The use of click chemistry in sphingolipid research.

Sphingolipid dysregulation is involved in a range of rare and fatal diseases as well as common pathologies including cancer, infectious diseases or neurodegeneration. Gaining insights into how sphingolipids are involved in these diseases would contribute much to our understanding of human physiology, as well as the pathology mechanisms. However, scientific progress is hampered by a lack of suitable tools that can be used in intact systems. To overcome this, efforts have turned to engineering modified lipids with small clickable tags and to harnessing the power of click chemistry to localize and follow these minimally modified lipid probes in cells. We hope to inspire the readers of this Review to consider applying existing click chemistry tools for their own aspects of sphingolipid research. To this end, we focus here on different biological applications of clickable lipids, mainly to follow metabolic conversions, their visualization by confocal or superresolution microscopy or the identification of their protein interaction partners. Finally, we describe recent approaches employing organelle-targeted and clickable lipid probes to accurately follow intracellular sphingolipid transport with organellar precision.

Lysosome-targeted multifunctional lipid probes reveal the sterol transporter NPC1 as a sphingosine interactor.

Lysosomes are catabolic organelles involved in macromolecular digestion, and their dysfunction is associated with pathologies ranging from lysosomal storage disorders to common neurodegenerative diseases, many of which have lipid accumulation phenotypes. The mechanism of lipid efflux from lysosomes is well understood for cholesterol, while the export of other lipids, particularly sphingosine, is less well studied. To overcome this knowledge gap, we have developed functionalized sphingosine and cholesterol probes that allow us to follow their metabolism, protein interactions, and their subcellular localization. These probes feature a modified cage group for lysosomal targeting and controlled release of the active lipids with high temporal precision. An additional photocrosslinkable group allowed for the discovery of lysosomal interactors for both sphingosine and cholesterol. In this way, we found that two lysosomal cholesterol transporters, NPC1 and to a lesser extent LIMP-2/SCARB2, bind to sphingosine and showed that their absence leads to lysosomal sphingosine accumulation which hints at a sphingosine transport role of both proteins. Furthermore, artificial elevation of lysosomal sphingosine levels impaired cholesterol efflux, consistent with sphingosine and cholesterol sharing a common export mechanism.

The Glucosylceramide Synthase Inhibitor PDMP Causes Lysosomal Lipid Accumulation and mTOR Inactivation.

For many years, the biology of glycosphingolipids was elucidated with the help of glucosylceramide synthase (GCS) inhibitors such as 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP). Additionally, PDMP gained interest because of its chemosensitizing effects. Several studies have successfully combined PDMP and anti-cancer drugs in the context of cancer therapy. However, the mechanism of action of PDMP is not fully understood and seems to go beyond glycolipid inhibition. Here, we used a functionalized sphingosine analogue (pacSph) to investigate the acute effects of PDMP on cellular sphingolipid distribution and found that PDMP, but not other GCS inhibitors, such as ND-DNJ (also called Miglustat), induced sphingolipid accumulation in lysosomes. This effect could be connected to defective export from lysosome, as monitored by the prolonged lysosomal staining of sphingolipids as well as by a delay in the metabolic conversion of the pacSph precursor. Additionally, other lipids such as lysobisphosphatidic acid (LBPA) and cholesterol were enriched in lysosomes upon PDMP treatment in a time-dependent manner. We could further correlate early LBPA enrichment with dissociation of the mechanistic target of rapamycin (mTOR) from lysosomes followed by nuclear translocation of its downtream target, transcription factor EB (TFEB). Altogether, we report here a timeline of lysosomal lipid accumulation events and mTOR inactivation arising from PDMP treatment.

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.

A Click Cage: Organelle-Specific Uncaging of Lipid Messengers.

Lipid messengers exert their function on short time scales at distinct subcellular locations, yet most experimental approaches for perturbing their levels trigger cell-wide concentration changes. Herein, we report on a coumarin-based photocaging group that can be modified with organelle-targeting moieties by click chemistry and thus enables photorelease of lipid messengers in distinct organelles. We show that caged arachidonic acid and sphingosine derivatives can be selectively delivered to mitochondria, the ER, lysosomes, and the plasma membrane. By comparing the cellular calcium transients induced by localized uncaging of arachidonic acid and sphingosine, we show that the precise intracellular localization of the released second messenger is crucial for the signaling outcome. Ultimately, we anticipate that this new class of caged compounds will greatly facilitate the study of cellular processes on the organelle level.

Caged lipids as tools for investigating cellular signaling.

Lipid derivatives that can be activated by light, often referred to as 'caged' lipids, are useful tools to manipulate intact cells non-invasively. Here we focus on experimental approaches that have made use of caged lipids. Apart from summarizing the recent advances and available tools in the field, we strive to highlight the experimental challenges that arise from lipid-specific biophysical properties and the abundance of an enormous diversity of distinct molecular lipid species in cells. This article is part of a Special Issue entitled Tools to study lipid functions.

The sterol transporter STARD3 transports sphingosine at ER-lysosome contact sites.

Sphingolipids are important structural components of membranes. Additionally, simple sphingolipids such as sphingosine are highly bioactive and participate in complex subcellular signaling. Sphingolipid deregulation is associated with many severe diseases including diabetes, Parkinson's and cancer. Here, we focus on how sphingosine, generated from sphingolipid catabolism in late endosomes/lysosomes, is reintegrated into the biosynthetic machinery at the endoplasmic reticulum (ER). We characterized the sterol transporter STARD3 as a sphingosine transporter acting at lysosome-ER contact sites. Experiments featuring crosslinkable sphingosine probes, supported by unbiased molecular dynamics simulations, exposed how sphingosine binds to the lipid-binding domain of STARD3. Following the metabolic fate of pre-localized lysosomal sphingosine showed the importance of STARD3 and its actions at contact sites for the integration of sphingosine into ceramide in a cellular context. Our findings provide the first example of interorganellar sphingosine transfer and pave the way for a better understanding of sphingolipid - sterol co-regulation.

