Methods for Studying Lipid-Protein Interactions and Studies of Ceramide 1-Phosphate

Lipids are important for supporting the homeostasis of cellular life. They play primary roles in energy storage, formation of cellular membranes, and acting as secondary messengers. Many of these roles occur through specific lipid-protein interactions. The first part of this thesis will describe different techniques and assays currently used to study lipid-protein interactions. In addition, it will describe the optimization of lipid-protein overlay assays.

The second part of this thesis will discuss a class of lipids called, sphingolipids. They play major roles as secondary messengers in health and disease. Specifically, ceramide 1-phosphate (C1P) is an important signaling sphingolipid that has been shown to regulate physiological processes such as cell proliferation, inflammation, apoptosis, and macrophage chemotaxis. However, despite this information, little is known about the biophysical properties of C1P, including many of its specific protein effectors.

Here, we set out to discover new C1P binding proteins through several immunoprecipitation assays followed by mass spectrometry. In addition, we describe in detail how a fluorescently modified C1P could be used to reveal new biophysical information regarding this lipid.