in a rotated orientation that interchanges the oxygen atoms at the 1- and 6-positions, thereby allowing the hydroxyl group at the 1-position to act as a leaving group. The reorientation of the intermediate is driven by formation of favorable interactions of the enzyme active site with the nonbridging oxygen in the trans intermediate.
| S-EPMC3733342 | biostudies-literature
Project description:Sprouty (Spry) proteins are negative regulators of receptor tyrosine kinase signaling; however, their exact mechanism of action remains incompletely understood. We identified phosphatidylinositol-specific phospholipase C (PLC)-? as a partner of the Spry1 and Spry2 proteins. Spry-PLC? interaction was dependent on the Src homology 2 domain of PLC? and a conserved N-terminal tyrosine residue in Spry1 and Spry2. Overexpression of Spry1 and Spry2 was associated with decreased PLC? phosphorylation and decreased PLC? activity as measured by production of inositol (1,4,5)-triphosphate (IP(3)) and diacylglycerol, whereas cells deficient for Spry1 or Spry1, -2, and -4 showed increased production of IP(3) at baseline and further increased in response to growth factor signals. Overexpression of Spry 1 or Spry2 or small-interfering RNA-mediated knockdown of PLC?1 or PLC?2 abrogated the activity of a calcium-dependent reporter gene, suggesting that Spry inhibited calcium-mediated signaling downstream of PLC?. Furthermore, Spry overexpression in T-cells, which are highly dependent on PLC? activity and calcium signaling, blocked T-cell receptor-mediated calcium release. Accordingly, cultured T-cells from Spry1 gene knockout mice showed increased proliferation in response to T-cell receptor stimulation. These data highlight an important action of Spry, which may allow these proteins to influence signaling through multiple receptors.
| S-EPMC2947483 | biostudies-literature
Project description:Phosphatidylinositol(4,5) bisphosphate (PI(4,5)P2) has become a major focus in biochemistry, cell biology and physiology owing to its diverse functions at the plasma membrane. As a result, the functions of PI(4,5)P2 can be explored in two separate and distinct roles - as a substrate for phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K) and as a primary messenger, each having unique properties. Thus PI(4,5)P2 makes contributions in both signal transduction and cellular processes including actin cytoskeleton dynamics, membrane dynamics and ion channel regulation. Signalling through plasma membrane G-protein coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and immune receptors all use PI(4,5)P2 as a substrate to make second messengers. Activation of PI3K generates PI(3,4,5)P3 (phosphatidylinositol(3,4,5)trisphosphate), a lipid that recruits a plethora of proteins with pleckstrin homology (PH) domains to the plasma membrane to regulate multiple aspects of cellular function. In contrast, PLC activation results in the hydrolysis of PI(4,5)P2 to generate the second messengers, diacylglycerol (DAG), an activator of protein kinase C and inositol(1,4,5)trisphosphate (IP3/I(1,4,5)P3) which facilitates an increase in intracellular Ca2+. Decreases in PI(4,5)P2 by PLC also impact on functions that are dependent on the intact lipid and therefore endocytosis, actin dynamics and ion channel regulation are subject to control. Spatial organisation of PI(4,5)P2 in nanodomains at the membrane allows for these multiple processes to occur concurrently.
| S-EPMC7517351 | biostudies-literature
Project description:1. Rat livers were dissociated into their constituent cells by perfusion through the portal vein with a medium containing collagenase, and hepatocytes separated from non-parenchymal cells. 2. It is shown that the procedure described by Wisher & Evans [(1975) Biochem. J. 146, 375-388] for preparation of plasma membranes from liver tissue when applied to isolated hepatocytes also yielded subfractions of similar morphology and marker-enzyme distribution. 3. Thus the distribution of alkaline phosphodiesterase, 5'-nucleotidase and the basal and glucagon-stimulated adenylate cyclase among two 'light' vesicular and one 'heavy' junction-containing plasma-membrane subfractions paralleled that reported for tissue-derived plasma-membrane subfractions. 4. Increased recoveries and specific activities of plasma-membrane marker enzymes were obtained when soya-bean trypsin inhibitor was included in the collagenase-containing perfusion media used to dissociate the liver. 5. Polyacrylamide-gel-electrophoretic analysis of the corresponding plasma-membrane subfractions prepared from liver tissue and isolated hepatocytes were generally similar. 6. The results indicate that the functional polarity of the hepatocyte's plasma membrane is retained after tissue dissociation. The damage occurring to plasma-membrane ectoenzymes by the collagenase-perfusion procedure is discussed.
| S-EPMC1164807 | biostudies-other
Project description:IntroductionOverexpression on plasma membrane of human epidermal growth factor receptor 2 (HER2) is reported in 25% to 30% of breast cancers. Heterodimer formation with cognate members of the epidermal growth factor receptor (EGFR) family, such as HER3 and EGFR, activates abnormal cell-signalling cascades responsible for tumorigenesis and further transcriptional HER2 gene upregulation. Targeting the molecular mechanisms controlling HER2 overexpression and recycling may effectively deactivate this feedback-amplification loop. We recently showed that inactivation of phosphatidylcholine-specific phospholipase C (PC-PLC) may exert a pivotal role in selectively modulating the expression on the membrane of specific receptors or proteins relevant to cell function. In the present study, we investigated the capability of PC-PLC inhibition to target the molecular mechanisms controlling HER2 overexpression on the membrane of breast cancer cells by altering the rates of its endocytosis and lysosomal degradation.MethodsLocalization on the membrane and interaction of PC-PLC with HER2, EGFR, and HER3 were investigated on HER2-overexpressing and HER2-low breast cancer cell lines, by using confocal laser scanning microscopy, flow cytometry, cell-surface biotinylation, isolation of lipid rafts, and immunoprecipitation experiments. The effects of the PC-PLC inhibitor tricyclodecan-9-yl-potassium xanthate (D609) on HER2 expression on the membrane and on the levels of overall HER2, HER2-HER3, and HER2-EGFR contents were monitored in the HER2-overexpressing SKBr3 cells, after either transient or continuous receptor engagement with anti-HER2 monoclonal antibodies, including trastuzumab. Changes of HER2 expression and cell proliferation were examined in SKBr3, BT-474, and MDA-MB-453 cells continuously exposed to D609 alone or combined with trastuzumab.ResultsPC-PLC selectively accumulates on the plasma membrane of HER2-overexpressing cells, where it colocalizes and associates with HER2 in raft domains. PC-PLC inhibition resulted in enhanced HER2 internalization and lysosomal degradation, inducing downmodulation of HER2 expression on the membrane. Moreover, PC-PLC inhibition resulted in strong retardation of HER2 reexpression on the membrane and a decrease in the overall cellular contents of HER2, HER2-HER3, and HER2-EGFR heterodimers. The PC-PLC inhibitor also induced antiproliferative effects, especially in trastuzumab-resistant cells.ConclusionsThe results pointed to PC-PLC inhibition as a potential means to counteract the tumorigenic effects of HER2 amplification and complement the effectiveness of current HER2-targeting therapies.
| S-EPMC2917016 | biostudies-literature