Sphingolipid subtypes differentially control proinsulin processing and systemic glucose homeostasis.

Impaired proinsulin-to-insulin processing in pancreatic ß-cells is a key defective step in both type 1 diabetes and type 2 diabetes (T2D) (refs. 1,2), but the mechanisms involved remain to be defined. Altered metabolism of sphingolipids (SLs) has been linked to development of obesity, type 1 diabetes and T2D (refs. 3-8); nonetheless, the role of specific SL species in ß-cell function and demise is unclear. Here we define the lipid signature of T2D-associated ß-cell failure, including an imbalance of specific very-long-chain SLs and long-chain SLs. ß-cell-specific ablation of CerS2, the enzyme necessary for generation of very-long-chain SLs, selectively reduces insulin content, impairs insulin secretion and disturbs systemic glucose tolerance in multiple complementary models. In contrast, ablation of long-chain-SL-synthesizing enzymes has no effect on insulin content. By quantitatively defining the SL-protein interactome, we reveal that CerS2 ablation affects SL binding to several endoplasmic reticulum-Golgi transport proteins, including Tmed2, which we define as an endogenous regulator of the essential proinsulin processing enzyme Pcsk1. Our study uncovers roles for specific SL subtypes and SL-binding proteins in ß-cell function and T2D-associated ß-cell failure.

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 type 1 conventional DCs (cDC1s) within the spleen. This maturation process could be mimicked by engulfment of empty, nonadjuvanted lipid nanoparticles (LNPs), was marked by intracellular accumulation of cholesterol, and was highly specific to cDC1s. Engulfment of either apoptotic cells or cholesterol-rich LNPs led to the activation of the liver X receptor (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 tolerogenic versus immunogenic cDC1s and suggest that administration of nonadjuvanted cholesterol-rich LNPs may be an approach for inducing tolerogenic DC maturation.

Cellular cholesterol and how to find it.

Cholesterol is an essential component of eukaryotic cellular membranes. Information about its subcellular localization and transport pathways inside cells are key for the understanding and treatment of cholesterol-related diseases. In this review we give an overview over the most commonly used methods that contributed to our current understanding of subcellular cholesterol localization and transport routes. First, we discuss methods that provide insights into cholesterol metabolism based on readouts of downstream effects such as esterification. Subsequently, we focus on the use of cholesterol-binding molecules as probes that facilitate visualization and quantification of sterols inside of cells. Finally, we explore different analogues of cholesterol which, when taken up by living cells, are integrated and transported in a similar fashion as endogenous sterols. Taken together, we highlight the challenges and advantages of each method such that researchers studying aspects of cholesterol transport may choose the most pertinent approach for their problem.

Bi- and Trifunctional Lipids for Visualization of Sphingolipid Dynamics within the Cell.

Investigations into lipid localization and transport are often hampered by a lack of methods and tools to faithfully visualize lipids in the context of living cells, since fluorescent modifications drastically change lipid properties. Here, we describe the use of bifunctional as well as trifunctional sphingosine to reveal its subcellular localization via crosslinking, fixation, and specific staining by click reaction with a fluorophore. Additionally, these probes allow investigations into lipid metabolism as revealed by thin-layer chromatography.

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-cross-linking 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 (pacSph). 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.

Pathogenic mycobacteria achieve cellular persistence by inhibiting the Niemann-Pick Type C disease cellular pathway.

BACKGROUND: Tuberculosis remains a major global health concern. The ability to prevent phagosome-lysosome fusion is a key mechanism by which intracellular mycobacteria, including Mycobacterium tuberculosis, achieve long-term persistence within host cells. The mechanisms underpinning this key intracellular pro-survival strategy remain incompletely understood. Host macrophages infected with persistent mycobacteria share phenotypic similarities with cells taken from patients suffering from Niemann-Pick Disease Type C (NPC), a rare lysosomal storage disease in which endocytic trafficking defects and lipid accumulation within the lysosome lead to cell dysfunction and cell death. We investigated whether these shared phenotypes reflected an underlying mechanistic connection between mycobacterial intracellular persistence and the host cell pathway dysfunctional in NPC. METHODS: The induction of NPC phenotypes in macrophages from wild-type mice or obtained from healthy human donors was assessed via infection with mycobacteria and subsequent measurement of lipid levels and intracellular calcium homeostasis. The effect of NPC therapeutics on intracellular mycobacterial load was also assessed. RESULTS: Macrophages infected with persistent intracellular mycobacteria phenocopied NPC cells, exhibiting accumulation of multiple lipid types, reduced lysosomal Ca2+ levels, and defects in intracellular trafficking. These NPC phenotypes could also be induced using only lipids/glycomycolates from the mycobacterial cell wall. These data suggest that persistent intracellular mycobacteria inhibit the NPC pathway, likely via inhibition of the NPC1 protein, and subsequently induce altered acidic store Ca2+ homeostasis. Reduced lysosomal calcium levels may provide a mechanistic explanation for the reduced levels of phagosome-lysosome fusion in mycobacterial infection. Treatments capable of correcting defects in NPC mutant cells via modulation of host cell calcium were of benefit in promoting clearance of mycobacteria from infected host cells. CONCLUSION: These findings provide a novel mechanistic explanation for mycobacterial intracellular persistence, and suggest that targeting interactions between the mycobacteria and host cell pathways may provide a novel avenue for development of anti-TB therapies.

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.