<p>The two lipid kinases, diacylglycerol kinase (DGK) and phosphatidylinositol 4-phosphate 5-kinase (PIP5K), are vital players of the phosphatidylinositol cycle. DGK regulates the intracellular balance between two important lipid signaling molecules, diacylglycerol and phosphatidic acid. PIP5K produces another key signal messenger, phosphatidylinositol 4,5-bisphosphate. We studied several fundamental aspects of DGK and PIP5K properties. We investigated the topology of the hydrophobic segment of FLAG-tagged DGK epsilon, and showed that a single amino acid mutation P32A caused the hydrophobic segment to favor a transmembrane orientation. We demonstrated that DGKε is localized in both the plasma membrane and endoplasmic reticulum. Our work helped to better elucidate the substrate specificity of DGKε and PIP5K isoforms, and it lead us to discover the motif that is common for several enzymes that exhibit specificity for substrates containing polyunsaturated fatty acids. We studied the organ distribution of murine DGK isoforms, and also expanded our knowledge of DGK expression in diabetic animals, showing that the expression profiles of several DGK isoforms are altered in adipocytes isolated from diabetic mice. Moreover, DGK expression profiles change dramatically during adipocyte differentiation. Taken together, our findings contribute to the growing knowledge about two enzymes, DGK and PIP5K, by providing the fundamental information about the structural and functional properties of these lipid kinases. Both PIP5K and DGK enzymes have a strong potential for use as drug targets. Although at present their clinical importance has not been completely assessed, we believe that their significance as drug targets will be recognized in the nearest future. <strong></strong></p> / Doctor of Philosophy (PhD)

Interactions of simvastatin with zwitterionic dipalmitoyl phosphotidylcholine (DPPC) multilamellar liposomes were investigated as a function of temperature and simvastatin concentration. And acyl chain length effect on the simvastatin-model membrane interactions was monitored with DPPC and dimyristoyl phosphotidylcholine (DMPC) lipids. All studies were carried out by two non-invasive techniques, namely Fourier transform infrared (FTIR) spectroscopy, and differential scanning calorimetry (DSC). The results showed that as simvastatin concentration increased, the main phase transition temperature decreased, the main phase transition curve broadened, and the characteristic pretransition was disappeared for both DMPC and DPPC model membranes. All concentrations of simvastatin disordered and decreased the fluidity of phospholipid membranes. Analysis of C=O stretching band showed that simvastatin either strengthen the existing hydrogen bonds of the glycerol skeleton closer to the head groups or caused the formation of new hydrogen bonds. A dehydration effect caused by simvastatin around the PO2- functional groups in the polar part of the lipids was monitored. This dehydration effect in the gel phase was more profound than in the liquid crystalline phase for 1, 6, and 12 mol% of simvastatin concentrations. DSC peaks broadened and shifted to lower temperature values by increasing the simvastatin concentration. For both lipids, simvastatin-induced lateral phase separation was observed in the DSC thermograms. Any change caused by the acyl chain length difference of DMPC and DPPC lipids was not observed on the simvastatin-membrane interactions. Also, for both of the lipids similar trends were observed in the FTIR and DSC results. More profound effects of simvastatin on the less stable DMPC membranes were observed.

The role of membrane-intrinsic enzymes of lipid metabolism in complex biological processes is being realized through comprehensive structure function studies. Detailed analysis of substrate-enzyme interactions occurring within the restrictive membrane environment has proved to be exceedingly challenging. Using detergent micelles, we describe a detailed model for substrate recognition and binding by the outer-membrane intrinsic enzyme PagP from Escherichia coli. PagP is an 8-stranded antiparallel β-barrel that transfers a palmitoyl group from a phospholipid molecule to lipid A, the endotoxin component of lipopolysaccharide. This simple modification provides bacterial resistance to host antimicrobial peptides and attenuates the inflammatory response signalled through the host toll-like receptor 4 pathway. We describe a molecular embrasure and a crenel, which display weakened transmembrane β-strand hydrogen bonding, to provide site-specific routes for lateral entry of substrates into the PagP active site. A Tyr147 localized to the L4 loop gates the entry of the phospholipid substrate through the crenel, while lipid A enters via the embrasure. The side chains of the catalytic residues that are located in the extracellular loops point towards the central axis of the enzyme, directly above the active site. An acyl-chain binding pocket known as the hydrocarbon ruler is buried within the transmembrane β-barrel structure, and is optimized to accommodate a 16-carbon saturated palmitate chain. The hydrocarbon ruler, therefore, accounts for PagP's stringent selectivity for a palmitate chain. Substituting Gly88 lining the floor of the hydrocarbon ruler with residues possessing linear, unbranched, aliphatic side chains changes the selectivity of PagP to utilize shorter acyl chains. The serendipitous discovery of an exciton interaction between Trp66 and Tyr26 at the floor of the hydrocarbon ruler provides an intrinsic spectroscopic probe to monitor the methylene unit acyl-chain resolution of PagP. A compromised acyl chain resolution of the Gly88Cys mutant is attributed to an unexpected decrease of the Cys sulfhydryl group pKa within the β-barrel interior, resulting in a burying of a charged thiolate within the PagP core. The structural perturbation associated with the Cys thiolate extinguishes the exciton and expands the acyl-chain selectivity. These molecular details of lateral lipid diffusion and acyl-chain selection provide the first such example for any membrane-intrinsic enzyme of lipid metabolism. / Thesis / Doctor of Philosophy (PhD)

<p>Phosphatidylinositol (PI) is a phospholipid that constitutes only a minor component of eukaryotic membranes. However, they are critical in many fundamental cellular processes, such as signal transduction pathways, vesicular trafficking and actin cytoskeletal dynamics. PI is highly enriched in specific acyl chains at both the <em>sn-1</em> and <em>sn-2</em> positions, the major species being 1-stearoyl-2-arachidonoyl. Enzymes required for PI synthesis are believed to play a major role in this enrichment through the selective catalysis of specific substrates. We have studied several aspects of two enzymes involved in PI synthesis, Diacylglycerol kinase ε (DGKε) and CDP-Diacylglycerol synthases (CDS). We have studied the role of the ATP-binding motif of DGKε and showed that this enzyme is not only required for enzymatic activity, but substrate specificity and sub-cellular localization. We have also looked at the region adjacent to the catalytic site, containing a cholesterol recognition motif, and determined that this also affects the enzymes activity and substrate specificity. Finally, we have characterized the enzymatic properties of two CDS isoforms <em>in vitro</em> and demonstrated that these isoforms exhibit different substrate specificities. Taken together, our results serve to further our understanding of both DGKε and CDS1/2 and their roles in PI synthesis and enrichment with specific acyl chains.</p> / Master of Science (MSc)