Thesis advisor: Jianmin Gao / Phosphatidylinositol-specific phospholipase C (PI-PLC) cleaves the substrate phosphatidylinositol through two steps: the first step occurs in the interface between lipid and solution, while the second step only takes place in water soluble environment. For interfacial catalysis, the enzyme should bind to the lipid surface first before engaging its substrate, therefore interfacial kinetics include both interfacial binding and an interfacial catalytic step. The Bacillus thuringiensis PI-PLC is activated by binding to zwitterionic surfaces; phosphatidylcholine (PC) and two tryptophan residues (Trp47 in the two-turn helix B and Trp242 in a disordered loop) at the rim of the barrel structure, in particular, are critical for this interaction. The helix B region in PI-PLC orients the side chains of Ile43 and Trp47 so that they form a hydrophobic protrusion from the protein surface that likely facilitates initial membrane binding. An earlier crystal structure of the dimeric W47A/W242A mutant, which is unable to bind to PC, showed that the helix B region was reorganized into an extended loop. Whether this conformational change occurred in the wild type PI-PLC was tested with a series of mutations targeting helix B residues and surrounding regions. 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 likely to play a role in the activated PI-PLC conformation. Besides the helix B residues, a number of hydrophobic residues along the rim of the <em>f</em>Ñ<em>f</em>Ò-barrel and close to both helix B and the active site were also altered to assess their contribution to membrane binding and kinetic activation. Results showed that Tyr86 and Tyr88, but not Tyr118, contribute to the protein binding to PC vesicles. These residues are capable of cation-<em>f</em>à interactions with the choline headgroup of the phospholipid PC. Although mammalian PLC<em>f</em>Ô1 is a complex multidomain protein, the catalytic domain resembles the bacterial PI-PLC enzymes. Little work has been done to characterize the extent to which this domain contributes to membrane binding. A mutated protein that removes the very anionic X/Y linker region that covers the active site was constructed. The interfacial binding and the corresponding enzyme activity of this mutant against WT were measured in both micelles and large unilamellar vesicles. The results showed at <em>f</em>ÝM protein concentration there was no large difference between the PLC<em>f</em>Ô1 and the deletion mutant in terms of vesicle binding. However, the deletion mutant showed much higher membrane binding affinity at nM concentrations. These results shed some light on the activation or inhibition role of the catalytic domain and pointed to a possible direction of future studies, for example examining specific mutant enzymes in the interfacial loop region. / Thesis (PhD) — Boston College, 2011. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Thesis advisor: Mary F. Roberts / Phosphatidylinositol-specific phospholipase C (PI-PLC) from B. thuringiensis is activated by phosphatidylcholine (PC) surfaces for both phosphatidylinositol (PI) cleavage to inositol 1,2-(cyclic)-phosphate (cIP) and subsequent hydrolysis of cIP to inositol-1-phosphate. These enzyme kinetics strongly suggest that this PI-PLC has two discrete binding sites for phospholipids - the active site binding PI (or substrate competitors) and an activator site specific for PC. However, it is difficult to determine the orientation and conformation of peripheral membrane proteins when docked to target membranes, let alone where sites for these might be on the protein. In this thesis, various biophysical techniques were applied to this bacterial PI-PLC to obtain structural information in the absence and presence of membranes to characterize specific conformational changes that occur when the protein binds to activating membranes. The crystal structures of an interfacially impaired double mutant of PI-PLC, W47A/W242A, was solved and showed the protein as a homodimer. The major interactions came from four clustered surface tyrosine residues from each monomer. This structure suggested the possibility of PI-PLC dimerization on membrane surfaces as part of the mechanism for interfacial activation. Mutations of these tyrosines showed a loss of activity and membrane binding. Crystal structures of these mutant proteins showed no significant change in the proteins, consistent with either disruption of a dimerization interface of a specific PC binding motif. FRET was used to try and monitor oligomerization of PI-PLC, derivatized on a cysteine introduced at residue 280 (W280C) with either a donor or acceptor fluorophore, on vesicle surfaces. The results suggested some specific aggregation could occur on very PC-rich surfaces but not on phospholipid vesicles with at least 50 mol% anionic phospholipids, strongly suggesting that a stable dimer was not forming when the enzyme was bound to vesicles mimicking conditions where enzyme specific activity is high. If dimerization occurs on surfaces, it must be transient. To examine which portions of the PI-PLC are interacting with membrane and to further explore if there is any evidence for PI-PLC dimerization on membrane surface, deuterium exchange coupled by mass spectrometry experiments were carried out with wild type PI-PLC, W47A/W242A and a covalent dimer formed from W242C that is more active than wild type enzyme. Results showed (i) a stable short helix B (containing an exposed tryptophan thought to insert into membranes) in wild type PI-PLC and its complete destabilization in W47A/W242C, (ii) a flexible surface loop (containing another tryptophan thought to partition into the membrane) that became protected when the protein was bound to vesicles, and (iii) reduced deuterium exchange for the peptide containing the tyrosines that either mediate transient dimerization or form a PC binding site.. These observations modify how we envision the protein anchoring to substrate-containing membranes. / Thesis (PhD) — Boston College, 2010. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

