The roles of sphingosine in calcium signaling and Niemann-Pick disease type C

Although several lipids have been shown to participate in intracellular signal transduction events and to influence central cellular processes, the bioactive actions of most lipids remain unexplored. This lack of knowledge is mainly due to a shortage of tools to manipulate lipid levels within living cells in a non-invasive way and to identify new protein interactors of single lipid species. This work presents the development of two methods to overcome these drawbacks applied to sphingosine (Sph). The origin of calcium signaling properties of Sph and its involvement in the pathophysiological development of the lysosomal storage disease Niemann-Pick type C (NPC) are reported. First, ‘caged’ variants of sphingosine were synthesized which enable the precise elevation of Sph levels in single living cells within seconds using light. This acute increase in Sph concentration led to an immediate release of lysosomal calcium through the actions of the two-pore channel 1 (TPC1). In cells derived from NPC patients, an accumulation of Sph in the endolysosomal compartments was visualized for the first time. Additionally, NPC cells exhibited reduced calcium signals upon Sph uncaging, indicating that Sph accumulation is upstream of a calcium defect in this disease. Sph-induced calcium release also initiated the nuclear translocation of transcription factor EB, which positively regulates the expression of autophagic and lysosomal biogenesis genes, further underlining the importance of lysosomal calcium release in direct lysosome-to-nucleus signaling pathways. In order to capture Sph-interacting proteins, a trifunctional Sph (TFS) was developed. TFS facilitates the release and immediate crosslinking of Sph to its interacting partners within the living cell. Mass-spectrometric analyses identified known Sph-binding proteins such as the ceramide synthase, as well as novel putative Sph-interactors. The general applicability of this method was proven by using trifunctional diacylglycerol as well as a trifunctional fatty acid. TFS was further employed in investigations of the subcellular localization and transport of Sph through the cell. NPC patient fibroblasts showed a striking accumulation of Sph in late endosomes and lysosomes. Sph transport out of these vesicles was severely hindered in the NPC condition. The kinetics of Sph efflux correlated with the severity of symptoms in different NPC patients, so this assay could potentially be used for monitoring and prognosis of NPC disease severity

Trifunctional lipid probes for comprehensive studies of single lipid species in living cells

Lipid-mediated signaling events regulate many cellular processes. Investigations of the complex underlying mechanisms are difficult because several different methods need to be used under varying conditions. Here we introduce multifunctional lipid derivatives to study lipid metabolism, lipid−protein interactions, and intracellular lipid localization with a single tool per target lipid. The probes are equipped with two photoreactive groups to allow photoliberation (uncaging) and photo–cross-linking in a sequential manner, as well as a click-handle for subsequent functionalization. We demonstrate the versatility of the design for the signaling lipids sphingosine and diacylglycerol; uncaging of the probe for these two species triggered calcium signaling and intracellular protein translocation events, respectively. We performed proteomic screens to map the lipid-interacting proteome for both lipids. Finally, we visualized a sphingosine transport deficiency in patient-derived Niemann−Pick disease type C fibroblasts by fluorescence as well as correlative light and electron microscopy, pointing toward the diagnostic potential of such tools. We envision that this type of probe will become important for analyzing and ultimately understanding lipid signaling events in a comprehensive manner. The roles of lipids in cells go far beyond providing the structural backbone of cellular membranes. Certain lipid species are powerful signaling molecules. Examples include the roles of sphingosine (Sph) and the diacylglycerol (DAG) variant, stearoyl-arachidonylglycerol (SAG) in intracellular calcium signaling (1, 2). The study of such signaling lipids is often complicated by the fact that they are under tight metabolic control and that they occur only in very low concentrations. Overexpression of metabolic enzymes for manipulation of signaling lipid levels is a slow process compared with the rapid turnover of those lipids and may therefore produce not only the target lipid but also multiple downstream metabolites. Chemical dimerizer and optogenetic approaches are options to manipulate lipid contents more rapidly, but they depend on cytosolic lipid-metabolizing enzymes. In the past, many applications therefore focused on phosphoinositides (3, 4). A more general way to rapidly increase lipid concentration is the use of caged lipids. These are equipped with a photocleavable protecting group (caging group), which blocks biological activity and renders them resistant to metabolic turnover before the active lipid is released using a flash of light (2, 5⇓–7). The sudden increase in target lipid concentration facilitates analysis of downstream lipid signaling events as well as lipid metabolism within living cells in pulse−chase experiments. To correctly interpret such signaling events, underlying processes such as lipid−protein interactions, intracellular lipid localization, and kinetics of lipid metabolism need to be considered. To date, lipid metabolism is typically monitored using isotope-labeled or alkyne-modified lipids (8⇓–10). Fluorescent lipids, lipid-binding antibodies, or lipid biosensors are mainly used to study lipid localization (11, 12). Most assays for studying lipid−protein interactions rely on reconstituted membranes/liposomes and are therefore largely restricted to soluble proteins (13⇓⇓–16). The plethora of methods used to investigate these different processes makes it difficult to compare or validate their respective results. A promising approach to integrate the study of lipid metabolism, lipid localization, and lipid−protein interactions has emerged in recent years; bifunctional lipids feature a small diazirine group to allow photo–cross-linking with interacting proteins in the intact cellular environment and a terminal alkyne for subsequent functionalization (17). Biotinylation of cross-linked lipid−protein conjugates enables their enrichment and identification of lipid-interacting proteins. To date, bifunctional lipids are one of the few methods to screen for lipid−protein interactions in living cells (18⇓⇓–21). Alternatively, bifunctional lipids can be used to visualize lipid localization by click reaction with a fluorophore (1, 18, 20). The application of the bifunctional lipid principle to signaling lipids, however, is handicapped by their tight metabolic control. Any precursor is rapidly incorporated into downstream lipids, complicating the interpretation of resulting data. The ability to liberate a single, well-defined signaling lipid species within cells and to immediately capture its interacting partners, investigate downstream signaling, and study its subcellular localization would enable much-needed insight into the regulation of lipid-dependent signaling. Here, we present “trifunctional” lipids as tools, combining the advantages of caged and bifunctional lipids in a single molecule to allow for a wide range of studies in living cells with tight temporal control. Applied to Sph and DAG, we show that trifunctional lipids enable (i) acute alteration of signaling lipid concentration, (ii) measurement of lipid metabolism on a population-wide as well as on a single-cell level, (iii) screening for lipid−protein interactions, and (iv) direct visualization of lipid localization by light and correlative light and electron microscopy (CLEM) in comparable experimental settings

