title: Limonoid compounds inhibit sphingomyelin biosynthesis by preventing CERT protein-dependent extraction of ceramides from the endoplasmic reticulum.
abstract: To identify novel inhibitors of sphingomyelin (SM) metabolism, a new and selective high throughput microscopy-based screening based on the toxicity of the SM-specific toxin, lysenin, was developed. Out of a library of 2011 natural compounds, the limonoid, 3-chloro-8β-hydroxycarapin-3,8-hemiacetal (CHC), rendered cells resistant to lysenin by decreasing cell surface SM. CHC treatment selectively inhibited the de novo biosynthesis of SM without affecting glycolipid and glycerophospholipid biosynthesis. Pretreatment with brefeldin A abolished the limonoid-induced inhibition of SM synthesis suggesting that the transport of ceramide (Cer) from the endoplasmic reticulum to the Golgi apparatus is affected. Unlike the Cer transporter (CERT) inhibitor HPA-12, CHC did not change the transport of a fluorescent short chain Cer analog to the Golgi apparatus or the formation of fluorescent and short chain SM from the corresponding Cer. Nevertheless, CHC inhibited the conversion of de novo synthesized Cer to SM. We show that CHC specifically inhibited the CERT-mediated extraction of Cer from the endoplasmic reticulum membranes in vitro. Subsequent biochemical screening of 21 limonoids revealed that some of them, such as 8β-hydroxycarapin-3,8-hemiacetal and gedunin, which exhibits anti-cancer activity, inhibited SM biosynthesis and CERT-mediated extraction of Cer from membranes. Model membrane studies suggest that 8β-hydroxycarapin-3,8-hemiacetal reduced the miscibility of Cer with membrane lipids and thus induced the formation of Cer-rich membrane domains. Our study shows that certain limonoids are novel inhibitors of SM biosynthesis and suggests that some biological activities of these limonoids are related to their effect on the ceramide metabolism.
authors.affiliation: Lipid Biology Laboratory, RIKEN Advanced Science Institute, Wako, Saitama 351-0198, Japan. hullin-matsuda@riken.jp
authors.forename: Françoise
authors.initials: F
authors.lastname: Hullin-Matsuda
authors.forename: Nario
authors.initials: N
authors.lastname: Tomishige
authors.forename: Shota
authors.initials: S
authors.lastname: Sakai
authors.forename: Reiko
authors.initials: R
authors.lastname: Ishitsuka
authors.forename: Kumiko
authors.initials: K
authors.lastname: Ishii
authors.forename: Asami
authors.initials: A
authors.lastname: Makino
authors.forename: Peter
authors.initials: P
authors.lastname: Greimel
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Abe
authors.forename: Elad L
authors.initials: EL
authors.lastname: Laviad
authors.forename: Michel
authors.initials: M
authors.lastname: Lagarde
authors.forename: Hubert
authors.initials: H
authors.lastname: Vidal
authors.forename: Tamio
authors.initials: T
authors.lastname: Saito
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Osada
authors.forename: Kentaro
authors.initials: K
authors.lastname: Hanada
authors.forename: Anthony H
authors.initials: AH
authors.lastname: Futerman
authors.forename: Toshihide
authors.initials: T
authors.lastname: Kobayashi
mesh_terms: D000818:Animals; D016466:CHO Cells; D002152:Calorimetry, Differential Scanning; D002518:Ceramides / Q000378:metabolism*; D006224:Cricetinae; D004721:Endoplasmic Reticulum / Q000187:drug effects* / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D036701:Limonins / Q000494:pharmacology*; D050356:Lipid Metabolism / Q000187:drug effects; D018613:Microscopy, Confocal; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D013107:Sphingolipids / Q000378:metabolism; D013109:Sphingomyelins / Q000096:biosynthesis*
chemical_list: D002518:Ceramides; D036701:Limonins; D013107:Sphingolipids; D013109:Sphingomyelins; C119090:CERT1 protein, human; D017346:Protein Serine-Threonine Kinases