Phosphatidylcholine Cation-Tyrosine π Complexes: Motifs for Membrane Binding by a Bacterial Phospholipase C.

Phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes are a virulence factor in many Gram-positive organisms. The specific activity of the Bacillus thuringiensis PI-PLC is significantly increased by adding phosphatidylcholine (PC) to vesicles composed of the substrate phosphatidylinositol, in part because the inclusion of PC reduces the apparent Kd for the vesicle binding by as much as 1000-fold when comparing PC-rich vesicles to PI vesicles. This review summarizes (i) the experimental work that localized a site on BtPI-PLC where PC is bound as a PC choline cation-Tyr-π complex and (ii) the computational work (including all-atom molecular dynamics simulations) that refined the original complex and found a second persistent PC cation-Tyr-π complex. Both complexes are critical for vesicle binding. These results have led to a model for PC functioning as an allosteric effector of the enzyme by altering the protein dynamics and stabilizing an 'open' active site conformation.

Morphologic Patterns Formed by the Anomalous Fibers Occurring Along the Anterior Capsule of the Crystalline Lens in People With the Long Anterior Zonule Trait.

People with the long anterior zonule (LAZ) trait, which may have prevalence near 2%, have zonular fibers that extend more central than usual along the anterior capsule of the crystalline lens. The anomalous fibers can be observed in vivo with clinical slit lamp biomicroscopy after pharmacologic pupil dilation, and although minimally studied, the LAZ trait may have importance to glaucoma, retinal degeneration, and cataract surgery. To further characterize LAZ morphology, a custom computer program was used to trace LAZ fibers seen on retro-illumination photos acquired during previous study at an academic, urban eye care facility in Chicago, IL. There were 59 African-Americans (54 female; median age = 70 years, 53-91 years) included in the analysis. After initial review of the zonule tracings, we identified three basic LAZ patterns. We called one pattern (47% of right eyes) a "non-segmental LAZ pattern," which was predominated by fibers that could be visually traced to the dilated pupil border where they became obscured by the iris. Another pattern (35% of right eyes), the "segmental LAZ pattern," was predominated by fibers that appeared to terminate abruptly without detectable extension to the pupil border. The third pattern (18% of right eyes), the "mixed LAZ pattern," had a more equivalent mixture of the other two fiber morphologies. Compared to the "non-segmental" group, the "segmental" LAZ eyes had smaller central zonule-free zones (P < 0.0001), and they tended to exhibit fewer LAZ fibers (P = 0.07). These data improve understanding of LAZ clinical anatomy and may be helpful to future investigation. Anat Rec, 300:1336-1347, 2017. © 2017 Wiley Periodicals, Inc.

d-3-Deoxy-dioctanoylphosphatidylinositol induces cytotoxicity in human MCF-7 breast cancer cells via a mechanism that involves downregulation of the D-type cyclin-retinoblastoma pathway.

Phosphatidylinositol analogs (PIAs) were originally designed to bind competitively to the Akt PH domain and prevent membrane translocation and activation. d-3-Deoxy-dioctanoylphosphatidylinositol (d-3-deoxy-diC8PI), but not compounds with altered inositol stereochemistry (e.g., l-3-deoxy-diC8PI and l-3,5-dideoxy-diC8PI), is cytotoxic. However, high resolution NMR field cycling relaxometry shows that both cytotoxic and non-toxic PIAs bind to the Akt1 PH domain at the site occupied by the cytotoxic alkylphospholipid perifosine. This suggests that another mechanism for cytotoxicity must account for the difference in efficacy of the synthetic short-chain PIAs. In MCF-7 breast cancer cells, with little constitutively active Akt, d-3-deoxy-diC8PI (but not l-compounds) decreases viability concomitant with increased cleavage of PARP and caspase 9, indicative of apoptosis. d-3-Deoxy-diC8PI also induces a decrease in endogenous levels of cyclins D1 and D3 and blocks downstream retinoblastoma protein phosphorylation. siRNA-mediated depletion of cyclin D1, but not cyclin D3, reduces MCF-7 cell proliferation. Thus, growth arrest and cytotoxicity induced by the soluble d-3-deoxy-diC8PI occur by a mechanism that involves downregulation of the D-type cyclin-pRb pathway independent of its interaction with Akt. This ability to downregulate D-type cyclins contributes, at least in part, to the anti-proliferative activity of d-3-deoxy-diC8PI and may be a common feature of other cytotoxic phospholipids.

