Indiana University-Purdue University Indianapolis (IUPUI)

Phosphoinositide-specific phospholipase C (PLC) is involved in Ca2+ mediated signalling events that lead to altered cellular status. PLC activation causes hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) and generates two second messengers, inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol. Each has distinct role in depending on the cell type in mammalian cells, IP3 binds to intracellular receptors, stimulating the release of sequestered Ca2+. DAG remains in the membrane, where it can activate members of the protein kinase C (PKC) family. In plant absence of PKC keeps the question open as to what is the role of DAG in plants. The role of IP3 apart form triggering calcium release is not known, although the phosphorylated product of IP3 by groups of kinases has been implicated in certain nuclear signalling pathway. Using various sequence-analysis methods on plant PLC sequences, we identified two conserved motifs in known PLC sequences. The identified motifs are located in the C2 domain of plant PLCs and are not found in any other protein. These motifs are specifically found in the Ca2+ binding loops and form adjoining beta strands. Further, we identified certain conserved residues that are highly distinct from corresponding residues of animal PLCs. The motifs reported here could be used to annotate plant-specific phospholipase C sequences. Furthermore, we demonstrated that the C2 domain alone is capable of targeting PLC to the membrane in response to a Ca2+ signal. We also showed that the binding event results from a change in the hydrophobicity of the C2 domain upon Ca2+ binding. Bioinformatic analyses revealed that all PLCs from Arabidopsis and rice lack a transmembrane domain, myristoylation and GPI-anchor protein modifications. Our bioinformatic study indicates that plant PLCs are located in the cytoplasm, the nucleus and the mitochondria. Our results suggest that there are no distinct isoforms of plant PLCs, as have been proposed to exist in the soluble and membrane associated fractions. The same isoform could potentially be present in both subcellular fractions, depending on the calcium level of the cytosol. we have used Saccharomyces cerevisiae as a model system to investigate physiological function of PLC in regulation of lipid metabolism. S. cerevisiae synthesizes membrane phospholipids via a pathway which appears to be similar to that of higher eukaryotes. The synthesis of glycerolipid begins with the formation of phosphatidic acid which is quantitatively a minor lipid but is responsible for the repression of UNAINO-containing phospholipid biosynthetic gene by governing localization of Opi1. When the levels of phosphatidic acid are lowered which causes translocation of Opi1 from endoplasmic reticulum membrane to nucleus, where it binds to INO2 of the INO2-INO4 activator complex thereby attenuating transcriptional activation. The expression of phospholipid biosynthetic gene is affected by many conditions which include carbon source, nutrient availability, growth stage, pH and temperature. The well studied conditions which regulate phospholipid biosynthetic genes transcription are through exogenous supplementation of inositol, which is achieved by lowering of phosphatidic acid levels by its utilization for the synthesis of phosphatidylinositol. Since inositol was able to change regulates phospholipid biosynthetic gene we proposed to investigate inositol triphosphate role in such regulation. We overexpressed a plant phospholipase C in yeast to study its effect on lipid biosynthesis. The overexpressed yeast cells were subjected to microarray analysis and the result were confirmed by Q-PCR. The result obtained indicated that there was decrease in the expression of UNAINO-containing genes. To further validate our observation we carried out an in vivo assay to determined activity of enzyme involved in phospholipid biosynthesis. These results were in accordance with our expression analysis further supporting our hypothesis. Our study indicates that phospholipase c regulates phospholipid biosynthesis at transcription level in response to various stimuli. Overall, these data suggest that the C2 domain of plant PLC plays a vital role in calcium signalling. Further it can be inferred from this study that PI-PLC regulates lipid metabolism in S. cerevisiae.

