Bioconjugation Strategies for Revealing the Roles of Lipids in Living Cells

Abstract

Conspectus The structural boundaries of living cells are composed of numerous membrane-forming lipids. Lipids not only are crucial for the cellular compartmentalization but also are involved in cell signaling as well as energy storage. Abnormal lipid levels have been linked to severe human diseases such as cancer, multiple sclerosis, neurodegenerative diseases, as well as lysosomal storage disorders. Given their biological significance, there is immense interest in studying lipids and their effect on cells. However, limiting factors include the low solubility of lipids, their structural complexity, and the challenge of using genetic techniques to directly manipulate lipid structure. Current methods to study lipids rely mostly on lipidomics, which analyzes the composition of lipid extracts using mass spectrometry. Although, these efforts have successfully catalogued and profiled a great number of lipids in cells, many aspects about their exact functional role and subcellular distribution remain enigmatic. In this Account, we outline how our laboratory developed and applied different bioconjugation strategies to study the role of lipids and lipid modifications in cells. Inspired by our ongoing work on developing lipid bioconjugation strategies to generate artificial cell membranes, we developed a ceramide synthesis method in live cells using a salicylaldehyde ester that readily reacts with sphingosine in form of a traceless ceramide ligation. Our study not only confirmed existing knowledge about the association of ceramides with cell death, but also gave interesting new findings about the structure–function relationship of ceramides in apoptosis. Our initial efforts led us to investigate probes that detect endogenous sphingolipids using live cell imaging. We describe the development of a fluorogenic probe that reacts chemoselectively with sphingosine in living cells, enabling the detection of elevated endogenous levels of this biomarker in human disease. Building on our interest in the fluorescence labeling of lipids, we have also explored the use of bioorthogonal reactions to label chemically synthesized lipid probes. We discuss the development of photocaged dihydrotetrazine lipids, where the initiation of the bioorthogonal reaction can be triggered by visible light, allowing for live cell modification of membranes with spatiotemporal control. Finally, proteins are often post-translationally modified by lipids, which have important effects on protein subcellular localization and function. Controlling lipid modifications with small molecule probes could help reveal the function of lipid post-translational modifications and could potentially inspire novel therapeutic strategies. We describe how our previous studies on synthetic membrane formation inspired us to develop an amphiphilic cysteine derivative that depalmitoylates membrane-bound S-acylated proteins in live cells. Ultimately, we applied this amphiphile mediated depalmitoylation (AMD) in studies investigating the palmitoylation of cancer relevant palmitoylat...

Incorporation, fate, and turnover of free fatty acids in cyanobacteria

“…Such new methodologies have led to considerable progress in understanding the evolution and development of early life forms (Bhattacharya et al 2019 ). These have also allowed the study of lipid post-translational modifications and have inspired the development of novel therapeutic strategies (Knittel and Devaraj 2022 , Wang and Chen 2022 ). Finally, a better understanding of cyanobacterial FA metabolism-associated pathways and their associated enzymes would equally pave the way toward the discovery of new biocatalysts and bioactive secondary metabolites of potential biotechnological interest.…”

Photoreactive bioorthogonal lipid probes and their applications in mammalian biology

“…Over the past two decades, coupled with the advent in multiplexing mass spectrometry based chemoproteomics, ligandphoto-affinity based protein profiling techniques have provided an edge over the aforementioned conventional approaches in mapping protein-ligand interactions, in that these multifunctional lipid probes enable the screening and identification of hitherto unknown protein ligands of lipids at a proteome wide scale under various physiological conditions in mammalian cells and tissues. While lipid probes studied in the past have employed multiple strategies, [54][55][56][57] such as appending a photoreactive group to a radioactive 58,59 or fluorophore conjugated lipid tail, [60][61][62][63] in this section, we aim to specifically discuss lipid probes that possess both a photoreactive group and a bioorthogonal handle, and how they have been profiled in mammalian systems to discover and characterize protein-lipid interactions. Commonly in such chemoproteomics experiments involving a lipid probe that has a photoreactive group and a bioorthogonal handle, as the first step, after incubation with the probe, the cellular lysates or live cells are irradiated with UV light of an appropriate wavelength and for a suitable exposure time, to ensure efficient crosslinking of the lipid probe with the protein(s) of interest.…”

“…While lipid probes studied in the past have employed multiple strategies, 54–57 such as appending a photoreactive group to a radioactive 58,59 or fluorophore conjugated lipid tail, 60–63 in this section, we aim to specifically discuss lipid probes that possess both a photoreactive group and a bioorthogonal handle, and how they have been profiled in mammalian systems to discover and characterize protein–lipid interactions. Commonly in such chemoproteomics experiments involving a lipid probe that has a photoreactive group and a bioorthogonal handle, as the first step, after incubation with the probe, the cellular lysates or live cells are irradiated with UV light of an appropriate wavelength and for a suitable exposure time, to ensure efficient crosslinking of the lipid probe with the protein(s) of interest.…”

NTA-Cholesterol Analogue for the Nongenetic Liquid-Ordered Phase-Specific Functionalization of Lipid Membranes with Proteins

“…In particular, hydrophobic insertion has the advantage of being a fast and simple modification process that does not require cellular machinery. To date, hydrophobic tags based on alkyl chains or lipid scaffolds have provided straightforward ways of decorating cell membranes with all kinds of (bio-)­molecules including proteins. − Another important aspect to consider when functionalizing biological membranes is the specificity of membrane functionalization for different membrane domains, e.g., lipid rafts . The division of biological membranes into liquid-disordered (Ld) and liquid-ordered (Lo) phases plays a crucial role in membrane organization and function but is rarely regarded in the context of cell surface functionalization.…”