Bcl2L13 is a ceramide synthase inhibitor in glioblastoma

Jensen, Samuel A.; Calvert, Andrea E.; Volpert, Giora; Kouri, Fotini M.; Hurley, Lisa; Luciano, Janina P.; Wu, Yongfei; Chalastanis, Alexandra; Futerman, Anthony H.; Stegh, Alexander H.

doi:10.1073/pnas.1316700111

Cited by 93 publications

(76 citation statements)

References 30 publications

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“…Taken together, these results suggest that transcriptional activation of acid ceramidase in our model is not mediated by ceramide itself and does not depend on the catalytic activity of CerS. Recently it has been demonstrated that ectopically expressed Bcl2L13 binds to CerS6 (51). Therefore, one possibility is that elevated expression of acid ceramidase in response to CerS6 overexpression occurs as a consequence of altered protein-protein interactions with non-CerS proteins.…”

Section: Discussionsupporting

confidence: 59%

Expression of Ceramide Synthase 6 Transcriptionally Activates Acid Ceramidase in a c-Jun N-terminal Kinase (JNK)-dependent Manner

Tirodkar¹,

Lü²,

Bai³

et al. 2015

Journal of Biological Chemistry

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Background:Ceramide is important for cellular signaling. Results: Increasing the expression of ceramide synthase 6 (CerS6) results in transcriptional activation of acid ceramidase independent of catalytic CerS6 activity. Conclusion: Modulation of a single member of the ceramide synthase family impacts on sphingolipid composition and ceramide metabolizing enzymes. Significance: Understanding how CerS impacts gene expression and signaling is important for the development of novel therapeutic approaches.

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Section: Discussionsupporting

confidence: 59%

Expression of Ceramide Synthase 6 Transcriptionally Activates Acid Ceramidase in a c-Jun N-terminal Kinase (JNK)-dependent Manner

Tirodkar¹,

Lü²,

Bai³

et al. 2015

Journal of Biological Chemistry

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“…1 ) has been used previously to assay CerS ( 17,18,21 ), no attempts have been made to minimize reaction volumes to allow the use of small amounts of biological material or to improve the mode of separating NBD-lipid substrates and products. We have now been able to decrease the reaction volume to a minimum of 20 µl, rather than the 100-250 µl used previously with [4, H]Sph ( 22 ) or with NBD-Sph ( 21 ), permitting the use of small amounts of protein for the assay.…”

Section: Optimizing Conditions For Use Of Nbd-sph In the Cers Assaymentioning

confidence: 99%

“…Because CerSs appear to be involved in the pathology of a number of human diseases (15)(16)(17)(18)(19), the assay that we have described in this study might prove useful in a clinical setting. Furthermore, SPE column chromatography could be were obtained using both methods with all three CerSs.…”

Section: Spe Chromatography To Separate Nbd Lipidsmentioning

confidence: 99%

“…Mammals contain six distinct CerSs, with each using a subset of fatty acylCoAs for the N -acylation of sphinganine (Sph) to generate dihydroceramides or of sphingosine to generate ceramides ( 2, 3 ). CerSs have become the focus of great interest due to the realization that ceramides with different acyl chain lengths play distinct roles in cell physiology ( 4-6 ), with studies focusing on biochemical analysis of the mode of CerS regulation ( 7-13 ), on biological approaches using CerS-null mice ( 4,6,14 ), and on pathophysiological studies using tissues from patients in which CerS activity changes during the development and progression of pathology (15)(16)(17)(18)(19). …”

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confidence: 99%

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A rapid ceramide synthase activity using NBD-sphinganine and solid phase extraction

