Stefka D. Spassieva

Necessary Role for the Lag1p Motif in (Dihydro)ceramide Synthase Activity

Lag1 (longevity assurance gene 1) homologues, a family of transmembrane proteins found in all eukaryotes, have been shown to be necessary for (dihydro)ceramide synthesis. All Lag1 homologues contain a highly conserved stretch of 52 amino acids known as the Lag1p motif. However, the functional significance of the conserved Lag1p motif for (dihydro)ceramide synthesis is currently unknown. In this work, we have investigated the function of the motif by introducing eight point mutations in the Lag1p motif of the mouse LASS1 (longevity assurance homologue 1 of yeast Lag1). The (dihydro)ceramide synthase activity of the mutants was tested using microsomes in HeLa cells and in vitro. Six of the mutations resulted in loss of activity in cells and in vitro. In addition, our results showed that C18:0 fatty acid CoA (but not cis-C18:1 fatty acid CoAs) are substrates for LASS1 and that LASS1 in HeLa cells is sensitive to fumonisin B1, an in vitro inhibitor of (dihydro)ceramide synthase. Moreover, we mutated the Lag1p motif of another Lag homologue, human LASS5. The amino acid substitutions in the human LASS5 were the same as in mouse LASS1, and had the same effect on the in vitro activity of LASS5, suggesting the Lag1p motif appears to be essential for the enzyme activity of all Lag1 homologues.

A deficiency of ceramide biosynthesis causes cerebellar purkinje cell neurodegeneration and lipofuscin accumulation.

Sphingolipids, lipids with a common sphingoid base (also termed long chain base) backbone, play essential cellular structural and signaling functions. Alterations of sphingolipid levels have been implicated in many diseases, including neurodegenerative disorders. However, it remains largely unclear whether sphingolipid changes in these diseases are pathological events or homeostatic responses. Furthermore, how changes in sphingolipid homeostasis shape the progression of aging and neurodegeneration remains to be clarified. We identified two mouse strains, flincher (fln) and toppler (to), with spontaneous recessive mutations that cause cerebellar ataxia and Purkinje cell degeneration. Positional cloning demonstrated that these mutations reside in the Lass1 gene. Lass1 encodes (dihydro)ceramide synthase 1 (CerS1), which is highly expressed in neurons. Both fln and to mutations caused complete loss of CerS1 catalytic activity, which resulted in a reduction in sphingolipid biosynthesis in the brain and dramatic changes in steady-state levels of sphingolipids and sphingoid bases. In addition to Purkinje cell death, deficiency of CerS1 function also induced accumulation of lipofuscin with ubiquitylated proteins in many brain regions. Our results demonstrate clearly that ceramide biosynthesis deficiency can cause neurodegeneration and suggest a novel mechanism of lipofuscin formation, a common phenomenon that occurs during normal aging and in some neurodegenerative diseases.

Disruption of ceramide synthesis by CerS2 down-regulation leads to autophagy and the unfolded protein response

Ceramide metabolism has come under recent scrutiny because of its role in cellular stress responses. CerS2 (ceramide synthase 2) is one of the six mammalian isoforms of ceramide synthase and is responsible for the synthesis of VLC (very-long-chain) ceramides, e.g. C24, C24:1. To study the role of CerS2 in ceramide metabolism and cellular homoeostasis, we down-regulated CerS2 using siRNA (small interfering RNA) and examined several aspects of sphingolipid metabolism and cell stress responses. CerS2 down-regulation had a broad effect on ceramide homoeostasis, not just on VLC ceramides. Surprisingly, CerS2 down-regulation resulted in significantly increased LC (long-chain) ceramides, e.g. C14, C16, and our results suggested that the increase was due to a ceramide synthase-independent mechanism. CerS2-down-regulation-induced LC ceramide accumulation resulted in growth arrest which was not accompanied by apoptotic cell death. Instead, cells remained viable, showing induction of autophagy and activation of PERK [PKR (double-stranded-RNA-dependent protein kinase)-like endoplasmic reticulum kinase] and IRE1 (inositol-requiring 1) pathways [the latter indicating activation of the UPR (unfolded protein response)].

