A Small Molecule Fluorogenic Probe for the Detection of Sphingosine in Living Cells.

The single-chained sphingolipid sphingosine is an essential structural lipid and signaling molecule. Abnormal sphingosine metabolism is observed in several diseases, including cancer, diabetes, and Alzheimer's. Despite its biological importance, there is a lack of tools for detecting sphingosine in living cells. This is likely due to the broader challenge of developing highly selective and live-cell compatible affinity probes for hydrophobic lipid species. In this work, we have developed a small molecule fluorescent turn-on probe for labeling sphingosine in living cells. We demonstrate that this probe exhibits a dose-dependent response to sphingosine and is able to detect endogenous pools of sphingosine. Using our probe, we successfully detected sphingosine accumulation in cells from patients with Niemann-Pick type C1 (NPC1), a lipid transport disorder in which increased sphingosine mediates disease progression. This work provides a simple and accessible method for the detection of sphingosine and should facilitate study of this critical signaling lipid in biology and disease.

Traceless native chemical ligation of lipid-modified peptide surfactants by mixed micelle formation.

Biology utilizes multiple strategies, including sequestration in lipid vesicles, to raise the rate and specificity of chemical reactions through increases in effective molarity of reactants. We show that micelle-assisted reaction can facilitate native chemical ligations (NCLs) between a peptide-thioester - in which the thioester leaving group contains a lipid-like alkyl chain - and a Cys-peptide modified by a lipid-like moiety. Hydrophobic lipid modification of each peptide segment promotes the formation of mixed micelles, bringing the reacting peptides into close proximity and increasing the reaction rate. The approach enables the rapid synthesis of polypeptides using low concentrations of reactants without the need for thiol catalysts. After NCL, the lipid moiety is removed to yield an unmodified ligation product. This micelle-based methodology facilitates the generation of natural peptides, like Magainin 2, and the derivatization of the protein Ubiquitin. Formation of mixed micelles from lipid-modified reactants shows promise for accelerating chemical reactions in a traceless manner.

Inhibition of NRAS Signaling in Melanoma through Direct Depalmitoylation Using Amphiphilic Nucleophiles.

Activating mutations in the small GTPase NRAS are responsible for driving tumor growth in several cancers. Unfortunately, the development of NRAS inhibitors has proven difficult due to the lack of hydrophobic binding pockets on the protein's surface. To overcome this limitation, we chose to target the post-translational S-palmitoyl modification of NRAS, which is required for its signaling activity. Utilizing an amphiphile-mediated depalmitoylation (AMD) strategy, we demonstrate the ability to directly cleave S-palmitoyl groups from NRAS and inhibit its function. C8 alkyl cysteine causes a dose-dependent decrease in NRAS palmitoylation and inhibits downstream signaling in melanoma cells with an activating mutation in NRAS. This compound reduces cell growth in NRAS-driven versus non-NRAS-driven melanoma lines and inhibits tumor progression in an NRAS-mutated melanoma xenograft mouse model. Our work demonstrates that AMD can effectively suppress NRAS activity and could represent a promising new avenue for discovering lead compounds for treatment of NRAS-driven cancers.

Designer Palmitoylation Motif-Based Self-Localizing Ligand for Sustained Control of Protein Localization in Living Cells and Caenorhabditis elegans.

Inducing protein translocation to the plasma membrane (PM) is an important approach for manipulating diverse signaling molecules/pathways in living cells. We previously devised a new chemogenetic system, in which a protein fused to Escherichia coli dihydrofolate reductase (eDHFR) can be rapidly translocated from the cytoplasm to the PM using a trimethoprim (TMP)-based self-localizing ligand (SL), mgcTMP. However, mgcTMP-induced protein translocation turned out to be transient and spontaneously reversed within 1 h, limiting its application. Here, we first demonstrated that the spontaneous reverse translocation was caused by cellular degradation of mgcTMP, presumably by proteases. To address this problem, we newly developed a proteolysis-resistant SL, mDcTMP. This mDcTMP now allows sustained PM localization of eDHFR-fusion proteins (over several hours to a day), and it was applicable to inducing prolonged signal activation and cell differentiation. mDcTMP also worked in live nematodes, making it an attractive new tool for probing and controlling living systems.

Amphiphile-Mediated Depalmitoylation of Proteins in Living Cells.

Post-translational S-palmitoylation plays a central role in protein localization, trafficking, stability, aggregation, and cell signaling. Dysregulation of palmitoylation pathways in cells can alter protein function and is the cause of several diseases. Considering the biological and clinical importance of S-palmitoylation, tools for direct, in vivo modulation of this lipid modification would be extremely valuable. Here, we describe a method for the cleavage of native S-palmitoyl groups from proteins in living cells. Using a cell permeable, cysteine-functionalized amphiphile, we demonstrate the direct depalmitoylation of cellular proteins. We show that amphiphile-mediated depalmitoylation (AMD) can effectively cleave S-palmitoyl groups from the native GTPase HRas and successfully depalmitoylate mislocalized proteins in an infantile neuronal ceroid lipofuscinosis (INCL) disease model. AMD enables direct and facile depalmitoylation of proteins in live cells and has potential therapeutic applications for diseases such as INCL, where native protein thioesterase activity is deficient.

