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.

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.

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.