<p>The neuropeptides orexin-A and -B act as endogenous ligands for G-protein-coupled receptors (GPCRs) called OX₁ and OX₂ receptors. Previous observations have established that orexin receptors have an ability to couple to different G-proteins and signaling pathways and induce Ca²⁺ elevations via both receptor-operated Ca²⁺ channels (ROCs) and store-operated Ca²⁺ channels (SOCs). This thesis further elucidates the intracellular signaling mechanisms of orexin receptors.</p><p>Orexin receptors were shown to activate ERK (extracellular signal-regulated kinase) via Ras, protein kinase C, phosphatidylinositol-3 kinase and Src. Ca²⁺ influx was shown to be obligatory for the activation of ERK and adenylyl cyclase, wherewith a hypothesis was formed that submembrane Ca²⁺ elevation is of central importance for the regulation of orexin receptors' coupling to different signaling pathways. This was further investigated with respect to OX₁R-mediated activation of phospholipase C (PLC) showing that ROC influx was of more central importance for the OX₁R signaling, but also SOCs amplified PLC activity. A technique to block OX₁R-induced IP₃increase and subsequent Ca²⁺ release was devised, leaving ROC influx as the only source of Ca²⁺ elevation upon OX₁R activation. This block had no effect on OX₁R-mediated activation of ERK, showing that ROC-dependent influx is the most central Ca²⁺ elevating process in OX₁R signaling. OX₁Rs' coupling to PLC was further investigated by measuring the metabolites generated, inositol phosphates and diacylglycerol (DAG). The results indicate involvement of two different PLC activities with different substrate specificities, which results in, at low orexin-A concentrations, DAG production without concomitant production of IP₃. At even lower orexin-A concentrations, OX₁Rs generate DAG by activating phospholipase D. In conclusion, the results strengthen the hypothesis that ROCs have a central role in orexin receptor signaling and DAG may be the signal of preference.</p>

The 5-HT2A receptor (5-HT2AR) is implicated in neuropsychiatric disorders such as schizophrenia and is thought to mediate the actions of a number of hallucinogenic and antipsychotic drugs. Additionally, certain polymorphic variants of the receptor, such as an allele resulting in substitution of amino acid 452 histidine (H) with tyrosine (Y), have been linked to schizophrenia or altered therapeutic response to antipsychotics. The 5-HT2AR utilises various intracellular signalling pathways, including the activation of phospholipase C (PLC) and phospholipase D (PLD) via recruitment of the small G-protein ADP-Ribosylation Factor (ARF). This thesis focusses on protein:protein interactions and signalling mechanisms of the 5-HT2AR and H452Y-5-HT2AR receptor variant. Both ARF1 and PLD1 have previously been shown to bind to the carboxy-terminal tail (ct) of the 5-HT2AR. In chapter three it is demonstrated that the 5-HT2AR can activate PLD in an ARF-dependent manner, primarily through the PLD1 isoform. GST-fusion proteins of truncated and mutated variants of the receptor ct are used to show that ARF1 and PLD1 independently bind to distinct sites. Coimmunoprecipitation, GST-fusion protein studies and PLD activity assays demonstrate that the introduction of the H452Y mutation decreases the physical interactions between the receptor and PLD1, as well as decreasing 5-HT2ARmediated PLD activation. In chapter four, potential mechanisms of wild-type and H452Y-5-HT2AR desensitisation are explored. It is shown that β-arrestin 2 (β-arr 2) confers a decrease in H452Y-5-HT2AR-mediated PLC activity, despite having no significant effect upon wild-type 5-HT2AR responses. The H452Y-5-HT2AR variant is also shown, by GST-fusion protein studies, to bind β-arr 2 more strongly. The H452Y-5-HT2AR additionally mediates increased levels of 5-HT-induced ERK phosphorylation compared to the wild type 5-HT2AR, potentially through increased scaffolding of ERK activation complexes by receptor-bound β-arr 2. Chapter five focusses on possible interactions of the 5-HT2AR with the Ca2+-binding proteins annexin A2, S100B and the annexin A2 partner p11, together with the functional consequences of these interactions. Co-immunoprecipiation and GST-fusion protein studies show that annexin A2 binds specifically to the 5-HT2AR ct. Furthermore, annexin A2 (but not S100B or p11) is shown to result in an amplification of 5-HT2AR-mediated PLC responses. These findings provide a greater insight into some of the signal transduction mechanisms of the 5-HT2AR and their perturbation in the H452Y polymorphic form of the receptor, and understanding of the molecular mechanisms underlying neuropsychiatric diseases in patient subgroups, potentially leading to improved therapeutic treatments.