A Role for Weak Electrostatic Interactions in Peripheral Membrane Protein Binding.

Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (BtPI-PLC) is a secreted virulence factor that binds specifically to phosphatidylcholine (PC) bilayers containing negatively charged phospholipids. BtPI-PLC carries a negative net charge and its interfacial binding site has no obvious cluster of basic residues. Continuum electrostatic calculations show that, as expected, nonspecific electrostatic interactions between BtPI-PLC and membranes vary as a function of the fraction of anionic lipids present in the bilayers. Yet they are strikingly weak, with a calculated ΔGel below 1 kcal/mol, largely due to a single lysine (K44). When K44 is mutated to alanine, the equilibrium dissociation constant for small unilamellar vesicles increases more than 50 times (∼2.4 kcal/mol), suggesting that interactions between K44 and lipids are not merely electrostatic. Comparisons of molecular-dynamics simulations performed using different lipid compositions reveal that the bilayer composition does not affect either hydrogen bonds or hydrophobic contacts between the protein interfacial binding site and bilayers. However, the occupancies of cation-π interactions between PC choline headgroups and protein tyrosines vary as a function of PC content. The overall contribution of basic residues to binding affinity is also context dependent and cannot be approximated by a rule-of-thumb value because these residues can contribute to both nonspecific electrostatic and short-range protein-lipid interactions. Additionally, statistics on the distribution of basic amino acids in a data set of membrane-binding domains reveal that weak electrostatics, as observed for BtPI-PLC, might be a less unusual mechanism for peripheral membrane binding than is generally thought.

Fluorinated Aromatic Amino Acids Distinguish Cation-π Interactions from Membrane Insertion.

Cation-π interactions, where protein aromatic residues supply π systems while a positive-charged portion of phospholipid head groups are the cations, have been suggested as important binding modes for peripheral membrane proteins. However, aromatic amino acids can also insert into membranes and hydrophobically interact with lipid tails. Heretofore there has been no facile way to differentiate these two types of interactions. We show that specific incorporation of fluorinated amino acids into proteins can experimentally distinguish cation-π interactions from membrane insertion of the aromatic side chains. Fluorinated aromatic amino acids destabilize the cation-π interactions by altering electrostatics of the aromatic ring, whereas their increased hydrophobicity enhances membrane insertion. Incorporation of pentafluorophenylalanine or difluorotyrosine into a Staphylococcus aureus phosphatidylinositol-specific phospholipase C variant engineered to contain a specific PC-binding site demonstrates the effectiveness of this methodology. Applying this methodology to the plethora of tyrosine residues in Bacillus thuringiensis phosphatidylinositol-specific phospholipase C definitively identifies those involved in cation-π interactions with phosphatidylcholine. This powerful method can easily be used to determine the roles of aromatic residues in other peripheral membrane proteins and in integral membrane proteins.

Avoidance of autophagy mediated by PlcA or ActA is required for Listeria monocytogenes growth in macrophages.

Listeria monocytogenes is a facultative intracellular pathogen that escapes from phagosomes and grows in the cytosol of infected host cells. Most of the determinants that govern its intracellular life cycle are controlled by the transcription factor PrfA, including the pore-forming cytolysin listeriolysin O (LLO), two phospholipases C (PlcA and PlcB), and ActA. We constructed a strain that lacked PrfA but expressed LLO from a PrfA-independent promoter, thereby allowing the bacteria to gain access to the host cytosol. This strain did not grow efficiently in wild-type macrophages but grew normally in macrophages that lacked ATG5, a component of the autophagy LC3 conjugation system. This strain colocalized more with the autophagy marker LC3 (42% ± 7%) at 2 h postinfection, which constituted a 5-fold increase over the colocalization exhibited by the wild-type strain (8% ± 6%). While mutants lacking the PrfA-dependent virulence factor PlcA, PlcB, or ActA grew normally, a double mutant lacking both PlcA and ActA failed to grow in wild-type macrophages and colocalized more with LC3 (38% ± 5%). Coexpression of LLO and PlcA in a PrfA-negative strain was sufficient to restore intracellular growth and decrease the colocalization of the bacteria with LC3. In a cell-free assay, purified PlcA protein blocked LC3 lipidation, a key step in early autophagosome biogenesis, presumably by preventing the formation of phosphatidylinositol 3-phosphate (PI3P). The results of this study showed that avoidance of autophagy by L. monocytogenes primarily involves PlcA and ActA and that either one of these factors must be present for L. monocytogenes growth in macrophages.