Selective knockdown of ceramide synthases reveals complex interregulation of sphingolipid metabolism

Mammalian ceramide synthases 1 to 6 (CerS1–6) generate Cer in an acyl-CoA-dependent manner, and expression of individual CerS has been shown to enhance the synthesis of ceramides with particular acyl chain lengths. However, the contribution of each CerS to steady-state levels of specific Cer species has not been evaluated. We investigated the knockdown of individual CerS in the MCF-7 human breast adenocarcinoma cell line by using small-interfering RNA (siRNA). We found that siRNA-induced downregulation of each CerS resulted in counter-regulation of nontargeted CerS. Additionally, each CerS knockdown produced unique effects on the levels of multiple sphingolipid species. For example, downregulation of CerS2 decreased very long-chain Cer but increased levels of CerS4, CerS5, and CerS6 expression and upregulated long-chain and medium-long-chain sphingolipids. Conversely, CerS6 knockdown decreased C16:0-Cer but increased CerS5 expression and caused non-C16:0 sphingolipids to be upregulated. Knockdown of individual CerS failed to decrease total sphingolipids or upregulate sphingoid bases. Treatment with siRNAs targeting combined CerS, CerS2, CerS5, and CerS6, did not change overall Cer or sphingomyelin mass but caused upregulation of dihydroceramide and hexosylceramide and promoted endoplasmic reticulum stress. These data suggest that sphingolipid metabolism is robustly regulated by both redundancy in CerS-mediated Cer synthesis and counter-regulation of CerS expression.

CERT depletion predicts chemotherapy benefit and mediates cytotoxic and polyploid‐specific cancer cell death through autophagy induction

Chromosomal instability (CIN) has been implicated in multidrug resistance and the silencing of the ceramide transporter, CERT, promotes sensitization to diverse cytotoxics. An improved understanding of mechanisms governing multidrug sensitization might provide insight into pathways contributing to the death of CIN cancer cells. Using an integrative functional genomics approach, we find that CERT‐specific multidrug sensitization is associated with enhanced autophagosome–lysosome flux, resulting from the expression of LAMP2 following CERT silencing in colorectal and HER2+ breast cancer cell lines. Live cell microscopy analysis revealed that CERT depletion induces LAMP2‐dependent death of polyploid cells following exit from mitosis in the presence of paclitaxel. We find that CERT is relatively over‐expressed in HER2+ breast cancer and CERT protein expression acts as an independent prognostic variable and predictor of outcome in adjuvant chemotherapy‐treated patients with primary breast cancer. These data suggest that the induction of LAMP2‐dependent autophagic flux through CERT targeting may provide a rational approach to enhance multidrug sensitization and potentiate the death of polyploid cells following paclitaxel exposure to limit the acquisition of CIN and intra‐tumour heterogeneity. Copyright

Guggulsterone and bexarotene induce secretion of exosome-associated breast cancer resistance protein and reduce doxorubicin resistance in MDA-MB-231 cells

Many breast cancer cells acquire multidrug resistance (MDR) mediated by ABC transporters such as breast cancer resistance protein (BCRP/ABCG2). Here we show that incubation of human breast cancer MDA‐MB‐231 cells with farnesoid X receptor antagonist guggulsterone (gug) and retinoid X receptor agonist bexarotene (bex) elevated ceramide, a sphingolipid known to induce exosome secretion. The gug+bex combination reduced cellular levels of BCRP to 20% of control cells by inducing its association and secretion with exosomes. Exogenous C6 ceramide also induced secretion of BCRP‐associated exosomes, while siRNA‐mediated knockdown or GW4869‐mediated inhibition of neutral sphingomyelinase 2 (nSMase2), an enzyme generating ceramide, restored cellular BCRP. Immunocytochemistry showed that ceramide elevation and concurrent loss of cellular BCRP was prominent in Aldefluor‐labeled breast cancer stem‐like cells. These cells no longer excluded the BCRP substrate Hoechst 33342 and showed caspase activation and apoptosis induction. Consistent with reduced BCRP, ABC transporter assays showed that gug+bex increased doxorubicin retention and that the combination of gug+bex with doxorubicin enhanced cell death by more than fivefold. Taken together, our results suggest a novel mechanism by which ceramide induces BCRP secretion and reduces MDR, which may be useful as adjuvant drug treatment for sensitizing breast cancer cells and cancer stem cells to chemotherapy.