<p>Temporal lobe epilepsy is a common and devastating disorder that features recurrent seizures and is often associated with pathologic anxiety and hippocampal sclerosis. An episode of prolonged seizures (status epilepticus) is thought to promote development of human temporal lobe epilepsy years later. A chemical-genetic approach established proof of concept that transiently inhibiting the receptor tyrosine kinase, TrkB, following status epilepticus prevented epilepsy, anxiety-like behavior and hippocampal damage in a mouse model, providing rationale for developing a therapeutic targeting TrkB signaling. To circumvent the undesirable consequence that global inhibition of TrkB exacerbates neuronal degeneration following status epilepticus, we sought to identify both the TrkB-activated signaling pathway mediating these pathologies and a compound that uncouples TrkB from the responsible signaling effector. To accomplish these goals, we used genetically modified mice and a model of seizures and epilepsy induced by a chemoconvulsant. Genetic inhibition of TrkB-mediated phospholipase C gamma 1 (PLC gamma 1) signaling suppressed seizures induced by a chemoconvulsant, leading to design of a peptide (pY816) that inhibited the interaction of TrkB with PLC gamma 1. We demonstrate that pY816 selectively inhibits TrkB-mediated activation of PLC gamma 1 both in vitro and in vivo. Treatment with pY816 prior to administration of a chemoconvulsant suppressed seizures in a dose- and time-dependent manner. Treatment with pY816 initiated after chemoconvulsant-evoked status epilepticus and continued for just three days suppressed seizure-induction of epilepsy, anxiety-like behavior and hippocampal damage assessed months later. This study elucidates the signaling pathway by which TrkB activation produces diverse neuronal activity-driven pathologies and demonstrates therapeutic benefits of an inhibitor of this pathway in an animal model in vivo. A strategy of uncoupling a receptor tyrosine kinase from a signaling effector may prove useful in diverse diseases in which excessive receptor tyrosine kinase signaling contributes.</p> / Dissertation

One purpose of this investigation is to determine the fatty acid and lipid content of typical Vibrio cholerae cells. The comparison of cholera lipid constituents with those of closely-related bacteria might be of taxonomic value. Furthermore, chemical characterization of the cholera vibrio could provide useful criteria for identification of these disease-producing microorganisms.

Thesis advisor: Mary F. Roberts / Phosphatidylinositol-specific phospholipase C (PI-PLC) is an enzyme important in membrane-associated signal transduction in eukaryotes, and pathogenic factors in bacteria. It catalyzes the conversion of PI to DAG and cIP, which is further converted to I-1-P. The phospholipid PC has been shown to activate cIP hydrolysis. EPR and NMR were used to examine PI-PLC binding to PC and PMe vesicles through the use of spin labels attached to cysteine mutants. It was concluded that the spin label interacted more significantly with the phosphorus of PC than that of PMe. The results also suggested the -OCH3 group was preferred over the -N(CH3)3 group, and that the protein penetrated into the bulk methylene region of the phospholipid bilayer. / Thesis (BS) — Boston College, 2005. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Chemistry. / Discipline: College Honors Program.

Pathogenic bacteria secrete various virulence factors as their biochemical weapons to gain access to and destroy the target cells. They can directly interact with the outer lipid bilayer membrane of eukaryotic cells, inducing the premature cell death by either apoptosis or necrosis. Such virulence factors account for much of the toxic actions associated with bacterial infection; therefore the detection of such proteins could provide a methodology for sensing/detection of pathogenic bacteria in, for example, food or human tissue. Detection and identification of pathogenic bacteria by conventional methods such as plating and counting in laboratory is expensive and time consuming. With growing concerns over emergence and re-emergence of pathogenic bacteria with high resistant to current antibiotics, there is a potential need for effective detection of pathogenic toxins invitro. On the other hand, artificially prepared lipid bilayer membrane on planar metallic surfaces provides the cell membrane mimics which are extremely useful in exploring the cellular functions and processes at the molecular level. Therefore in this work, an application of planar tethered bilayer lipid membrane (pTBLM) as a biomimetic sensing platform for the detection of clinically important pathogens, Staphylococcus aureus and Pseudomonas aeruginosa via their secreted virulence factors was presented. Planar TBLM was modified by incorporation of cholesterol and detection of bacterial toxins at human body temperature was examined by impedance and surface plasmon resonance methods. The results of pathogenic bacterial toxin detection were compared with those of Escherichia coli (DH5α), the human gut normal flora with non-pathogenic strain, as a control. Additionally pTBLM was transferred onto single nanoporous Si3N4 membrane to enhance the toxin sensitivity and extend the lifetime for the possible realization of future membrane chips for ion channel characterizations and drug screenings. Then the single ion channel measurement was demonstrated with nanopore-suspended TBLM (Nano-psTBLM) using α-toxin of S. aureus. The results presented in this work therefore, may pave the more effective and efficient ways for future pathogenic bacterial detection in which the sensing mechanism was solely based on the nature of interactions as well as modes of action between bacterial toxins and artificial lipid bilayer membranes.

