Fatty acid transport protein 2 interacts with ceramide synthase 2 to promote ceramide synthesis.

Dihydroceramide is a lipid molecule generated via the action of (dihydro)ceramide synthases (CerSs), which use two substrates, namely sphinganine and fatty acyl-CoAs. Sphinganine is generated via the sequential activity of two integral membrane proteins located in the endoplasmic reticulum. Less is known about the source of the fatty acyl-CoAs, although a number of cytosolic proteins in the pathways of acyl-CoA generation modulate ceramide synthesis via direct or indirect interaction with the CerSs. In this study, we demonstrate, by proteomic analysis of immunoprecipitated proteins, that fatty acid transporter protein 2 (FATP2) (also known as very long-chain acyl-CoA synthetase) directly interacts with CerS2 in mouse liver. Studies in cultured cells demonstrated that other members of the FATP family can also interact with CerS2, with the interaction dependent on both proteins being catalytically active. In addition, transfection of cells with FATP1, FATP2, or FATP4 increased ceramide levels although only FATP2 and 4 increased dihydroceramide levels, consistent with their known intracellular locations. Finally, we show that lipofermata, an FATP2 inhibitor which is believed to directly impact tumor cell growth via modulation of FATP2, decreased de novo dihydroceramide synthesis, suggesting that some of the proposed therapeutic effects of lipofermata may be mediated via (dihydro)ceramide rather than directly via acyl-CoA generation. In summary, our study reinforces the idea that manipulating the pathway of fatty acyl-CoA generation will impact a wide variety of down-stream lipids, not least the sphingolipids, which utilize two acyl-CoA moieties in the initial steps of their synthesis.

16pdel lipid changes in iPSC-derived neurons and function of FAM57B in lipid metabolism and synaptogenesis.

The complex 16p11.2 deletion syndrome (16pdel) is accompanied by neurological disorders, including epilepsy, autism spectrum disorder, and intellectual disability. We demonstrated that 16pdel iPSC differentiated neurons from affected people show augmented local field potential activity and altered ceramide-related lipid species relative to unaffected. FAM57B, a poorly characterized gene in the 16p11.2 interval, has emerged as a candidate tied to symptomatology. We found that FAM57B modulates ceramide synthase (CerS) activity, but is not a CerS per se. In FAM57B mutant human neuronal cells and zebrafish brain, composition and levels of sphingolipids and glycerolipids associated with cellular membranes are disrupted. Consistently, we observed aberrant plasma membrane architecture and synaptic protein mislocalization, which were accompanied by depressed brain and behavioral activity. Together, these results suggest that haploinsufficiency of FAM57B contributes to changes in neuronal activity and function in 16pdel syndrome through a crucial role for the gene in lipid metabolism.

Dependence of ABCB1 transporter expression and function on distinct sphingolipids generated by ceramide synthases-2 and -6 in chemoresistant renal cancer.

Oncogenic multidrug resistance is commonly intrinsic to renal cancer based on the physiological expression of detoxification transporters, particularly ABCB1, thus hampering chemotherapy. ABCB1 activity is directly dependent on its lipid microenvironment, localizing to cholesterol- and sphingomyelin (SM)-rich domains. As ceramides are the sole source for SMs, we hypothesized that ceramide synthase (CerS)-derived ceramides regulate ABCB1 activity. Using data from RNA-Seq databases, we found that patient kidney tumors exhibited increased CerS2 mRNA, which was inversely correlated with CerS6 mRNA in ABCB1+ clear cell carcinomas. Endogenous elevated CerS2 and lower CerS5/6 mRNA and protein resulted in disproportionately higher CerS2 to CerS5/6 activities (approximately twofold) in chemoresistant ABCB1high (A498, Caki-1) compared with chemosensitive ABCB1low (ACHN, normal human proximal convoluted tubule cell) cells. In addition, lipidomics analyses by HPLC-MS/MS showed bias toward CerS2-associated C20:0/C20:1-ceramides compared with CerS5/6-associated C14:0/C16:0-ceramides (2:1). SMs were similarly altered. We demonstrated that chemoresistance to doxorubicin in ABCB1high cells was partially reversed by inhibitors of de novo ceramide synthesis (l-cycloserine) and CerS (fumonisin B1) in cell viability assays. Downregulation of CerS2/6, but not CerS5, attenuated ABCB1 mRNA, protein, plasma membrane localization, rhodamine 123+ efflux transport activity, and doxorubicin resistance. Similar findings were observed with catalytically inactive CerS6-H212A. Furthermore, CerS6-targeting siRNA shifted ceramide and SM composition to ultra long-chain species (C22-C26). Inhibitors of endoplasmic reticulum-associated degradation (eeyarestatin I) and the proteasome (MG132, bortezomib) prevented ABCB1 loss induced by CerS2/6 downregulation. We conclude that a critical balance in ceramide/SM species is prerequisite to ABCB1 expression and functionalization, which could be targeted to reverse multidrug resistance in renal cancers.