Traceless synthesis of ceramides in living cells reveals saturation-dependent apoptotic effects.

Mammalian cells synthesize thousands of distinct lipids, yet the function of many of these lipid species is unknown. Ceramides, a class of sphingolipid, are implicated in several cell-signaling pathways but poor cell permeability and lack of selectivity in endogenous synthesis pathways have hampered direct study of their effects. Here we report a strategy that overcomes the inherent biological limitations of ceramide delivery by chemoselectively ligating lipid precursors in vivo to yield natural ceramides in a traceless manner. Using this method, we uncovered the apoptotic effects of several ceramide species and observed differences in their apoptotic activity based on acyl-chain saturation. Additionally, we demonstrate spatiotemporally controlled ceramide synthesis in live cells through photoinitiated lipid ligation. Our in situ lipid ligation approach addresses the long-standing problem of lipid-specific delivery and enables the direct study of unique ceramide species in live cells.

Nonenzymatic biomimetic remodeling of phospholipids in synthetic liposomes.

Cell membranes have a vast repertoire of phospholipid species whose structures can be dynamically modified by enzymatic remodeling of acyl chains and polar head groups. Lipid remodeling plays important roles in membrane biology and dysregulation can lead to disease. Although there have been tremendous advances in creating artificial membranes to model the properties of native membranes, a major obstacle has been developing straightforward methods to mimic lipid membrane remodeling. Stable liposomes are typically kinetically trapped and are not prone to exchanging diacylphospholipids. Here, we show that reversible chemoselective reactions can be harnessed to achieve nonenzymatic spontaneous remodeling of phospholipids in synthetic membranes. Our approach relies on transthioesterification/acyl shift reactions that occur spontaneously and reversibly between tertiary amides and thioesters. We demonstrate exchange and remodeling of both lipid acyl chains and head groups. Using our synthetic model system we demonstrate the ability of spontaneous phospholipid remodeling to trigger changes in vesicle spatial organization, composition, and morphology as well as recruit proteins that can affect vesicle curvature. Membranes capable of chemically exchanging lipid fragments could be used to help further understand the specific roles of lipid structure remodeling in biological membranes.

SNAP-Tag-Reactive Lipid Anchors Enable Targeted and Spatiotemporally Controlled Localization of Proteins to Phospholipid Membranes.

The natural mechanisms that direct proteins to membranes are typically complex, requiring multiple steps and accessory components. It would be advantageous to develop simplified methods to direct proteins of interest to phospholipid membranes in a single step. Here we report a modular method for membrane localization of proteins by using chemically modified phospholipid anchors capable of covalent attachment to O(6)-methylguanine DNA methyltransferase (SNAP-tag) fusion proteins. To our knowledge, this is the first use of SNAP-tag reactions to modify benzylguanine-functionalized lipid membranes. We demonstrate that photocaged lipid precursors enable light-triggered spatial and temporal control over protein localization. The anchoring system is compatible with cell-free expression, allowing for genetic targeting of proteins to lipid membranes of giant unilamellar vesicles. This technique can be used to control membrane curvature effects, similar to what has been previously observed with certain membrane-bound proteins. This work addresses a current need in synthetic biology for simplified and robust methods to control membrane localization of expressed proteins and shows promise as a general tool for protein targeting to lipid vesicles and cellular membranes.

Surface features of the lipid droplet mediate perilipin 2 localization.

All eukaryotic organisms store excess lipid in intracellular lipid droplets. These dynamic structures are associated with and regulated by numerous proteins. Perilipin 2, an abundant protein on most lipid droplets, promotes neutral lipid accumulation in lipid droplets. However, the mechanism by which perilipin 2 binds to and remains anchored on the lipid droplet surface is unknown. Here we identify features of the lipid droplet surface that influence perilipin 2 localization. We show that perilipin 2 binding to the lipid droplet surface requires both hydrophobic and electrostatic interactions. Reagents that disrupt these interactions also decrease binding. Moreover, perilipin 2 binding does not depend on other lipid droplet-associated proteins but is influenced by the lipid composition of the surface. Perilipin 2 binds to synthetic vesicles composed of dioleoylphosphatidylcholine, a phospholipid with unsaturated acyl chains. Decreasing the temperature of the binding reaction, or introducing phospholipids with saturated acyl chains, decreases binding. We therefore demonstrate a role for surface lipids and acyl chain packing in perilipin 2 binding to lipid droplets. The ability of the lipid droplet phospholipid composition to impact protein binding may link changes in nutrient availability to lipid droplet homeostasis.