A Chemical Biology Approach to Phosphoinositide Metabolic Analysis Public Deposited

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Last Modified
  • March 21, 2019
Creator
  • Waybright, Jarod
    • Affiliation: Eshelman School of Pharmacy, Division of Chemical Biology and Medicinal Chemistry
Abstract
  • Lipids serve a diverse array of functions including maintaining cellular structure and compartmentalization, regulating post-translational modifications of proteins, and as members of complex signaling networks. Despite their importance there is minimal understanding of how bioactive lipids and lipid pathways systematically regulate biological processes. Phosphoinositides (PIPs) are a diverse class of lipid signaling molecule implicated in nearly all facets of cell signaling including migration, proliferation, and apoptosis. Mutations in numerous PIP modifying enzymes occur frequently in human disease, especially cancers, though the effects of these mutations on the global metabolic system have been poorly defined. Likewise, heterogeneous protein expression and undefined feedback loops further complicate obtaining a global view of metabolism and understanding the PIP metabolic pathway’s role in disease. Current approaches have been unsuccessful in obtaining a systems-wide analysis due to various technical challenges including low sensitivity, use of indirect measurements of activity, and a lack of validated reporters and delivery methods. We have developed a new approach to systematic PIP analysis including synthesis and validation of fluorescent reporters, novel PIP delivery methods, and applying these methods to analyze PIP metabolism in breast cancer cell lines. We have shown that hydrophobicity is a key determinant in the ability of fluorescent reporters to serve as substrates for phosphoinositides modifying enzymes, which proceed via interfacial catalysis, through chemical synthesis of fluorescent phosphoinositides with varied hydrophobicity in the diacylglycerol (DAG) side chain and examined their propensity to serve as substrates towards various enzymes. We used these findings to develop a suite of fluorescent reporters that could subsequently be used as standards and reporters in future experimentation. To achieve our goal of systematically analyzing cellular metabolism, we examined a variety of delivery methods for fluorescent PIPs as previously reported techniques have failed to achieve a level of delivery sufficient for metabolic analysis. We further developed a fluorescent model phospholipid that allowed us to explore novel delivery techniques including intramolecular charge masking and photocaging methods while simultaneously exploring previously published methods including liposomes and charge altering releasable transporters (CARTs) for the delivery of PIPs. A PIP2:CART complex was demonstrated to efficiently deliver fluorescent PIP2 to cells, which were then analyzed for their metabolite distribution. This method was effective in achieving conversion of the fluorescent reporter to a variety of PIP substrates as judged by comparison with synthetic standards. We further applied this system to preliminary studies on breast cancer cell lines possessing unique mutations in PIP modifying enzymes. We have thus far been successful in demonstrating a platform using fluorescent PIP reporters, novel delivery techniques that lead to a variety of metabolites than can be analyzed by capillary electrophoresis (CE) or thin layer chromatography (TLC). This system could be a powerful tool in future studies of PIP metabolism in human disease with potential diagnostic applications.
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Rights statement
  • In Copyright
Advisor
  • Lawrence, David
  • Sondek, John
  • Allbritton, Nancy
  • Zhang, Qisheng
  • Jarstfer, Michael
Degree
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill Graduate School
Graduation year
  • 2017
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