A novel C-terminal DxRSDxE motif in ceramide synthases involved in dimer formation.

Ceramide is a lipid moiety synthesized via the enzymatic activity of ceramide synthases (CerSs), six of which have been identified in mammalian cells, and each of which uses a unique subset of acyl-CoAs for ceramide synthesis. The CerSs are part of a larger gene family, the Tram-Lag-CLN8 domain family. Here, we identify a unique, C-terminal motif, the DxRSDxE motif, which is only found in CerSs and not in other Tram-Lag-CLN8 family members. Deletion of this motif in either CerS2 or in CerS6 did not affect the ability of either enzyme to generate ceramide using both an in vitro assay and metabolic labeling, but deletion of this motif did affect the activity of CerS2 when coexpressed with CerS6. Surprisingly, transfection of cells with either CerS2 or CerS6 lacking the motif did not result in changes in cellular ceramide levels. We found that CerS2 and CerS6 interact with each other, as shown by immunoprecipitation, but deletion of the DxRSDxE motif impeded this interaction. Moreover, proteomics analysis of cells transfected with CerS6Δ338-344 indicated that deletion of the C-terminal motif impacted cellular protein expression, and in particular, the levels of ORMDL1, a negative regulator of sphingolipid synthesis. We suggest that this novel C-terminal motif regulates CerS dimer formation and thereby impacts ceramide synthesis.

The Complex Tail of Circulating Sphingolipids in Atherosclerosis and Cardiovascular Disease.

Sphingolipids (SLs) are critical players in a number of cellular processes and have recently been implicated in a large number of human diseases, including atherosclerosis and cardiovascular disease (CVD). SLs are generated intracellularly in a stepwise manner, starting with the generation of the sphingoid long chain base (LCB), followed by N-acylation of the LCB to form ceramide, which can be subsequently metabolized to sphingomyelin and glycosphingolipids. Fatty acids, which are taken up by cells prior to their activation to fatty acyl-CoAs, are used in 2 of these enzymatic steps, including by ceramide synthases, which use fatty acyl-CoAs of different chain lengths to generate ceramides with different N-acyl chain lengths. Recently, alterations in plasma ceramides with specific N-acyl chain lengths and degrees of saturation have emerged as novel biomarkers for the prediction of atherosclerosis and overall cardiovascular risk in the general population. We briefly review the sources of plasma SLs in atherosclerosis, the roles of SLs in CVD, and the possible use of the "ceramide score" as a prognostic marker for CVD.

Ceramide synthases: Reflections on the impact of Dr. Lina M. Obeid.

Sphingolipids are a family of lipids that are critical to cell function and survival. Much of the recent work done on sphingolipids has been performed by a closely-knit family of sphingolipid researchers, which including our colleague, Dr. Lina Obeid, who recently passed away. We now briefly review where the sphingolipid field stands today, focusing in particular on areas of sphingolipid research to which Dr. Obeid made valued contributions. These include the 'many-worlds' view of ceramides and the role of a key enzyme in the sphingolipid biosynthetic pathway, namely the ceramide synthases (CerS). The CerS contain a number of functional domains and also interact with a number of other proteins in lipid metabolic pathways, fulfilling Dr. Obeid's prophecy that ceramides, and the enzymes that generate ceramides, form the critical hub of the sphingolipid metabolic pathway.

The role of ceramide in regulating endoplasmic reticulum function.

Sphingolipids (SLs) are an important class of membrane lipids containing a long chain sphingoid base backbone. SL synthesis is compartmentalized between two major cell organelles, the endoplasmic reticulum (ER) and the Golgi apparatus. The initial steps of sphingolipid synthesis take place in the ER, where the simplest SL, ceramide, is synthesized. Although ceramide is a critical membrane component, an imbalance of ceramide levels can have significant deleterious effects on cell properties leading to events such as apoptosis. For this reason and others, ER ceramide levels must be tightly regulated. Here, we describe the biological and biophysical properties of ceramide and discuss how this might impact the ER membrane. This article is part of a special issue entitled: ER Platforms for Membrane Lipid Dynamics.