Tidhar

Sims

Rosenfeld-Gur

et al. 2015

Journal of Lipid Research

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For many years, CerS activity was assayed using radioactive substrates [either 3 H-Sph ( 7 ) or 14 C-labeled acyl-CoAs ( 20 )], with separation of the substrate and products performed by TLC. Recently, a fl uorescent Sph analog has become available, (7-nitro-2-1,3-benzoxadiazol-4-yl) (2 S ,3 R )-2-aminooctadecane-1,3-diol (NBD-Sph), which is a substrate for CerS ( 18,21 ). Clearly, the use of fl uorescent substrates is preferable to use of radioactive substrates as it avoids radiation hazards ( 21 ).We now describe an assay using NBD-Sph, in which the fl uorescent lipid products are separated by solid phase extraction (SPE) C18 chromatography using a 96-well plate. This assay has a number of advantages over other assays, including short assay times, the use of small reaction volumes (20 µl) and small amounts of protein. It alleviates the use of TLC for lipid separation, which often results in degradation of Sph on the TLC plate. We suggest that this assay will permit the rapid assay of CerS activity in large numbers of samples and will prove particularly useful for laboratories that do not have access to facilities available in more lipid-oriented laboratories. MATERIALS AND METHODS MaterialsMethanol gradient grade for liquid chromatography, water for chromatography, and silica gel 60 TLC plates were from Merck (Darmstadt, Germany). Formic acid (purity >98%) was from Sigma-Aldrich (St. Louis, MO). Chloroform for spectrophotometry was from J. T. Baker (Center Valley, PA). Ammonium acetate was from Macron Chemicals (Center Valley, PA). Strata end-capped silica-based C18-E (15 mg/well) 96-well plates and the vacuum manifold for SPE with vacuum gauge and cover mat were from Phenomenex (Torrance, CA). The vacuum pump was an Alcatel 2002 from Ideal Vacuum Products (Albuquerque, NM).Abstract Ceramides are synthesized by six mammalian ceramide synthases (CerSs), each of which uses fatty acylCoAs of different chain lengths for N -acylation of the sphingoid long-chain base. We now describe a rapid and reliable CerS assay that uses a fl uorescent N-[6-[(7-nitrobenzo-2-oxa-1,3-diazol-4-yl) (NBD ) sphinganine substrate followed by separation of the NBD-lipid substrate and products using solid phase extraction (SPE) C18 chromatography. SPE chromatography is a quick and reliable alternative to TLC, and moreover, there is no degradation of either NBD-sphinganine or NBD-ceramide. We have optimized the assay for use with minimal amounts of protein in a minimal volume. This assay will prove useful for the analysis of CerS activity, which is of particular importance in light of the growing involvement of CerS in cell regulation and in the pathology of human diseases. ( 1 ), is synthesized by the N -acylation of sphingoid long-chain bases by ceramide synthases (CerSs). Mammals contain six distinct CerSs, with each using a subset of fatty acylCoAs for the N -acylation of sphinganine (Sph) to generate dihydroceramides or of sphingosine to generate ceramides ( 2, 3 ). CerSs have become the focus of great interest due to the realizatio...

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“…Curcumin has been reported to stimulate de novo ceramide synthesis by triggering dimerization and activation of ER-resident ceramide synthases (39,40) but also perturbs Ca 2ϩ homeostasis (25,41,42) and may act as a cross-linking agent (33). Hence, future studies should reveal whether a more specific and acute manipulation of ER ceramide levels influences SMSr oligomerization.…”

Section: Discussionmentioning

confidence: 99%

Monitoring Changes in the Oligomeric State of a Candidate Endoplasmic Reticulum (ER) Ceramide Sensor by Single-molecule Photobleaching

Cabukusta¹,

Köhlen²,

Richter

et al. 2016

Journal of Biological Chemistry

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Edited by George CarmanSingle-molecule photobleaching has emerged as a powerful non-invasive approach to extract the stoichiometry of multimeric membrane proteins in their native cellular environment. However, this method has mainly been used to determine the subunit composition of ion channels and receptors at the plasma membrane. Here, we applied single-molecule photobleaching to analyze the oligomeric state of an endoplasmic reticulum (ER) resident candidate ceramide sensor protein, SMSr/SAMD8. Coimmunoprecipitation and chemical cross-linking studies previously revealed that the N-terminal sterile alpha motif (or SAM) domain of SMSr drives self-assembly of the protein into oligomers and that SMSr oligomerization is promoted by curcumin, a drug known to perturb ER ceramide and calcium homeostasis. Application of cell spreading surface-active coating materials in combination with total internal reflection fluorescence (TIRF) microscopy allowed us to image GFP-tagged SMSr proteins as single fluorescent spots in the ER of HeLa cells in which expression of endogenous SMSr was abolished. In line with our biochemical analysis, we find that the number of bleaching steps in SMSr-GFP-positive spots displays a substantial drop after removal of the SAM domain. In contrast, treatment of cells with curcumin increased the number of bleaching steps. Our results document the first successful application of single-molecule photobleaching to resolve drug-induced and domain-dependent changes in the oligomeric state of an ER-resident membrane protein, hence establishing a complementary method to unravel the mechanism by which SMSr controls ceramide levels in the ER.

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Bcl2L13 is a ceramide synthase inhibitor in glioblastoma

Cited by 93 publications

References 30 publications

Expression of Ceramide Synthase 6 Transcriptionally Activates Acid Ceramidase in a c-Jun N-terminal Kinase (JNK)-dependent Manner

Expression of Ceramide Synthase 6 Transcriptionally Activates Acid Ceramidase in a c-Jun N-terminal Kinase (JNK)-dependent Manner

A rapid ceramide synthase activity using NBD-sphinganine and solid phase extraction

Monitoring Changes in the Oligomeric State of a Candidate Endoplasmic Reticulum (ER) Ceramide Sensor by Single-molecule Photobleaching

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