Toxoplasma ceramide synthases: a curious case of gene duplication, divergence and key functionality

Toxoplasma gondii is an obligate, intracellular eukaryotic apicomplexan protozoan parasite that can cause foetal damage and abortion in both animals and humans. Sphingolipids have indispensable functions as signaling molecules and are essential and ubiquitous components of eukaryotic membranes that are both synthesized and scavenged by the Apicomplexa. Ceramide is the precursor for all sphingolipids, and here we report the identification, localisation and analyses of the Toxoplasma ceramide synthases Tg CerS1 and Tg CerS2 and, using a conditional gene regulation approach, establish their roles in pathogenicity and parasite fitness. Interestingly, we observed that whilst Tg CerS1 was a fully functional orthologue of the yeast Lag1p capable of catalysing the conversion of sphinganine to ceramide, in contrast Tg CerS2 was catalytically inactive. Furthermore, genomic deletion of Tg CerS1 using CRISPR/Cas-9 led to viable but slow growing parasites indicating its importance but not indispensability. In contrast, genomic knock out of Tg CerS2 was only accessible utilising the rapamycin-inducible Cre recombinase system. Surprisingly, the results demonstrated that this ‘pseudo’ ceramide synthase, Tg CerS2, has an even greater role in parasite fitness than its catalytically active orthologue (Tg CerS1). Phylogenetic analyses indicated that, as in humans and plants, the ceramide synthase isoforms found in Toxoplasma and other Apicomplexa arose through gene duplication. However, in the Apicomplexa the duplicated copy subsequently evolved into a non-functional ‘pseudo’ ceramide synthase. This arrangement is unique to the Apicomplexa and further illustrates the unusual biology that characterize these protozoan parasites, a feature that could potentially be exploited in the development of new antiprotozoals.

A Lipidomics Atlas of Selected Sphingolipids in Multiple Mouse Nervous System Regions

Many lipids, including sphingolipids, are essential components of the nervous system. Sphingolipids play critical roles in maintaining the membrane structure and integrity and in cell signaling. We used a multi-dimensional mass spectrometry-based shotgun lipidomics platform to selectively analyze the lipid species profiles of ceramide, sphingomyelin, cerebroside, and sulfatide; these four classes of sphingolipids are found in the central nervous system (CNS) (the cerebrum, brain stem, and spinal cord) and peripheral nervous system (PNS) (the sciatic nerve) tissues of young adult wild-type mice. Our results revealed that the lipid species profiles of the four sphingolipid classes in the different nervous tissues were highly distinct. In addition, the mRNA expression of sphingolipid metabolism genes—including the ceramidase synthases that specifically acylate the N-acyl chain of ceramide species and sphingomyelinases that cleave sphingomyelins generating ceramides—were analyzed in the mouse cerebrum and spinal cord tissue in order to better understand the sphingolipid profile differences observed between these nervous tissues. We found that the distinct profiles of the determined sphingolipids were consistent with the high selectivity of ceramide synthases and provided a potential mechanism to explain region-specific CNS ceramide and sphingomyelin levels. In conclusion, we portray for the first time a lipidomics atlas of select sphingolipids in multiple nervous system regions and believe that this type of knowledge could be very useful for better understanding the role of this lipid category in the nervous system.

A STUDY OF ORM EXPRESSION CHANGES FUMONISIN B1-INDUCED PERTURBATIONS OF SPHINGOLIPID HOMEOSTASIS AND DIFFERENTIALLY TOUCHES CERAMIDE SYNTHASE ACTIVITIES

Sphingolipid synthesis is tightly regulated in eukaryotes. This regulation in plants ensures sufcient sphingolipids to support growth, while limiting accumulation of sphingolipid metabolites that induce programmed cell death (PCD). Serine palmitoyltransfersase (SPT) catalyzes the rst step in sphingolipid biosynthesis and is considered the primary sphingolipid homeostatic regulatory point. In this report, Arabidopsis putative SPT regulatory proteins, orosomucoid-like proteins AtORM1 and AtORM2 were found to physically interact with the Arabidopsis SPT and to suppress SPT activity when co-expressed with Arabidopsis SPT subunits LCB1 and LCB2 and the small subunit of SPT in a yeast SPT-decient mutant. Consistent with a role in SPT suppression, AtORM1 and AtORM2 overexpression lines displayed increased resistance to the PCD-inducing mycotoxin fumonisin B1 (FB1), with an accompanying reduced accumulation of longchain bases (LCBs) and C16-fatty acid-containing ceramide accumulation relative to wild type plants. Conversely, RNAi suppression lines of AtORM1 and AtORM2 displayed increased sensitivity to FB1 and an accompanying strong increase in LCBs and C16 fatty acid-containing ceramides relative to wild-type plants. Overexpression lines were also found to have reduced activity of the Class I ceramide synthase that uses C16-fatty acid acyl-CoA and dihydroxy LCB substrates, but increased activity of Class II ceramide synthases that use very long-chain fatty acylCoA and trihydroxy LCB substrates. RNAi suppression lines, in contrast, displayed increased Class I ceramide synthase activity, but reduced Class II ceramide synthase activity. These ndings indicate that ORM-mediation of SPT activity differentially regulates functionally distinct ceramide synthase activities as part of a broader sphingolipid homeostatic regulatory network.