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.

Thesis advisor: Mary F. Roberts / Secreted phosphatidylinositol-specific phospholipase Cs (PI-PLCs) are often virulence factors in pathogenic bacteria. Understanding how these enzymes interact with target membranes may provide novel methods to control bacterial infections. In this work, two typical PI-PLC enzymes, from Bacillus thuringiensis (Bt) and Staphylococcus aureus (Sa), were studied and their membrane binding properties were examined and correlated with enzymatic activity. BtPI-PLC is kinetically activated by allosteric binding of a phosphatidylcholine (PC) molecule. MD simulations of the protein in solution suggested correlated loop and helix motions around the active site could regulate BtPI-PLC activity. Vesicle binding and enzymatic studies of variants of two proline residues, Pro245 and Pro254, that were associated with these motions showed that loss of the correlated motions between the two halves of PI-PLC were more critical for enzymatic activity than for vesicle binding. Furthermore, loss of enzyme activity could be rescued to a large extent with PC present in a vesicle. This suggests that binding to PC changes the enzyme conformation to keep the active site accessible. SaPI-PLC shows 41.3% sequence similarity with BtPI-PLC but has very different ways its activity is regulated. While it is kinetically activated by PC it does not in fact bind to that phospholipid. Enzymatic and membrane interaction assays showed that SaPI-PLC has evolved a complex, apparently unique way to control its access to PI or GPI-anchored substrate. (i) An intramolecular cation-pi latch facilitates soluble product release under acidic conditions without dissociation from the membrane. (ii) There is a cationic pocket on the surface of enzyme that likely modulates the location of the protein. (iii) Dimerization of protein is enhanced in membranes containing phosphatidylcholine (PC), which acts not by specifically binding to the protein, but by reducing anionic lipid interactions with the cationic pocket that stabilizes monomeric protein. SaPI-PLC activity is modulated by competition between binding of soluble anions or anionic lipids to the cationic sensor and transient dimerization on the membrane depleted in anionic phospholipids. This protein also served as a way to test the hypothesis that a cation-pi box provides for PC recognition site. This structural motif was engineered into SaPI-PLC by forming N254Y/H258Y. This variant selectively binds PC-enriched vesicles and the enzyme binding behavior mimics that of BtPI-PLC. Itaconic acid (ITA) is a metabolite synthesized in macrophages and related cell lines by a cis-aconitate decarboxylase (cADC). cADC activity is dramatically increased upon macrophage stimulation. In this work, the cell line RAW264.7 was used to show that cADC activity upon stimulation requires de novo protein synthesis. MS analyses of partially purified RAW264.7 protein extracts from stimulated cells show a large increase for immunoresponsive gene 1 protein (IRG1) and siRNA knockdown of the IRG1 reduces cADC activity upon stimulation. Suspected active site residues of IRG1 were identified by mutagenesis studies of the recombinant protein based on a homology structure model of fungal cADC. The cloning and overexpression of this enzyme should help clarify the cofactor-independent decarboxylation mechanism of this mammalian enzyme as well as open up future studies into the specific role of ITA in the mammalian immune system and cancers. / Thesis (PhD) — Boston College, 2013. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

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.