Understanding phosphatidylserine and sphingolipid metabolism in macrophages

Abstract

Lipids encompass more than 45,000 species and are known to partake in several biological functions. Their diversity and low abundance in biological systems presents a challenge for studying them. In my doctoral research, I have capitalized on the power of mass spectrometry to dissect the biochemistry of lipid metabolism underlying certain signaling and cell biological processes operating in macrophages – cells of the immune system. I started off by studying the metabolism of oxidized phospholipids – oxidized phosphatidylserine in particular. Oxidative stress, through the elevation of Reactive Oxygen Species (ROS), damages cellular Phosphatidylserines (PS) and give rise to oxidized PS, which often get misread as an apoptotic signal. It was hypothesized that the cells must have a mechanism to counter this untimely apoptosis, by means of an enzyme capable of hydrolyzing oxidized PS. In an effort to find this enzyme(s), I developed methods to achieve stable and dose-dependent increase in intracellular ROS and subsequent elevation in oxidized PS, following which, a chemical-genetic screen for serine hydrolases then helped me pick out a few candidate lipases. My colleagues then proceeded to identify ABHD12 as a novel oxidized PS lipase. To further study lipid biochemisty in macrophages, I performed lipidomic analyses of phagosomes – organelles made of internalized and membrane bound particles. The formation, maturation, and subsequent degradation of a phagosome is an important immune response essential for protection against many pathogens. Yet, the global lipid profile of phagosomes was unknown, especially as a function of their maturation in immune cells. Through mass spectrometry based lipidomics, I find that ceramides and glucosylceramides get enriched as the phagosomes proceed from an Early (EP) stage to a matured Late (LP) stage for fusion with lysosomes. This was attributed to an interplay of the activities of ceramide synthase 2, pH dependant ceramidase and glucosylceramide synthase enzymes, through proteomic and biochemical assays. Taken together, these studies provide a comprehensive picture and possible new roles of sphingolipid metabolism during phagosomal maturation.