The Role of Ceramide Metabolism and Signaling in the Regulation of Mitophagy and Cancer Therapy

Sphingolipids are bioactive lipids responsible for regulating diverse cellular functions such as proliferation, migration, senescence, and death. These lipids are characterized by a long-chain sphingosine backbone amide-linked to a fatty acyl chain with variable length. The length of the fatty acyl chain is determined by specific ceramide synthases, and this fatty acyl length also determines the sphingolipid’s specialized functions within the cell. One function in particular, the regulation of the selective autophagy of mitochondria, or mitophagy, is closely regulated by ceramide, a key regulatory sphingolipid. Mitophagy alterations have important implications for cancer cell proliferation, response to chemotherapeutics, and mitophagy-mediated cell death. This review will focus on the alterations of ceramide synthases in cancer and sphingolipid regulation of lethal mitophagy, concerning cancer therapy.

Destabilization of β-cell FIT2 by saturated fatty acids contribute to ER stress and diabetes

ABSTRACTWestern type diets are linked to obesity and diabetes partly because of their high saturated fatty acid (SFA) content. We found that SFAs, but not unsaturated fatty acids (USFAs), reduced the number of lipid droplets (LDs) within pancreatic β-cells. Mechanistically, SFAs but not USFAs disabled LD biogenesis by inducing palmitoylation and subsequent ERAD-C mediated degradation of LD formation protein, Fat storage-Inducing Transmembrane protein 2 (FIT2). Targeted ablation of FIT2 reduced β-cell LD numbers, lowered β-cell ATP levels, reduced Ca2+ signaling, downregulated β-cell transcription factors (RNA sequencing analysis), and exacerbated diet-induced diabetes in mice. Subsequent mass spectrometry studies revealed increased C16:0 ceramide accumulation in islets of mice lacking β-cell FIT2 under lipotoxic conditions. Inhibition of ceramide synthases ameliorated the enhanced ER stress. Overexpression of FIT2 increased number of intracellular LDs and rescued SFA-induced ER-stress and apoptosis thereby highlighting the protective role of FIT2 and LDs against β-cell lipotoxicity and diet-induced diabetes.

Dissecting the regulatory roles of ORM proteins in the sphingolipid pathway of plants

Sphingolipids are a vital component of plant cellular endomembranes and carry out multiple functional and regulatory roles. Different sphingolipid species confer rigidity to the membrane structure, facilitate trafficking of secretory proteins, and initiate programmed cell death. Although the regulation of the sphingolipid pathway is yet to be uncovered, increasing evidence has pointed to orosomucoid proteins (ORMs) playing a major regulatory role and potentially interacting with a number of components in the pathway, including both enzymes and sphingolipids. However, experimental exploration of new regulatory interactions is time consuming and often infeasible. In this work, a computational approach was taken to address this challenge. A metabolic network of the sphingolipid pathway in plants was reconstructed. The steady-state rates of reactions in the network were then determined through measurements of growth and cellular composition of the different sphingolipids in Arabidopsis seedlings. The Ensemble modeling framework was modified to accurately account for activation mechanisms and subsequently used to generate sets of kinetic parameters that converge to the measured steady-state fluxes in a thermodynamically consistent manner. In addition, the framework was appended with an additional module to automate screening the parameters and to output models consistent with previously reported network responses to different perturbations. By analyzing the network’s response in the presence of different combinations of regulatory mechanisms, the model captured the experimentally observed repressive effect of ORMs on serine palmitoyltransferase (SPT). Furthermore, predictions point to a second regulatory role of ORM proteins, namely as an activator of class II (or LOH1 and LOH3) ceramide synthases. This activating role was found to be modulated by the concentration of free ceramides, where an accumulation of these sphingolipid species dampened the activating effect of ORMs on ceramide synthase. The predictions pave the way for future guided experiments and have implications in engineering crops with higher biotic stress tolerance.