Chemical Tools for Lipid Cell Biology

Abstract

Conspectus Lipids are key components of all organisms. We are well educated in their use as fuel and their essential role to form membranes. We also know much about their biosynthesis and metabolism. We are also aware that most lipids have signaling character meaning that a change in their concentration or location constitutes a signal that helps a living cell to respond to changes in the environment or to fulfill its specific function ranging from secretion to cell division. What is much less understood is how lipids change location in cells over time and what other biomolecules they interact with at each stage of their lifetime. Due to the large number of often quite similar lipid species and the sometimes very short lifetime of signaling lipids, we need highly specific tools to manipulate and visualize lipids and lipid–protein interactions. If successfully applied, these tools provide fabulous opportunities for discovery. In this Account, I summarize the development of synthetic tools from our lab that were designed to address crucial properties that allow them to function as tools in live cell experiments. Techniques to change the concentration of lipids by adding a small molecule or by light are described and complemented by examples of biological findings made when applying the tools. This ranges from chemical dimerizer-based systems to synthetic “caged” lipid derivatives. Furthermore, I discuss the problem of locating a lipid in an intact cell. Synthetic molecular probes are described that help to unravel the lipid location and to determine their binding proteins. These location studies require in-cell lipid tagging by click chemistry, photo-cross-linking to prevent further movement and the “caging” groups to avoid premature metabolism. The combination of these many technical features in a single tool allows for the analysis of not only lipid fluxes through metabolism but also lipid transport from one membrane to another as well as revealing the lipid interactome in a cell-dependent manner. This latter point is crucial because with these multifunctional tools in combination with lipidomics we can now address differences in healthy versus diseased cells and ultimately find the changes that are essential for disease development and new therapeutics that prevent these changes.

Trifunctional Sphinganine: A New Tool to Dissect Sphingolipid Function

“…13,14 Trifunctional lipids add three functional groups to the endogenous lipid structure: (1) a terminal alkyne, for click chemistry with affinity tags or fluorophores, (2) a diazirine for photocrosslinking with interacting proteins, and (3) a photocage to prevent premature metabolism and signaling. 13,14 Here, we investigated the metabolism and subcellular localization of both probes and showed that while the two sphingoid bases result in nearly identical pools of metabolites, the two lipids have subtle differences in their subcellular dynamics. Finally, we performed LC-MS/MS-based identification of probeinteracting proteins and described a network of lipidinteracting proteins with both distinct and overlapping interactions for each lipid base.…”

“…To begin to unravel the cell biological differences between Spa and Sph, we synthesized trifunctional sphinganine to compare to the previously described trifunctional sphingosine . Trifunctional lipids are designed to overcome several challenges in interrogating lipid biology: their fast metabolism, their weak and transient (but functionally important) interactions with proteins, and the difficulty in tagging them without disturbing the cellular location. , Trifunctional lipids add three functional groups to the endogenous lipid structure: (1) a terminal alkyne, for click chemistry with affinity tags or fluorophores, (2) a diazirine for photo-crosslinking with interacting proteins, and (3) a photocage to prevent premature metabolism and signaling. , Here, we investigated the metabolism and subcellular localization of both probes and showed that while the two sphingoid bases result in nearly identical pools of metabolites, the two lipids have subtle differences in their subcellular dynamics. Finally, we performed LC-MS/MS-based identification of probe-interacting proteins and described a network of lipid-interacting proteins with both distinct and overlapping interactions for each lipid base.…”

Lipophilic Anchors that Embed Bioconjugates in Bilayer Membranes: A Review

“…Topics that are beyond the scope of this article include molecular and nanoparticle designs that promote membrane permeation, 5 amphiphilic nanoparticles that alter membrane curvature, 6 T h i s c o n t e n t i s detailed biosynthetic pathways that produce membraneanchored proteins, 7 and molecular probes for the study of membrane diffusion and lipidomics. 8 In terms of presentation format, the article starts by briefly reviewing the various ways that Nature anchors proteins in a cell membrane, and then describes the strategies that chemical biologists use to fluorescently label a membrane protein in living cells. The latter sections describe bioconjugation methods that anchor synthetic conjugates in liposome membranes or cell plasma membranes and includes a section on switchable anchors.…”

“…The article focuses on the different biosynthetic, semisynthetic, and synthetic strategies that are used to prepare an anchored construct, and we summarize the overarching concepts that underlie the molecular designs with citations directing the interested reader to the specialized synthetic and technical publications. Topics that are beyond the scope of this article include molecular and nanoparticle designs that promote membrane permeation, amphiphilic nanoparticles that alter membrane curvature, detailed biosynthetic pathways that produce membrane-anchored proteins, and molecular probes for the study of membrane diffusion and lipidomics …”

A Protocol for Activated Bioorthogonal Fluorescence Labeling and Imaging of 4‐Hydroxyphenylpyruvate Dioxygenase in Plants

“…Numerous investigations have shown that both covalent and non-covalent fluorescent labeling is a highly sensitive strategy for studying a wide variety of biomolecules including enzymes, proteins, nucleic acids and glycans. [20][21][22][23][24][25][26][27][28] Several bioorthogonal reactions have been devised to enable fluorescence labeling to be performed under physiological conditions, within organisms, and without interfering with concurrent biochemical reactions or causing harm to organisms or target biomolecules. [29][30][31][32][33][34][35] Moreover, increasing attention has been given to applications of bioorthogonalbased fluorescent labeling to visualization cells in in vivo settings.…”