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