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Sphingolipids play key roles in membrane structure and cellular signaling. Ceramide synthase (CerS)-generated ceramide is implicated in cellular stress responses and induction of apoptosis. Ceramide and other sphingolipids are linked to the induction of ER stress response pathways. However, the mechanisms by which ceramide modulates ER stress signaling are not well understood. Here, we show that the ER stress inducer brefeldin A (BFA) causes increased glycosylation of CerS6, and that treatment with BFA causes increased endogenous ceramide accumulation. To our surprise, we found that CerS6 activity was not affected by BFA-induced glycosylation. Instead, our data show that basal glycosylation of CerS6 at Asn18 is required for CerS6 activity. We used a robust HCT116 CRISPR-Cas9 CerS6 KO with reintroduction of either WT CerS6 or a mutant CerS6 with a point mutation at asparagine-18 to an alanine (N18A) which abrogated glycosylation at that residue. Our data show that cells stably expressing the N18A mutant CerS6 had significantly lower activity in vitro and in situ as compared to WT CerS6 expressing cells. Further, the defective CerS6 with N18A mutation also had defects in GSK3β, AKT, JNK, and STAT3 signaling. Despite being required for CerS6 activity, Asn18 glycosylation did not influence ER stress response pathways. Overall, our study provides vital insight into the regulation of CerS6 activity by posttranslational modification at Asn18 and identifies glycosylation of CerS6 to be important for ceramide generation and regulation of downstream cellular signaling pathways.

Abstract Mechanisms that alter protein phosphatase 2A (PP2A)‐dependent lung tumour suppression via the I2PP2A/SET oncoprotein are unknown. We show here that the tumour suppressor ceramide binds I2PP2A/SET selectively in the nucleus and including its K209 and Y122 residues as determined by molecular modelling/simulations and site‐directed mutagenesis. Because I2PP2A/SET was found overexpressed, whereas ceramide was downregulated in lung tumours, a sphingolipid analogue drug, FTY720, was identified to mimick ceramide for binding and targeting I2PP2A/SET, leading to PP2A reactivation, lung cancer cell death, and tumour suppression in vivo. Accordingly, while molecular targeting of I2PP2A/SET by stable knockdown prevented further tumour suppression by FTY720, reconstitution of WT‐I2PP2A/SET expression restored this process. Mechanistically, targeting I2PP2A/SET by FTY720 mediated PP2A/RIPK1‐dependent programmed necrosis (necroptosis), but not by apoptosis. The RIPK1 inhibitor necrostatin and knockdown or genetic loss of RIPK1 prevented growth inhibition by FTY720. Expression of WT‐ or death‐domain‐deleted (DDD)‐RIPK1, but not the kinase‐domain‐deleted (KDD)‐RIPK1, restored FTY720‐mediated necroptosis in RIPK1−/− MEFs. Thus, these data suggest that targeting I2PP2A/SET by FTY720 suppresses lung tumour growth, at least in part, via PP2A activation and necroptosis mediated by the kinase domain of RIPK1.

Mehmet Demir, Ersin Oba, Efe Can, Mahmut Odabasi, Semra Tiryaki, Erhan Ozdal, Hakan SensozEye Clinic, Sisli Etfal Training and Research Hospital, Istanbul, TurkeyPurpose: The inspection of foveal and parafoveal thickness in healthy pregnant women in the last trimester.Materials and methods: This study included 40 healthy pregnant women in their last trimester (study group: 40 women, 80 eyes) and 37 nonpregnant women (control group: 37 women, 74 eyes). Visual acuity, intraocular pressure, slit lamp examination of anterior and posterior segments, and visual field examination with automated perimetry were performed in both groups. Foveal and parafoveal thickness in the four quadrants (upper, nasal, temporal, and inferior parafoveal) and peripapillary retinal nerve fiber layer were measured by optical coherence tomography. There were no systemic or ocular problems in either group. Findings were analyzed with statistical software. A P value

Membrane contact sites (MCS), close membrane apposition between organelles, are platforms for interorganellar transfer of lipids including cholesterol, regulation of lipid homeostasis, and co-ordination of endocytic trafficking. Sphingosine kinases (SphKs), two isoenzymes that phosphorylate sphingosine to the bioactive sphingosine-1-phosphate (S1P), have been implicated in endocytic trafficking. However, the physiological functions of SphKs in regulation of membrane dynamics, lipid trafficking and MCS are not known. Here, we report that deletion of SphKs decreased S1P with concomitant increases in its precursors sphingosine and ceramide, and markedly reduced endoplasmic reticulum (ER) contacts with late endocytic organelles. Expression of enzymatically active SphK1, but not catalytically inactive, rescued the deficit of these MCS. Although free cholesterol accumulated in late endocytic organelles in SphK null cells, surprisingly however, cholesterol transport to the ER was not reduced. Importantly, deletion of SphKs promoted recruitment of the ER-resident cholesterol transfer protein Aster-B (also called GRAMD1B) to the plasma membrane (PM), consistent with higher accessible cholesterol and ceramide at the PM, to facilitate cholesterol transfer from the PM to the ER. In addition, ceramide enhanced in vitro binding of the Aster-B GRAM domain to phosphatidylserine and cholesterol liposomes. Our study revealed a previously unknown role for SphKs and sphingolipid metabolites in governing diverse MCS between the ER network and late endocytic organelles versus the PM to control the movement of cholesterol between distinct cell membranes.

Enhanced ceramide glycosylation catalyzed by glucosylceramide synthase (GCS) limits therapeutic efficiencies of antineoplastic agents including doxorubicin in drug-resistant cancer cells. Aimed to determine the role of GCS in tumor response to chemotherapy, a new mixed-backbone oligonucleotide (MBO-asGCS) with higher stability and efficiency has been generated to silence human GCS gene. MBO-asGCS was taken up efficiently in both drug-sensitive and drug-resistant cells, but it selectively suppressed GCS overexpression, and sensitized drug-resistant cells. MBO-asGCS increased doxorubicin sensitivity by 83-fold in human NCI/ADR-RES, and 43-fold in murine EMT6/AR1 breast cancer cells, respectively. In tumor-bearing mice, MBO-asGCS treatment dramatically inhibited the growth of multidrug-resistant NCI/ADR-RE tumors, decreasing tumor volume to 37%, as compared with scrambled control. Furthermore, MBO-asGCS sensitized multidrug-resistant tumors to chemotherapy, increasing doxorubicin efficiency greater than 2-fold. The sensitization effects of MBO-asGCS relied on the decreases of gene expression and enzyme activity of GCS, and on the increases of C(18)-ceramide and of caspase-executed apoptosis. MBO-asGCS was accumulation in tumor xenografts was greater in other tissues, excepting liver and kidneys; but MBO-asGCS did not exert significant toxic effects on liver and kidneys. This study, for the first time in vivo, has demonstrated that GCS is a promising therapeutic target for cancer drug resistance, and MBO-asGCS has the potential to be developed as an antineoplastic agent.