Phospholipid-binding sites of phosphatase and tensin homolog (PTEN): exploring the mechanism of phosphatidylinositol 4,5-bisphosphate activation.

The lipid phosphatase activity of the tumor suppressor phosphatase and tensin homolog (PTEN) is enhanced by the presence of its biological product, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). This enhancement is suggested to occur via the product binding to the N-terminal region of the protein. PTEN effects on short-chain phosphoinositide (31)P linewidths and on the full field dependence of the spin-lattice relaxation rate (measured by high resolution field cycling (31)P NMR using spin-labeled protein) are combined with enzyme kinetics with the same short-chain phospholipids to characterize where PI(4,5)P2 binds on the protein. The results are used to model a discrete site for a PI(4,5)P2 molecule close to, but distinct from, the active site of PTEN. This PI(4,5)P2 site uses Arg-47 and Lys-13 as phosphate ligands, explaining why PTEN R47G and K13E can no longer be activated by that phosphoinositide. Placing a PI(4,5)P2 near the substrate site allows for proper orientation of the enzyme on interfaces and should facilitate processive catalysis.

Quantifying transient interactions between Bacillus phosphatidylinositol-specific phospholipase-C and phosphatidylcholine-rich vesicles.

Bacillus thuringiensis secretes the virulence factor phosphatidylinositol-specific phospholipase C (BtPI-PLC), which specifically binds to phosphatidylcholine (PC) and cleaves GPI-anchored proteins off eukaryotic plasma membranes. To elucidate how BtPI-PLC searches for GPI-anchored proteins on the membrane surface, we measured residence times of single fluorescently labeled proteins on PC-rich small unilamellar vesicles (SUVs). BtPI-PLC interactions with the SUV surface are transient with a lifetime of 379 ± 49 ms. These data also suggest that BtPI-PLC does not directly sense curvature, but rather prefers to bind to the numerous lipid packing defects in SUVs. Despite this preference for defects, all-atom molecular dynamics simulations of BtPI-PLC interacting with PC-rich bilayers show that the protein is shallowly anchored with the deepest insertions ∼18 Å above the bilayer center. Membrane partitioning is mediated, on average, by 41 hydrophobic, 8 hydrogen-bonding, and 2 cation-π (between PC choline headgroups and Tyr residues) transient interactions with phospholipids. These results lead to a quantitative model for BtPI-PLC interactions with cell membranes where protein binding is mediated by lipid packing defects, possibly near GPI-anchored proteins, and the protein diffuses on the membrane for ∼100-380 ms, during which time it may cleave ∼10 GPI-anchored proteins before dissociating. This combination of short two-dimensional scoots followed by three-dimensional hops may be an efficient search strategy on two-dimensional surfaces with obstacles.

Cytotoxic amphiphiles and phosphoinositides bind to two discrete sites on the Akt1 PH domain.

The mechanism of binding of two promising anticancer agents (the cytotoxic alkylphospholipids perifosine and miltefosine) to the Akt PH domain is investigated by high-resolution field-cycling (31)P nuclear magnetic resonance (NMR) spectroscopy using a spin-labeled recombinant PH domain. These results strongly indicate that there are two discrete amphiphile binding sites on the domain: (i) the cationic site that binds phosphoinositides and some alkylphospholipids and (ii) a second site that is occupied by only the alkylphospholipids. The identification of this second site for amphiphiles on the Akt1 PH domain provides a new target for drug development as well as insights into the regulation of the activity of the intact Akt1 protein. The field-cycling NMR methodology could be used to define discrete phospholipid or amphiphile binding sites on a wide variety of peripheral membrane proteins.

Does changing the predicted dynamics of a phospholipase C alter activity and membrane binding?

