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Abstract

Sphingolipids are essential lipid constituents of cellular membranes and their complex metabolic pathways interconnect amino acid, fatty acid, phosphoglycerolipid and carbohydrate metabolism. They influence the structure of cellular membranes, participate in signal transduction cascades and orchestrate cell-cell communication. Therefore, they contribute to the regulation of numerous cellular processes including proliferation, apoptosis and cellular differentiation. The core of sphingolipids is a sphingoid base, a long chain amino-alcohol, whose synthesis is initiated by the action of the enzyme serine palmitolytransferase (SPT). SPT is a complex of 3 polypeptides and catalyzes the rate-limiting step of the sphingolipid biosynthesis. Therefore SPT activity is regulated, e.g. by inhibiting ORMDL peptides and by feedback regulation through ceramide concentrations in the membrane of the endoplasmic reticulum. Opposite to that the small subunit of the SPT complex (SPTSSA or SPTSSB) enhances the activity of the enzyme and determines the chain length of the final sphingoid bases. The length of the sphingoid bases in ceramides has been shown to have an impact on the properties of the bilayer, especially in influencing the order and fluidity of membranes, which can result in inducing phase separation for the formation of microdomains. As microdomains act as a hub for cell signaling by selectively recruiting or excluding proteins and facilitating the interaction between interaction partners, they are essential for modulating signaling pathways and associated signaling events. It has been shown that the sphingoid base length of ceramide has a greater impact on initiating microdomain formation and intermolecular interactions within the ceramide-rich gel phase as compared to the length of the acyl chain. As longer sphingoid bases tend to enhance stability of the microdomains, this possibly influences the duration and amplitude of associated signaling events. As of now, the latter had not been studied and the physiological roles and biological importance of sphingoid bases with carbon chains exceeding 18 carbons remain unclear. To fill this gap, I successfully generated CRISPR-Cas9 edited SPTSSA knock out (KO) monoclonal neuroblastoma cells (SH-SY5Y). SPTSSA KO caused a decrease in SPT activity and an more than 7-fold increase in the amount of ceramides containing C20-sphingosine as compared to corresponding control cell clones, in line with recent reports in other cellular systems. However, steady state levels of total ceramides were not significantly affected. Compared to ceramides with C18-sphingosine, ceramides with C20-sphingosine were enriched in C22- and C24-acyl chains, while C16-acyl chains were strongly decreased. To study the impact of this shift in ceramide composition on cell homeostasis and function, I monitored the cellular changes in SPTSSA KO cells both at steady state and upon induction of differentiation into neuron-like cells. Through multiomics studies, I revealed that the changes in distribution of ceramide species altered a small subset of the proteome, phosho-proteome and the lipidome of the cell at a steady state. Most pronounced changes were observed in concentrations of soluble metabolites. Comparison of the proteome after generation of the SPTSSA KO clones and after more than 30 passages, reveal a few proteins, which had persistent differential expression between KO and control groups (AHNAK, SLC27A6, MVP, CNTNAP1 SH3PXD2A). However, those proteins associated with cell cycle and proliferation lost differential expression over time. This was paralleled by an initially reduced proliferation rate in KO clones, which vanished over time. Apparently, KO cells adopted to the genetic modification and transitioned back towards a more proliferative phenotype. Analysis of EGF receptor signaling by evaluating the alternations in the phosphorylation status of tyrosine 992 (Y992), tyrosine 1173 (Y1173) and tyrosine 1069 (Y1069) following 10 min of EGF stimulation did not reveal noticeable differences in the intensity of induced phosphorylation patterns at the specified EGFR phosphorylation sites and their respective downstream targets. Finally, I investigated the effect of SPTSSA KO on neuronal differentiation. Induction of differentiation reduced cell viability by inducing cell death specifically in SPTSSA KO. Furthermore, the SPTSSA KO caused a significant increase in the mitochondrial membrane potential as compared to that of control cells while not leading to significant differences in mitochondrial mass. The proteome analysis at steady state already indicated potential modifications in mitochondrial function, which were attributed to altered expression of the mitochondrial respiratory chain complex (I, III, and IV) components. Specifically, I confirmed the decreased levels of MT-CO2, the core catalytic subunit of complex IV, and the increased level of UQCRC2, a core subunit of complex III, in KO cells by Western blot analysis. In conclusion, this study revealed that alterations in the composition of the SPT complex and corresponding ratio of sphingolipids containing either C18- or C20-sphingosine could lead to changes in the proteome, phosphoprotome, metabolome, and lipidome of the cells as well as to differential fitness for cell transitions. These findings underscored the importance of maintaining ceramide homeostasis for fundamental cellular physiology, as exemplified here for cell differentiation and mitochondrial homeostasis.