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Cell membranes are integral for many biological processes. In addition to containing and protecting cellular contents and maintaining the chemical integrity of the cell, these interfaces host a variety of ligand-receptor interactions. These ligand-receptor interactions are important for cell signaling and transport and the ability to monitor them is key to understanding these processes. In addition, therapeutics and drug discovery is also aided by membrane-specific study, as the majority of drugs target receptors associated with the cell surface. The cell membrane can be effectively mimicked by the use of supported lipid bilayers, which provide a robust platform exhibiting the lateral fluidity and composition associated with cell membranes. The ability to study both ligand-receptor interactions as well as small molecule-membrane interactions on these model membranes is aided by the fact that these assays can be multiplexed and are amenable to use with low sample volumes with high throughput. Our laboratory has recently developed a strategy for fluorescent microscopy studies of ligand-receptor interactions on supported lipid bilayers without the use of fluorescently-labeled analytes. This technique involves the incorporation of pH-sensitive fluorophores into the composition of the supported lipid bilayer as embedded reporter dyes. It was determined that this assay can operate as either a “turn-on” or a “turn-off” sensor depending on the analyte to be detected. It was additionally found that modulating the ionic strength of the operating buffer allows for tuning the operating pH and sensitivity of the assay. This label-free technique can be utilized to monitor small peptide interactions with bilayers containing specific phospholipids. Basic amino acid sequences which are associated with transporting contents across membranes or anti-microbial activity can be monitored binding to negatively charged bilayers without the use of labels. Not only is this a sensitive technique for detecting small peptides, but thermodynamic data can be extracted as well. In a final set of experiments, the interaction of proteins with phosphatidylserine (PS) in supported lipid bilayers is observed by utilizing PS-Cu2+-induced quenching of fluorophores. Disruption of this metal-phospholipid, specifically by Ca2+-dependent protein kinases, results in a turn-on fluorescent assay, which can be used to monitor the binding of the protein to PS and the effects of other metal interference.

Annual lipid cycles were determined for adult male and female Cnemidophorus tigris collected near El Paso, Texas during 1970-1971.

Calcium/phospholipid-dependent protein kinase (PKC) was partially purified from P1798 lymphosarcoma. Phospholipid-dependence was specific for phosphatidylserine. PKC phosphorylated Histone 1, with an apparent K_m of 14.1 μM. Chlorpromazine, a lipid-binding drug, inhibited PKC activity by 100%. Further studies were undertaken to establish analytical conditions which could be applied to the study of PKC in intact cells. The conditions included (1) determining optimum cell concentration for measuring PKC activity, (2) recovering PKC into the soluble fraction of cell extracts, (3) evaluating calcium and phospholipid requirements of PKC in this fraction, and (4) inhibiting PKC in this fraction. Final studies involved treatment of intact cells with potential activators. Both phytohaemagglutinin and a phorbol ester increased PKC activation.