The enzymatic activity of secreted phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes is associated with bacterial virulence. Although the PI-PLC active site has no obvious lid, molecular-dynamics simulations suggest that correlated loop motions may limit access to the active site, and two Pro residues, Pro(245) and Pro(254), are associated with these correlated motions. Whereas the region containing both Pro residues is quite variable among PI-PLCs, it shows high conservation in virulence-associated, secreted PI-PLCs that bind to the surface of cells. These regions of the protein are also associated with phosphatidylcholine binding, which enhances PI-PLC activity. In silico mutagenesis of Pro(245) disrupts correlated motions between the two halves of Bacillus thuringiensis PI-PLC, and Pro(245) variants show significantly reduced enzymatic activity in all assay systems. PC still enhanced activity, but not to the level of wild-type enzyme. Mutagenesis of Pro(254) appears to stiffen the PI-PLC structure, but experimental mutations had minor effects on activity and membrane binding. With the exception of P245Y, reduced activity was not associated with reduced membrane affinity. This combination of simulations and experiments suggests that correlated motions between the two halves of PI-PLC may be more important for enzymatic activity than for vesicle binding.

Competition between anion binding and dimerization modulates Staphylococcus aureus phosphatidylinositol-specific phospholipase C enzymatic activity.

BACKGROUND: Bacterial phosphatidylinositol-specific phospholipase C targets PI and glycosylphosphatidylinositol-linked proteins of eukaryotic cells. RESULTS: Functional relevance of a homodimeric S. aureus PI-PLC crystal structure is supported by enzyme kinetics and mutagenesis. Nonsubstrate phosphatidylcholine increases activity by facilitating enzyme dimerization. CONCLUSION: Activating transient dimerization is antagonized by anions binding to a discrete site. SIGNIFICANCE: Interplay of protein oligomerization and anion binding controls enzyme activity. Staphylococcus aureus phosphatidylinositol-specific phospholipase C (PI-PLC) is a secreted virulence factor for this pathogenic bacterium. A novel crystal structure shows that this PI-PLC can form a dimer via helix B, a structural feature present in all secreted, bacterial PI-PLCs that is important for membrane binding. Despite the small size of this interface, it is critical for optimal enzyme activity. Kinetic evidence, increased enzyme specific activity with increasing enzyme concentration, supports a mechanism where the PI-PLC dimerization is enhanced in membranes containing phosphatidylcholine (PC). Mutagenesis of key residues confirm that the zwitterionic phospholipid acts not by specific binding to the protein, but rather by reducing anionic lipid interactions with a cationic pocket on the surface of the S. aureus enzyme that stabilizes monomeric protein. Despite its structural and sequence similarity to PI-PLCs from other Gram-positive pathogenic bacteria, S. aureus PI-PLC appears to have a unique mechanism where enzyme activity is modulated by competition between binding of soluble anions or anionic lipids to the cationic sensor and transient dimerization on the membrane.

Structure of the S. aureus PI-specific phospholipase C reveals modulation of active site access by a titratable π-cation latched loop.

Staphylococcus aureus secretes a phosphatidylinositol-specific phospholipase C (PI-PLC) as a virulence factor that is unusual in exhibiting higher activity at acidic pH values than other enzymes in this class. We have determined the crystal structure of this enzyme at pH 4.6 and pH 7.5. Under slightly basic conditions, the S. aureus PI-PLC structure closely follows the conformation of other bacterial PI-PLCs. However, when crystallized under acidic conditions, a large section of mobile loop at the αß-barrel rim in the vicinity of the active site shows ~10 Å shift. This loop displacement at acidic pH is the result of a titratable intramolecular π-cation interaction between His258 and Phe249. This was verified by a structure of the mutant protein H258Y crystallized at pH 4.6, which does not exhibit the large loop shift. The intramolecular π-cation interaction for S. aureus PI-PLC provides an explanation for the activity of the enzyme at acid pH and also suggests how phosphatidylcholine, as a competitor for Phe249, may kinetically activate this enzyme.

Itaconic acid is a mammalian metabolite induced during macrophage activation.

Itaconic acid (ITA), or methylenesuccinic acid, is not generally classified as a mammalian metabolite. Using NMR-based metabolomics and (13)C-labeling, we have detected ITA in both macrophage-like VM-M3 and RAW 264.7 tumor cell lines as well as stimulated and unstimulated primary murine macrophages. Macrophage activation by addition of lipopolysaccharide and IFN-γ markedly increased ITA production and secretion. Crude cell extracts synthesize ITA via decarboxylation of cis-aconitate, indicative of a novel mammalian cis-aconitic decarboxylase activity. Our results highlight a previously unidentified biosynthetic pathway related to TCA cycle metabolism in mammalian cells and a novel metabolite that likely plays a role in macrophage-based immune response.

