CHEMICAL APPROACH FOR STUDYING LIPID TRAFFICKING AND INTERACTIONS WITH THE PROTEOME
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Cellular membranes are multifunctional supramolecular assemblies encapsulating our cells and the organelles within them. Chemistry has much to offer to the world of lipid biology in the form of precision tools for visualizing lipid localization and abundance, manipulating lipid composition, and decoding the various functions of lipids in cells. Herein, I first provide an overview in Chapter 1 of the recent efforts from the Baskin lab in this space focused on imaging and editing the phospholipidome, by exploiting the transphosphatidylation activity of phospholipase D (PLD) with exogenous clickable alcohols followed by bioorthogonal tagging to generated functional lipids. In Chapter 2, I harness PLD-transphosphatidylation and describe a chemoenzymatic approach for selective visualization of organelle compartments. Detailed analysis of the trafficking kinetics of both the fluorophore-tagged phospholipid analogs and their non-fluorescent, azide-containing precursors revealed that the latter exhibit time-dependent differences in organelle selectivity, suggesting their use as probes for visualizing intracellular lipid transport pathways. In Chapter 3, I focus on investigation of the interactome of a rare phospholipid, N-acylphosphatidylethanolamine (NAPE). A photoaffinity labeling analog of NAPE enabled identification of CD147, CD44, and BZW2 as protein interactors of NAPE. E218 in CD147, key to recruitment of monocarboxylate transporters (MCTs), is required for photocrosslinking of the NAPE-based probe, implying that NAPE−CD147 interactions may be related to MCT functions. I then found that NAPE accumulation led to increased generation of reactive oxygen species, likely through clearance of lactate by MCTs. As well, heterodimerization of CD44 and BZW2 was found to be controlled by NAPE levels, suggesting potential roles for NAPE as a lipid-based molecular glue, and essential residues for this binding event were identified. Chapter 4 offers directions for extending the above findings, including local manipulation of enzymatic activity by the organelle-selective labeling strategy, and the utilization of split TurboID to elucidate the downstream effectors of a CD44–BZW2 complex.