Date of Award
Doctor of Philosophy
Michael D. Best
David C. Baker, Francisco N. Barrera Olivares, Tessa R. Calhoun
The protein signaling activities of the glycerolipid diacylglycerol (DAG) form the impetus for the projects described herein. DAG’s governance of cellular functions involves activation of peripheral membrane proteins (PMPs) at bilayer surfaces, which includes the activation of protein kinase C (PKC) to regulate oncogenesis. In addition to enzymatic signal transduction, DAG influences membrane mechanics and is a central lipid metabolite. Relatively little is known about DAG when compared to more common signaling lipids such as phosphatidylinositol polyphosphates (PIPns). This is due in part to the surreptitious nature of PMP operation and the complexity of natural bilayers. We developed a liposomal platform to identify PMP binding as a function of specific lipids. Synthetic, photocrosslinking lipids with clickable tags are incorporated into liposomes to capture and enrich proteins. Affinity-based protein profiling (AfBPP) experiments initially demonstrated proteome-wide increases in affinity when using DAG or phosphatidic acid (PA) as chase lipids. With the aid of collaborators at The Scripps Research Institute (TSRI), we optimized our AfBPP protocol to label select proteins as a function of liposomal DAG content when a generic lipid probe was also present in the membrane. The generic probe strategy varies natural lipid content with consistent probe concentration between liposomal treatments, this is called the lipomimetic approach. Lipid specific probes have also been applied to liposomal AfBPP, which is termed the lipospecific approach.In a separate project, we tested to see if DAG could potentiate the cell-association of a liposomal delivery system (LDS). LDSs are a rapidly expanding field; most existing nanodrugs are liposomal. Strategies for increasing LDS efficacies often undermine clinical translatability. Incorporating natural signaling lipids into nanodrugs architectures is a clinically viable targeting strategy. A polyethylene glycol (PEG) decorated (PEGylated) liposome bearing a cell penetrating peptide (CPP) was doped with DAG and/or PS and significant, dose-dependent increases in association to target cells were observed. We also advanced LDSs with new technologies for controlling vesicle release and fusion. Liposomes have limitless utility as theranostic tools and platforms for biochemical investigations. Herein, we bring liposomal technologies closer to their scientific and clinical potentials.
Mattern-Schain, Samuel Isaac, "Liposomal discovery of diacylglycerol (DAG)-binding proteins and the use of signaling lipids to improve nanodrug delivery. " PhD diss., University of Tennessee, 2018.