Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.4.3 (phospholipase C)
 18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis catalyzes the cleavage of the phosphorus-oxygen bond in phosphatidylinositol. The focus of this work is to dissect the roles of the carboxylate side chain of Asp(274) in the Asp(274)-His(32) dyad, where a short strong hydrogen bond (SSHB) was shown to exist based on NMR criteria. A regular hydrogen bond (HB) was observed in D274N, and no low field proton resonance was detected for D274E and D274A. Comparison of the activity of wild type, D274N, and D274A suggested that the regular HB contributes significantly (approximately 4 kcal/mol) to catalysis, whereas the SSHB contributes only an additional 2 kcal/mol. The mutant D274E displays high activity similar to wild type, suggesting that the negative charge is sufficient for the catalytic role of Asp(274). To further support this interpretation and rule out possible contribution of regular HB or SSHB in D274E, we showed that the activity of D274G can be rescued by exogenous chloride ions to a level comparable with that of D274E. Comparison between different anions suggested that the ability of an anion to rescue the activity is due to the size and the charge of the anion not the property as a HB acceptor. In conclusion, a major fraction of the functional role of Asp(274) in the Asp(274)-His(32) dyad can be attributed to a negative charge (as in D274E and D274G-Cl(-)), and the SSHB in the wild type enzyme provides minimal contribution to catalysis. These results represent novel insight for an Asp-His catalytic dyad and for the mechanism of phosphatidylinositol-specific phospholipase C.
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PMID:The catalytic role of aspartate in a short strong hydrogen bond of the Asp274-His32 catalytic dyad in phosphatidylinositol-specific phospholipase C can be substituted by a chloride ion. 1515 21

We have used high-resolution field-cycling 31P NMR spectroscopy to measure spin-lattice relaxation rates (R1 = 1/T1) of multicomponent phospholipid vesicle and micelle samples over a large field range, from 0.1 to 11.7 T. The shape of the curve for R1 as a function of field and a model-free analysis were used to extract tauc, a correlation time for each type of phospholipid molecule in the bilayer that is likely to reflect rotation of the molecule about the axis perpendicular to the membrane surface; Sc2, a chemical shift anisotropy (CSA) order parameter; and tauhf, a time constant reflecting faster internal motion. This 31P technique was also used to monitor association of a peripheral membrane protein, Bacillus thuringiensis phosphatidylinositol-specific phospholipase C, with both phosphatidylcholine and phosphatidylmethanol bilayers. Differences in phospholipid dynamics induced by the protein shed light on how zwitterionic phosphatidylcholine, and not the anionic phosphatidylmethanol, activates the enzyme toward its substrate.
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PMID:High-resolution 31p field cycling NMR as a probe of phospholipid dynamics. 1549 36

The signal transduction protein phospholipase C-gamma1 (PLC-gamma1) is activated when its C-terminal SH2 domain (PLCC) binds the phosphorylated Tyr-1021 site (pTyr-1021) in the beta-platelet-derived growth factor receptor (PDGFR). To better understand the contributions that dynamics make to binding, we have used NMR relaxation experiments to investigate the motional properties of backbone amide and side chain methyl groups in a peptide derived from the pTyr-1021 site of PDGFR, both free and in complex with the PLCC SH2 domain. The free peptide has relaxation properties that are typical for a small, unstructured polymer, while the backbone of the bound peptide is least flexible for residues in the central portion of the binding site with the amplitude of pico- to nanosecond time scale motions increasing toward the C-terminus of the peptide. The increase in large amplitude motion toward the end of the pY1021 peptide is consistent with the bound peptide existing as an ensemble of states with C-terminal residues having the broadest distribution of backbone conformations, while residues in the central binding site are the most restricted. Deuterium spin relaxation experiments establish that the protein-peptide interface is highly dynamic, and this mobility may play an important role in modulating the affinity of the interaction.
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PMID:NMR dynamics-derived insights into the binding properties of a peptide interacting with an SH2 domain. 1564 95

We report the results on the expression in Escherichia coli of a functional neurotoxin LqqV from the scorpion Leiurus quinquestriatus quinquestriatus. The gene for LqqV was synthesized using recursive PCR and expressed as a poly-histidine-tagged fusion protein in thioredoxin mutant E. coli strain [AD494(DE3)pLysS], thus permitting disulfide-bond formation. When cultured at 37 degrees C, about 50% of the expressed protein is contained as a monomer in the soluble fraction of the E. coli extract. The fusion protein from the soluble fraction was purified and the His-tag was cleaved by thrombin, resulting in a yield of about 1.5 mg/liter. The globular structure of the purified protein was confirmed by NMR and CD spectroscopy. Patch-clamp measurements using native sodium channels in guinea pig ventricular myocytes reveal (1) a slowing of inactivation and (2) a decrease in peak current upon application of toxin, thus confirming the alpha-toxin activity of the purified recombinant protein.
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PMID:Expression of functional scorpion neurotoxin Lqq-V in E.coli. 1615 May 14