"When cells are exposed to environmental stress, they respond by compartmentalizing mRNA and translation proteins in stress granulates to protect mRNA. However, the mechanism through which external stress is communicated into the cell to form stress granules is unknown. Phospholipase Cβ (PLCβ) is activated by Gq on the plasma membrane in response to sensory stimuli to initiate calcium signals resulting in a variety of cellular responses. Here, we show that PLCβ binds to major proteins that organize stress granules as well as the main component of the RNA-induced silencing machinery, Argonaute-2 (Ago2). Under stress, PLCβ moves from the plasma membrane to the cytosol to escort Ago2 into stress granules and potentially inhibit mRNA degradation by regulating microRNAs (miRs) expression. Using a model muscle cell line functionally adapted to handle stress, we find that upon osmotic stress, the movement of PLCβ into the cytosol to move Ago2 into stress granules changes the population and distribution of miRs, and in particular, members of the let family. The impact of changes in let is to acutely affect glucose metabolism allowing cells to adapt to stress conditions. Our studies present a model in which PLCβ relays information about external stress to promote stress granule formation and protect mRNAs."

Neutrophils (PMN) and group IIA phospholipase A2 (gIIA PLA2) are components of the innate immune system mobilized to sites of invasion by microorganisms such as Staphylococcus aureus. Although accumulating coincidentally in vivo, the in vitro anti-staphylococcal activities of PMN and gIIA PLA2 have thus far been separately studied. The goal of this thesis was to study the collaborative activity of PMN and gIIA PLA2 against S. aureus. We have identified and characterized the collaboration of PMN and gIIA PLA2 against S. aureus ingested by PMN. PMN induced conversion of bacterial phosphatidylglycerol into cardiolipin, but were unable to degrade S. aureus phospholipids without gIIA PLA2. PMN reduced by 10-fold the concentration of gIIA PLA2 needed to digest bacterial phospholipids alone. In addition to increased phospholipid degradation, collaboration of PMN and gIIA PLA2 caused greater bacterial killing and greater loss of bacterial green fluorescent protein fluorescence. The collaboration of PMN and gIIA PLA2 against S. aureus is dependent on catalytic activity and is specific to gIIA PLA2 as related secretory PLA2, groups IB, V, and X, show little or no phospholipid degradation of S. aureus either alone or in the presence of PMN. Synergy of PMN and gIIA PLA2 requires a functional NADPH oxidase and phagocytosis. Although addition of gIIA PLA2 after phagocytosis causes some bacterial phospholipid degradation, the greatest effect is observed when gIIA PLA2 is added before phagocytosis. An extracellular source of H2O2 can partially restore antibacterial activities to NADPH oxidase deficient PMN including the ability to collaborate with gIIA PLA2, supporting a role for reactive oxygen species in NADPH oxidase dependent antimicrobial functions of PMN. In contrast, iberiotoxin, an inhibitor of BK potassium channels had no effect of PMN antibacterial activities. Although H2O2 partially restored antibacterial activity to NADPH oxidase deficient PMN, extracellular H2O2 was not sufficient to increase S. aureus to gIIA PLA2 activity. In summary, PMN and gIIA PLA2 collaborate against S. aureus. These findings revealed collaboration between cellular oxygen-dependent and extracellular oxygen-independent host defense systems that may be important in the ultimate resolution of S. aureus infections.