Intracellular sphingosine releases calcium from lysosomes

To elucidate new functions of sphingosine (Sph), we demonstrate that the spontaneous elevation of intracellular Sph levels via caged Sph leads to a significant and transient calcium release from acidic stores that is independent of sphingosine 1-phosphate, extracellular and ER calcium levels. This photo-induced Sph-driven calcium release requires the two-pore channel 1 (TPC1) residing on endosomes and lysosomes. Further, uncaging of Sph leads to the translocation of the autophagy-relevant transcription factor EB (TFEB) to the nucleus specifically after lysosomal calcium release. We confirm that Sph accumulates in late endosomes and lysosomes of cells derived from Niemann-Pick disease type C (NPC) patients and demonstrate a greatly reduced calcium release upon Sph uncaging. We conclude that sphingosine is a positive regulator of calcium release from acidic stores and that understanding the interplay between Sph homeostasis, calcium signaling and autophagy will be crucial in developing new therapies for lipid storage disorders such as NPC

Bifunctional Sphingosine for Cell-Based Analysis of Protein-Sphingolipid Interactions

Sphingolipids are essential structural components of cellular membranes and are crucial regulators of cellular processes. While current high-throughput approaches allow for the systematic mapping of interactions of soluble proteins with their lipid-binding partners, photo-crosslinking is the only technique that enables for the proteome-wide mapping of integral membrane proteins with their direct lipid environment. Here we report the synthesis of a photoactivatable and clickable analog of sphingosine. When administered to sphingosine-1-phosphate lyase deficient cells, pacSph allows its metabolic fate and the subcellular flux of de novo synthesized sphingolipids to be followed in a time resolved manner. The chemoproteomic profiling yielded over 180 novel sphingolipid-binding proteins, of which we validated a number, demonstrating the unique value of this technique as a discovery tool. This work provides an important resource for the understanding of the global cellular interplay between sphingolipids and their interacting proteins.status: publishe

LXR signaling controls homeostatic dendritic cell maturation

Dendritic cells (DCs) mature in an immunogenic or tolerogenic manner depending on the context in which an antigen is perceived, preserving the balance between immunity and tolerance. Whereas the pathways driving immunogenic maturation in response to infectious insults are well-characterized, the signals that drive tolerogenic maturation during homeostasis are still poorly understood. We found that the engulfment of apoptotic cells triggered homeostatic maturation of conventional cDC1s within the spleen. This maturation process could be mimicked by engulfment of empty, non-adjuvanted lipid nanoparticles (LNPs), was marked by intracellular accumulation of cholesterol, and highly unique to type 1 DCs. Engulfment of either apoptotic cells or cholesterol-rich LNPs led to activation of the LXR pathway, which promotes the efflux of cellular cholesterol, and repressed genes associated with immunogenic maturation. In contrast, simultaneous engagement of TLR3 to mimic viral infection via administration of poly(I:C)-adjuvanted LNPs repressed the LXR pathway, thus delaying cellular cholesterol efflux and inducing genes that promote T cell-mediated immunity. These data demonstrate that conserved cellular cholesterol efflux pathways are differentially regulated in in tolerogenic versus immunogenic cDC1s and suggest that administration of non-adjuvanted cholesterol-rich LNPs may be an approach for inducing tolerogenic DC maturation