Invadolysin (INV) is a member of the M8 family of metzincin metalloproteases. The gene was discovered in the Heck laboratory. Based on studies in Drosophila, INV is important for mitotic progression, nuclear envelope protein dynamics, and germ cell migration. INV-like immunoreactivity has shown its association with lipid droplets (LDs), which are intracellular organelles for lipid and protein storage. INV is the first metalloprotease found on LDs. Thus, INV’s role and LD-associated pathways are the puzzles we would like to investigate. The formation of LDs is dependent on the nutritional status of cells and starvation can disrupt the generation of LDs. Based on this concept, I established a starvation / re-feeding system. When nutrition is sufficient, LDs were surrounded by INV, whereas no INV or LDs were found in the majority of starved cells. With a supply of oleic acid (OA), LDs re-appeared and so did INV localized to LDs. In this system, inhibition of protein kinase C (PKC) disrupts INV’s re-localization to LDs. As I found INV to be phosphorylated by PKC in vitro (residues within the N-terminus might be phosphorylated by PKC), I conclude that PKC might regulate INV’s re-localization in the starvation / re-feeding system. 3T3-L1 mouse fibroblasts can differentiate into adipocytes in vitro; this is termed adipogenesis. Since INV is a LD associated protein, the role of INV in adipogenesis is of interest. INV localized on LDs in the early stage of differentiation but disassociated from LDs in mature adipocytes. The levels of INV mRNA and protein were significantly increased upon differentiation to adipocytes. On the other hand, INV decreased when adipocyte differentiation was inhibited by PKC and PI3K inhibitors, suggesting that the increase of INV is required for adipocyte differentiation. I was interested to examine the possible role of INV in InR/PI3K/Akt signalling, and therefore compared wild type with mutant INV (Drosophila INV4Y7). Decreased levels of phospho-Akt and phospho-S6K, and increased mRNA levels of d4E-BP were observed in INV4Y7 mutant larvae, suggesting that INV may be required for InR/PI3K/Akt signalling. In addition, a decreased level of Lsd2 (LD binding protein) was found in INV4Y7 mutants. These correlations between INV and molecules important for signaling suggest that INV might be a mediator of nutritional metabolism. In light of these data, I speculate that INV plays a homeostatic role, possibly by affecting the InR/PI3K/Akt signaling pathway. In conclusion, the localization of INV to LDs is dependent on the activity of PKC. An increase in invadolysin accompanies adipogenesis, in which PKC and PI3K may be mediators. Examining mutant Drosophila, I found INV to be involved in InR/PI3K/Akt signalling. Collectively, I conclude that INV may serve as a regulator in adipogenesis and the InR/PI3K/Akt signaling pathway.

Genetic variation may explain the heterogeneity in the lipid response to dietary change. A systematic literature review found 79 articles on dietary intervention studies, 14 articles on observational studies, and 22 reviews on diet-gene interactions. The evidence suggests that variation within the genes for apolipoprotein (apo) AI, AIV, B and E may influence the lipid response to dietary change. This study assessed the influence of six polymorphisms within the genes for apo B, apo E and lipoprotein lipase (LPL) on the association between habitual diet and lipid levels in 239 healthy men and women (91 men and 148 women) aged 18-54 years, including 110 twin pairs, who were recruited for a population-based study of coronary heart disease (CHD) risk factors. Diet was assessed by a food frequency questionnaire, which was compared with 4-day weighed records in 41 men and 40 women aged 19-58 years. The nutrients of interest had either a correlation coefficient ≥0.5, ≥50/≤10% in the same/opposite third, a KW30.04. Genotypes were determined by the polymerase chain reaction and digestion with the appropriate enzyme. Significant diet-gene interactions were observed at each of the polymorphic sites, suggesting that genetic variation contributes to the framework within which diet, especially n-3 PUFAs, the P:S ratio and NSP can influence lipid levels. In particular, individuals with the apo B XbaI X+ allele, the apo B signal peptide insertion/deletion D allele, the apo &egr;4 allele, the LPL PvuII P- allele and the LPL S447X X allele may be at greater risk of developing CHD due to their poorer lipid profiles and/or poorer response to diet. At present, it is premature to recommend the use of genotyping in the design of therapeutic diets, however investigating diet-gene interactions will increase our knowledge of the mechanisms involved in the role of diet in reducing CHD risk.

