OPTOGENETICS AND ENZYME ENGINEERING TO DISSECT PHOSPHATIDIC ACID SIGNALING

220 pagesThe simple structure of phosphatidic acid (PA) belies its complex biological functions as both a key phospholipid biosynthetic intermediate and a potent signaling molecule. In the latter role, PA controls processes including vesicle trafficking, actin dynamics, cell growth, and migration. However, experimental methods to decode the pleiotropy of PA are sorely lacking, and because of rapid PA metabolism and trafficking in cells, approaches to manipulate its levels require high spatiotemporal precision. Here, we describe the development and applications of molecular tools that enable perturbation of PA signaling. Photoswitchable PA analogs contain azobenzene photoswitches in their acyl tails, enabling molecular shape and bioactivity to be controlled by light. Optogenetic phospholipase Ds (optoPLDs) use light-mediated heterodimerization to recruit a bacterial PLD, which exchanges phospholipid head groups through hydrolysis or transphosphatidylation of phosphatidylcholine with water or exogenous alcohols, to desired organelle membranes. These tools demonstrate that PA regulates two oncogenic signaling pathways, mTOR signaling and Hippo signaling, in a location- and acyl chain structure-specific manner. We broaden the utility of optoPLD by engineering its catalytic activity using directed evolution, obtaining a series of so-called superPLDs with greatly enhanced stability in the intracellular environment and catalytic efficiencies of up to 100-fold higher than wild-type PLD. SuperPLDs are efficient tools for both optogenetics-enabled editing of phospholipids within specific organelle membranes in live cells and biocatalytic synthesis of natural and unnatural designer phospholipids in vitro. Lipidomics analysis using superPLD reveals the tight homeostasis of PA in mammalian cells, achieved by striking conversion of excess PA molecules to CDP-diacylglycerol. To map PA interactome and identify molecular mechanisms of this PA homeostasis, we incorporate superPLD-enabled membrane editing to a proximity dependent labeling system. This “feeding-and-fishing” approach captures protein interactors that are recruited to target membranes upon PA production. Subsequent proteomics analysis led us to propose mechanism whereby PA homeostasis is cooperatively achieved through upregulated inter-organelle PA transport by lipid transfer proteins, upregulated PA degradation, and downregulated PA synthesis. Collectively, our studies developing and utilizing optoPLD and superPLD highlight the importance of and offer a potent approach for designable membrane editing to decode the pleiotropic functions of lipids.2024-09-0