The membrane binding affinity of the pleckstrin homology (PH) domain of phospholipase C (PLC)-delta1 was investigated using a vesicle coprecipitation assay and the structure of the membrane-associated PH domain was probed using solid-state (13)C NMR spectroscopy. Twenty per cent phosphatidylserine (PS) in the membrane caused a moderate but significant reduction of the membrane binding affinity of the PH domain despite the predicted electrostatic attraction between the PH domain and the head groups of PS. Solid-state NMR spectra of the PH domain bound to the phosphatidylcholine (PC)/PS/phosphatidylinositol 4,5-bisphosphate (PIP(2)) (75 : 20 : 5) vesicle indicated loss of the interaction between the amphipathic alpha2-helix of the PH domain and the interface region of the membrane which was previously reported for the PH domain bound to PC/PIP(2) (95 : 5) vesicles. Characteristic local conformations in the vicinity of Ala88 and Ala112 induced by the hydrophobic interaction between the alpha2-helix and the membrane interface were lost in the structure of the PH domain at the surface of the PC/PS/PIP(2) vesicle, and consequently the structure becomes identical to the solution structure of the PH domain bound to d-myo-inositol 1,4,5-trisphosphate. These local structural changes reduce the membrane binding affinity of the PH domain. The effects of PS on the PH domain were reversed by NaCl and MgCl(2), suggesting that the effects are caused by electrostatic interaction between the protein and PS. These results generally suggest that the structure and function relationships among PLCs and other peripheral membrane proteins that have similar PH domains would be affected by the local lipid composition of membranes.
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PMID:Phosphatidylserine induces functional and structural alterations of the membrane-associated pleckstrin homology domain of phospholipase C-delta1. 1722 80

Alkyl-lysophospholipids (ALPs), developed initially to be antitumor agents, have proved highly effective in the treatment of visceral leishmaniasis, a disease caused by the species making up the protozoan complex Leishmania donovani. Although their effectiveness is known, the mode of action against this parasite is not completely understood. In the present work, we have studied the effect of 3 derivatives, edelfosine, miltefosine, and ilmofosine. Using nuclear magnetic resonance spectroscopy ('H-NMR), we have examined the excreted catabolites from glucose metabolism in the promastigote forms treated with these compounds. The ALPs at concentrations of 19 and 38 microM inhibit the excretion of acetate, succinate, and pyruvate. The effect of edelfosine, miltefosine, and ilmofosine on the activity of the enzymes hexokinase, glycerolkinase 3-PD, phosphoglucose isomerase, superoxide dismutase, and phospholipase C were also examined. Glycerolkinase 3-PD and phosphoglucose isomerase are generally insensitive to the compounds, whereas hexokinase and superoxide dismutase are inhibited by miltefosine and ilmofosine. The ALPs exhibited an activated effect against the phospholipase C activity. Alkyl-lysophospholipids were shown to have a significant effect on several enzymes in important biochemical pathways indispensable for the survival of L. donovani promasigotes.
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PMID:Effect of alkyl-lysophospholipids on some aspects of the metabolism of Leishmania donovani. 1816 58

Clostridium perfringens alpha-toxin induces the hemolysis of sheep erythrocytes by activating the metabolism of sphingomyelin (SM) via a GTP binding protein in membranes. alpha-Toxin stimulated the formation of 15-N-nervonoyl sphingosine (C24:1-ceramide), which was identified by positive ion fast atom bombardment-MS and 1H-NMR spectroscopy. C24:1-ceramide stimulated the toxin-induced hemolysis of saponin-pretreated sheep erythrocytes and increased the production of sphingosine 1-phosphate (S1P) in the cells, but N-lignoceroyl sphingosine did not. These events elicited by the toxin in the presence of C24:1-ceramide were significantly attenuated by treatment with dihydrosphingosine, a sphingosine kinase inhibitor. TLC showed that the level of C24:1-ceramide was highest among the ceramides with an unsaturated bond in the fatty acyl chain in the detergent-resistant membranes (DRMs). The toxin specifically bound to DRMs rich in cholesterol, resulting in the hydrolysis of N-nervonoic sphingomyelin (C24:1-SM) in DRMs. Treatment of the cells with pertussis toxin (PT) inhibited the alpha-toxin-induced formation of C24:1-ceramide from C24:1-SM in DRMs and hemolysis, indicating that endogenous sphingomyelinase, which hydrolyzes C24:1-SM to C24:1-ceramide, is controlled by PT-sensitive GTP binding protein in membranes. These results show that the toxin-induced metabolism of C24:1-SM to S1P in DRMs plays an important role in the toxin-induced hemolysis of sheep erythrocytes.
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PMID:The relationship between the metabolism of sphingomyelin species and the hemolysis of sheep erythrocytes induced by Clostridium perfringens alpha-toxin. 1826 51

The Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC), an interfacial enzyme associated with prokaryotic infectivity, is activated by binding to zwitterionic surfaces, particularly phosphatidycholine (PC). Two tryptophan residues (Trp47 in the two-turn helix B and Trp242 in a disordered loop) at the rim of the barrel structure are critical for this interaction. The helix B region (Ile43 to Gly48) in wild-type PI-PLC orients the side chains of Ile43 and Trp47 so that they pack together and form a hydrophobic protrusion from the protein surface that likely facilitates initial membrane binding. In previous studies we reported that in the crystal structure of the dimeric W47A/W242A mutant, which is unable to bind to PC, the helix B region has been reorganized by the mutation into an extended loop. Here we report the construction and characterization (catalytic activity, fluorescence, and NMR studies) of a series of PI-PLC mutants targeting helix B residues and surrounding regions to explore what is needed to stabilize the "membrane-active" conformation of the helix B region. Results strongly suggest that, while hydrophobic groups and presumably an intact helix B are critical for the initial binding of PI-PLC to membranes, disruption of helix B to allow enzyme dimerization is what leads to the activated PI-PLC conformation.
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PMID:Role of helix B residues in interfacial activation of a bacterial phosphatidylinositol-specific phospholipase C. 1834 43

D-3-deoxyphosphatidylinositol (D-3-deoxy-PI) derivatives have cytotoxic activity against various human cancer cell lines. These phosphatidylinositols have a potentially wide array of targets in the phosphatidylinositol-3-kinase (PI3K)/Akt signaling network. To explore the specificity of these types of molecules, we have synthesized D-3-deoxydioctanoylphosphatidylinositol (D-3-deoxy-diC8PI), D-3,5-dideoxy-diC8PI, and D-3-deoxy-diC8PI-5-phosphate and their enantiomers, characterized their aggregate formation by novel high-resolution field cycling (31)P NMR, and examined their susceptibility to phospholipase C (PLC), their effects on the catalytic activities of PI3K and PTEN against diC8PI and diC8PI-3-phosphate substrates, respectively, and their ability to induce the death of U937 human leukemic monocyte lymphoma cells. Of these molecules, only D-3-deoxy-diC8PI was able to promote cell death; it did so with a median inhibitory concentration of 40 microM, which is much less than the critical micelle concentration of 0.4 mM. Under these conditions, little inhibition of PI3K or PTEN was observed in assays of recombinant enzymes, although the complete series of deoxy-PI compounds did provide insights into ligand binding by PTEN. D-3-deoxy-diC8PI was a poor substrate and not an inhibitor of the PLC enzymes. The in vivo results are consistent with the current thought that the PI analogue acts on Akt1, since the transcription initiation factor eIF4e, which is a downstream signaling target of the PI3K/Akt pathway, exhibited reduced phosphorylation on Ser209. Phosphorylation of Akt1 on Ser473 but not Thr308 was reduced. Since the potent cytotoxicity for U937 cells was completely lost when L-3-deoxy-diC8PI was used as well as when the hydroxyl group at the inositol C5 in D-3-deoxy-diC8PI was modified (by either replacing this group with a hydrogen or phosphorylating it), both the chirality of the phosphatidylinositol moiety and the hydroxyl group at C5 are major determinants of the binding of 3-deoxy-PI to its target in cells.
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PMID:Insights into the structural specificity of the cytotoxicity of 3-deoxyphosphatidylinositols. 1849 65

Several isoforms of phospholipase C (PLC) are regulated through interactions with Ras superfamily GTPases, including Rac proteins. Interestingly, of two closely related PLCgamma isoforms, only PLCgamma(2) has previously been shown to be activated by Rac. Here, we explore the molecular basis of this interaction as well as the structural properties of PLCgamma(2) required for activation. Based on reconstitution experiments with isolated PLCgamma variants and Rac2, we show that an unusual pleckstrin homology (PH) domain, designated as the split PH domain (spPH), is both necessary and sufficient to effect activation of PLCgamma(2) by Rac2. We also demonstrate that Rac2 directly binds to PLCgamma(2) as well as to the isolated spPH of this isoform. Furthermore, through the use of NMR spectroscopy and mutational analysis, we determine the structure of spPH, define the structural features of spPH required for Rac interaction, and identify critical amino acid residues at the interaction interface. We further discuss parallels and differences between PLCgamma(1) and PLCgamma(2) and the implications of our findings for their respective signaling roles.
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PMID:rac regulates its effector phospholipase Cgamma2 through interaction with a split pleckstrin homology domain. 1872 11


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