A molecular-imprint nanosensor for ultrasensitive detection of proteins.

Molecular imprinting is a technique for preparing polymer scaffolds that function as synthetic receptors. Imprinted polymers that can selectively bind organic compounds have proven useful in sensor development. Although creating synthetic molecular-imprinting polymers that recognize proteins remains challenging, nanodevices and nanomaterials show promise in this area. Here, we show that arrays of carbon-nanotube tips with an imprinted non-conducting polymer coating can recognize proteins with subpicogram per litre sensitivity using electrochemical impedance spectroscopy. We have developed molecular-imprinting sensors specific for human ferritin and human papillomavirus derived E7 protein. The molecular-imprinting-based nanosensor can also discriminate between Ca(2+)-induced conformational changes in calmodulin. This ultrasensitive, label-free electrochemical detection of proteins offers an alternative to biosensors based on biomolecule recognition.

Understanding the stereospecific interactions of 3-deoxyphosphatidylinositol derivatives with the PTEN phosphatase domain.

PTEN is an important control element of PI3K/AKT signaling involved in controlling the processes of embryonic development, cell migration and apoptosis. While its dysfunction is implicated in a large fraction of cancers, PTEN activity in the same pathway may also contribute to metabolic syndromes such as diabetes. In those cases, selective inhibitors of PTEN may be useful. A new class of chiral PTEN inhibitors based on the 3-deoxy-phosphatidylinositol derivatives was recently identified (Wang et al. [17]). However, lack of detailed understanding of protein-ligand interactions has hampered efforts to develop effective agonists or antagonists of PTEN. Here, we use computational modeling to characterize the interactions of the diverse 3-deoxyphosphatidylinositol inhibitors with the PTEN protein. We show that, while each of the compounds binds with the inositol headgroup inserting into the proposed active site of the PTEN phosphatase domain, hydrogen bonding restrictions lead to distinct binding geometries for ligand pairs of opposite chirality. We furthermore demonstrate that the binding modes differ primarily in the orientation of acyl tails of the ligands and that the activity of the compounds is primarily controlled by the effectiveness of tail-protein contacts. These findings are confirmed by binding affinity calculations which are in good agreement with experiment. Finally, we show that while more potent d-series ligands bind in a manner similar to that of the native substrate, an alternate hydrophobic pocket suitable for binding the opposite chirality l-series inhibitors exists, offering the possibility of designing highly selective PTEN-targeting compounds.

Enzymology with a spin-labeled phospholipase C: soluble substrate binding by 31P NMR from 0.005 to 11.7 T.

31P NMR relaxation studies from 0.005 to 11.7 T are used to monitor water-soluble inositol 1,2-(cyclic) phosphate (cIP) binding to phosphatidylinositol-specific phospholipase C spin-labeled at H82C, a position near the active site of the enzyme, and to determine how activating phosphatidylcholine (PC) molecules affect this interaction. We show that, in the absence of an interface, cIP binding to the protein is not rate-limiting, and that lower activation by PC vesicles as opposed to micelles is likely due to hindered product release. The methodology is general and could be used for determining distances in other weakly binding small molecule ligand-protein interactions.

Correlation of vesicle binding and phospholipid dynamics with phospholipase C activity: insights into phosphatidylcholine activation and surface dilution inhibition.

The enzymatic activity of the peripheral membrane protein, phosphatidylinositol-specific phospholipase C (PI-PLC), is increased by nonsubstrate phospholipids with the extent of enhancement tuned by the membrane lipid composition. For Bacillus thuringiensis PI-PLC, a small amount of phosphatidylcholine (PC) activates the enzyme toward its substrate PI; above 0.5 mol fraction PC (XPC), enzyme activity decreases substantially. To provide a molecular basis for this PC-dependent behavior, we used fluorescence correlation spectroscopy to explore enzyme binding to multicomponent lipid vesicles composed of PC and anionic phospholipids (that bind to the active site as substrate analogues) and high resolution field cycling 31P NMR methods to estimate internal correlation times (tauc) of phospholipid headgroup motions. PI-PLC binds poorly to pure anionic phospholipid vesicles, but 0.1 XPC significantly enhances binding, increases PI-PLC activity, and slows nanosecond rotational/wobbling motions of both phospholipid headgroups, as indicated by increased tauc. PI-PLC activity and phospholipid tauc are constant between 0.1 and 0.5 XPC. Above this PC content, PI-PLC has little additional effect on the substrate analogue but further slows the PC tauc, a motional change that correlates with the onset of reduced enzyme activity. For PC-rich bilayers, these changes, together with the reduced order parameter and enhanced lateral diffusion of the substrate analogue in the presence of PI-PLC, imply that at high XPC, kinetic inhibition of PI-PLC results from intravesicle sequestration of the enzyme from the bulk of the substrate. Both methodologies provide a detailed view of protein-lipid interactions and can be readily adapted for other peripheral membrane proteins.