BACKGROUND: Tuberous sclerosis complex (TSC) is a multisystem hamartomatous disease caused by inactivating mutations in the TSC1/TSC2 genes leading to hyperactivation of mammalian target of rapamycin complex 1 (mTORC1) in TSC tumors. Novel therapeutic regimens and imaging biomarkers remain critical unmet needs in TSC. Mitochondrial fatty acid oxidation (FAO) is a major cellular source of acetyl-CoA. Acetyl-CoA is a central metabolite in lipid anabolism and catabolism. The purpose of this study was to identify novel metabolic therapeutic targets, particularly involving lipid metabolism, to achieve durable responses in TSC. METHODS AND RESULTS: Fluoroacetate (FACE), an acetate derivative, is a surrogate biomarker of mitochondrial activity. It accumulates in the mitochondria without being oxidized to CO2, and it is converted to fluorocitrate, irreversibly inhibiting the TCA (tricarboxylic acid) cycle enzyme aconitase. Micro-positron emission tomography (PET) imaging of subcutaneous xenografts of TSC2-deficient Eker rat uterine leiomyoma-derived ELT3 cells showed rapid uptake of [18F]FACE that was maintained after 72-hour treatment with mTORC1 inhibitor rapamycin. This result suggests that mitochondrial oxidative metabolism is sustained in the presence of rapamycin. Consistent with this finding, treatment of TSC2-deficient cells with rapamycin led to a significant increase in FAO and a decrease in glucose oxidation in vitro as measured by a 14C-CO2 collection metabolic assay. Expression of the A isoform of carnitine palmitoyltransferase I (CPT1A; FAO rate-limiting enzyme) was selectively increased in TSC2-deficient cells. Pharmacological inhibition of CPT1A by ST1326 led to a decrease in FAO, as measured by a 14C-CO2 collection metabolic assay, and a decrease in mitochondrial ATP production, measured by the Seahorse analyzer. CONCLUSIONS: This study proposes a role for FAO in TSC tumor bioenergetics and for CPT1A as a potential therapeutic target in TSC.

The phosphoinositide species phosphatidylinositol 3,4,5, trisphosphate (PIP3) is an essential mediator of many vital cellular processes involved in cell growth, survival, and metabolism. The class I PI3Ks are responsible for production of PIP3, and their activity is tightly controlled through interactions with regulatory proteins and activating stimuli. The class IA PI3Ks are composed of three distinct p110 catalytic subunits (p110, p110, p110) and they play different roles in specific tissues due to disparities in both expression and engagement downstream of cell surface receptors. Disruption of PI3K regulation is a frequent driver of numerous human diseases. Growth of all cell types is dependent on PI3K signalling, and development of immune cells relies on a precise balance of PIP3 production. Activating mutations in the genes encoding the catalytic and regulatory subunits of PI3K lead to cancer and immunodeficiencies. The PIK3CA gene encoding the p110 catalytic subunit of class IA PI3K is one of the most frequently mutated genes in cancer, and mutations in the PIK3CD gene encoding the p110 catalytic subunit lead to primary immunodeficiency. All class IA p110 subunits interact with p85 regulatory subunits, and mutations/deletions in different p85 regulatory subunits (PIK3R1, PIK3R2, PIK3R3) have been identified in both cancer and primary immunodeficiencies. By asking how these mutations mediate activation and disease phenotypes, we can identify the natural regulatory molecular mechanisms of class IA PI3Ks. Fundamentally understanding how mutations in PI3K subunits mediate human disease will expand our knowledge of PI3K biology and is essential to the development of novel therapeutics. To identify the molecular mechanisms of class IA PI3K activating mutations, I employed a sophisticated combination of hydrogen-deuterium eXchange mass spectrometry (HDX-MS) with biochemical activity assays to probe the regulatory mechanisms of PI3Ks. HDX-MS measures the exchange rate of amide hydrogens in solution, which in turn can provide information on protein conformation and conformational changes between different states. By comparing PI3K mutants identified in primary immunodeficiency and cancer patients to wild-type enzymes, I have identified dynamic conformational changes induced by activating mutations. Biochemical and biophysical analysis of these mutants led us to generate a panel of engineered mutations to further characterise molecular mechanisms by which class IA PI3Ks are regulated. This thesis will consist of an introduction to class IA PI3K signalling and an introduction to the method of HDX-MS, followed by two data chapters wherein I investigate the mechanisms of activating mutations in PIK3CD followed by an investigation into activating mutations in PIK3R1. A conclusion and discussion of future directions will be presented in the final chapter. This work provides novel insight into the complex regulatory mechanisms of the class IA PI3Ks, which may lead to better understanding of human diseases that activate these enzymes. / Graduate / 2020-04-06