Primary cilia in stem cells and neural progenitors are regulated by neutral sphingomyelinase 2 and ceramide

Human embryonic stem and induced pluripotent stem cell–derived neuroprogenitors (NPs) develop primary cilia. Ciliogenesis depends on the sphingolipid ceramide and its interaction with atypical PKC, both of which distribute to the primary cilium and the apicolateral cell membrane in NP rosettes.

Combination of C17 Sphingoid Base Homologues and Mass Spectrometry Analysis as a New Approach to Study Sphingolipid Metabolism

In recent years, sphingolipid metabolites ceramide, sphingosine, and sphingosine-1-phosphate have emerged as important second messengers in addition to their role as precursors of biomembrane components. The investigation of these sphingolipid metabolites requires the development of new, more sensitive methods for assaying the enzymes involved in their production. This chapter describes the utilization of mass spectrometry technology in combination with nonnaturally occurring C(17) sphingoid bases in the in vitro assays of two of the enzymes of the sphingolipid pathway, ceramide synthase and sphingosine kinase. These new in vitro methods provide high sensitivity and extreme accuracy even when crude extracts are used as enzyme sources.

A lesion mimic phenotype in tomato obtained by isolating and silencing an Lls1 homologue

Lesion mimic phenotypes serve as a tool to study the regulation of cell death in plants. In order to obtain a tomato lesion mimic phenotype, we used the conservation of the lethal leaf spot 1 (Lls1) genes between plant species. The tomato Lls1 homologue was cloned, sequenced and analyzed. It showed high conservation at the protein and nucleotide level compared to the genes from maize and Arabidopsis. Using virus induced gene silencing, we obtained a phenotype resembling the lls1 mutant in maize. Lesion formation, lesion spreading and light dependence of the tomato lls1 phenotype are strong indications for the functional conservation of the Lls1 gene between the dicotyledonous tomato and the monocotyledonous maize

Elevation of 20-carbon long chain bases due to a mutation in serine palmitoyltransferase small subunit b results in neurodegeneration

Significance Sphingolipids are essential in eukaryotes and are particularly important in neural tissues. Generally, sphingolipids have an 18-carbon (C18) long chain base (LCB) backbone. However, low-abundance sphingolipids containing LCBs of 16 or 20 carbons have also been discovered. Yet their specific functions and biological significance is not known. This work demonstrates that elevation of 20-carbon LCBs and/or sphingolipids containing C20 LCBs has detrimental neurodegenerative effects in the brain and the retina, leading to perturbation of protein homeostasis. This work describes, for the first time to our knowledge, the specific pathological roles of a class of low-abundance LCBs in vivo. Sphingolipids typically have an 18-carbon (C18) sphingoid long chain base (LCB) backbone. Although sphingolipids with LCBs of other chain lengths have been identified, the functional significance of these low-abundance sphingolipids is unknown. The LCB chain length is determined by serine palmitoyltransferase (SPT) isoenzymes, which are trimeric proteins composed of two large subunits (SPTLC1 and SPTLC2 or SPTLC3) and a small subunit (SPTssa or SPTssb). Here we report the identification of an Sptssb mutation, Stellar (Stl), which increased the SPT affinity toward the C18 fatty acyl-CoA substrate by twofold and significantly elevated 20-carbon (C20) LCB production in the mutant mouse brain and eye, resulting in surprising neurodegenerative effects including aberrant membrane structures, accumulation of ubiquitinated proteins on membranes, and axon degeneration. Our work demonstrates that SPT small subunits play a major role in controlling SPT activity and substrate affinity, and in specifying sphingolipid LCB chain length in vivo. Moreover, our studies also suggest that excessive C20 LCBs or C20 LCB-containing sphingolipids impair protein homeostasis and neural functions.