Modulation of Bacillus thuringiensis phosphatidylinositol-specific phospholipase C activity by mutations in the putative dimerization interface.

Cleavage of phosphatidylinositol (PI) to inositol 1,2-(cyclic)-phosphate (cIP) and cIP hydrolysis to inositol 1-phosphate by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C are activated by the enzyme binding to phosphatidylcholine (PC) surfaces. Part of this reflects improved binding of the protein to interfaces. However, crystallographic analysis of an interfacially impaired phosphatidylinositol-specific phospholipase (W47A/W242A) suggested protein dimerization might occur on the membrane. In the W47A/W242A dimer, four tyrosine residues from one monomer interact with the same tyrosine cluster of the other, forming a tight dimer interface close to the membrane binding regions. We have constructed mutant proteins in which two or more of these tyrosine residues have been replaced with serine. Phospholipid binding and enzymatic activity of these mutants have been examined to assess the importance of these residues to enzyme function. Replacing two tyrosines had small effects on enzyme activity. However, removal of three or four tyrosine residues weakened PC binding and reduced PI cleavage by the enzyme as well as PC activation of cIP hydrolysis. Crystal structures of Y247S/Y251S in the absence and presence of myo-inositol as well as Y246S/Y247S/Y248S/Y251S indicate that both mutant proteins crystallized as monomers, were very similar to one another, and had no change in the active site region. Kinetic assays, lipid binding, and structural results indicate that either (i) a specific PC binding site, critical for vesicle activities and cIP activation, has been impaired, or (ii) the reduced dimerization potential for Y246S/Y247S/Y248S and Y246S/Y247S/Y248S/Y251S is responsible for their reduced catalytic activity in all assay systems.

Listeria monocytogenes phosphatidylinositol-specific phospholipase C: Kinetic activation and homing in on different interfaces.

The phosphatidylinositol-specific phospholipase C (PI-PLC) from Listeria monocytogenes forms aggregates with anionic lipids leading to low activity. The specific activity of the enzyme can be enhanced by dilution of the protein or by addition of both zwitterionic and neutral amphiphiles (e.g., diheptanoylphosphatidylcholine or Triton X-100) or 0.1-0.2 M inorganic salts. Activation by amphiphiles occurs with both micellar (phosphatidylinositol dispersed in detergents) and monomeric [dibutroylphosphatidylinositol (diC(4)PI)] phosphotransferase substrates and inositol 1,2-(cyclic)-phosphate (cIP), the phosphodiesterase substrate. The presence of zwitterionic and neutral amphiphiles (to which the protein binds weakly) dilutes the surface concentration of the interfacial anionic substrate and thereby reduces the level of enzyme-phospholipid particle aggregation. Zwitterionic amphiphiles also can bind directly to the protein and enhance catalysis since they enhance both diC(4)PI and cIP hydrolysis. In contrast to activation by amphiphiles, the rate enhancement by salt occurs for only the phosphotransferase step of the reaction. Added salt has a synergistic effect with zwitterionic phospholipids, leading to high specific activities for PI cleavage with only moderate dilution of the anionic substrate in the interface. This kinetic activation correlates with weakening of strong PI-PLC hydrophobic interactions with the interface as monitored by a decrease in the maximum monolayer surface pressure for insertion of the protein. Several point mutations of surface hydrophobic residues (W49A, L51A, L235A, and F237W) can dramatically alter the unusual kinetics of this secreted enzyme. The high affinity of PI-PLC for anionic phospholipids along with a strong hydrophobic interaction, which gives rise to the unusual kinetic behavior, is considered in terms of how it might contribute to the role of this phospholipase in L. monocytogenes infectivity.

Insights into the structural specificity of the cytotoxicity of 3-deoxyphosphatidylinositols.

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.