Multifunctionalized lipid derivatives

Summary It is well understood how lipids are synthesized and metabolized in cells and that many lipids exhibit signalling functions to regulate cellular processes in a spatially and temporally defined way. The latter requires the build- up and turnover of lipid species in membranes either in a site-specific fashion or, alternatively, a directed form of lipid transport. This work aims to investigate the intracellular transfer of lipids from one membrane to another by several proteins that we discovered to be involved in lipid transport. In the previous funding period, we synthesized multifunctional lipid derivatives of five phosphoinositides and four common glycerophospholipids. These feature a photo-activatable protecting group ("cage") to release the lipid derivative by light and a photo-crosslinking diazirine to covalently attach the lipid derivative to binding proteins. An alkyne group for click chemistry is useful for isolating lipid-protein conjugates or for determining the lipid location in cells by fluorescent tagging and microscopy. In published work, we identified specific lipid binding proteins for phosphatidylinositol 3,4,5-trisphosphate (PIP3), phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2], and phosphatidylinositol (PI) via proteomic analysis. We then used siRNAs to block lipid transport and validated two hits that were required for transporting PIP3 and PI(3,4)P2: cytosolic MPP6 and transmembrane ATP11A. This R35 application proposes the continuation of work described in the application of R01 GM127631, namely the characterization of the lipid transport by the two above mentioned proteins (Project 1). This includes the purification and characterization of recombinant proteins and their functional mutants. We will use purified proteins to determine the 3D structure of MPP6 and its mutants by cryo-electron microscopy with and without crosslinked lipid derivatives. In Project 2, we will synthesize lipid derivatives featuring the photo-crosslinking diazirine closer to the membrane interphase to reach more transiently binding proteins such as those with a PH domain. Comparative proteomic analysis of the lipid interactomes will then be used to identify proteins involved in signalling with and without receptor stimulation. In Project 3, we will use multifunctional lipid derivatives to investigate the lipid interactomes of healthy and virus-infected cells. We recently discovered that an RNA virus infection leads to massive changes in the host cell lipidome. One exciting aspect is that one group of cellular phosphoinositides featuring a particular fatty acid composition is strongly up regulated. We will measure the lipid interactomes of flavivirus- and COVID-infected cells and identify targets crucial for viral infection and replication. Hits will be validated by protein knock-down and the effect on virus infection will be studied. Our unique lipid tools will help to better understand the lipid and lipid binding components of a viral infection.