Masahiro Nishijima

Molecular machinery for non-vesicular trafficking of ceramide

Synthesis and sorting of lipids are essential for membrane biogenesis; however, the mechanisms underlying the transport of membrane lipids remain little understood. Ceramide is synthesized at the endoplasmic reticulum and translocated to the Golgi compartment for conversion to sphingomyelin. The main pathway of ceramide transport to the Golgi is genetically impaired in a mammalian mutant cell line, LY-A. Here we identify CERT as the factor defective in LY-A cells. CERT, which is identical to a splicing variant of Goodpasture antigen-binding protein, is a cytoplasmic protein with a phosphatidylinositol-4-monophosphate-binding (PtdIns4P) domain and a putative domain for catalysing lipid transfer. In vitro assays show that this lipid-transfer-catalysing domain specifically extracts ceramide from phospholipid bilayers. CERT expressed in LY-A cells has an amino acid substitution that destroys its PtdIns4P-binding activity, thereby impairing its Golgi-targeting function. We conclude that CERT mediates the intracellular trafficking of ceramide in a non-vesicular manner.

Cardiolipin provides specificity for targeting of tBid to mitochondria

Recent evidence supports the theory that mitochondrial homeostasis is the key regulatory step in apoptosis through the actions of members of the Bcl-2 family. Pro-apoptotic members of the family, such as Bax, Bad and Bid, can induce the loss of outer-membrane integrity with subsequent redistribution of pro-apoptotic proteins such as cytochrome c that are normally located in the intermembrane spaces of mitochondria. The anti-apoptotic members of the family, such as Bcl-2 and Bcl-XL, protect the integrity of the mitochondrion and prevent the release of death-inducing factors. Bid normally exists in an inactive state in the cytosol, but after cleavage by caspase 8, the carboxy-terminal portion (tBid) moves from cytosol to mitochondria, where it induces release of cytochrome c. Here we address the question of what mediates specific targeting of tBid to the mitochondria. We provide evidence that cardiolipin, which is present in mitochondrial membranes, mediates the targeting of tBid to mitochondria through a previously unkown three-helix domain in tBid. These findings implicate cardiolipin in the pathway for cytochrome c release.

Mouse Toll-like Receptor 4·MD-2 Complex Mediates Lipopolysaccharide-mimetic Signal Transduction by Taxol

Taxol, an antitumor agent derived from a plant, mimics the action of lipopolysaccharide (LPS) in mice but not in humans. Although Taxol is structurally unrelated to LPS, Taxol and LPS are presumed to share a receptor or signaling molecule. The LPS-mimetic activity of Taxol is not observed in LPS-hyporesponsive C3H/HeJ mice, which possess a point mutation in Toll-like receptor 4 (TLR4); therefore, TLR4 appears to be involved in both Taxol and LPS signaling. In addition, TLR4 was recently shown to physically associate with MD-2, a molecule that confers LPS responsiveness on TLR4. To determine whether TLR4·MD-2 complex mediates a Taxol-induced signal, we constructed transformants of the mouse pro-B cell line, Ba/F3, expressing mouse TLR4 alone, both mouse TLR4 and mouse MD-2, and both mouse MD-2 and mouse TLR4 lacking the cytoplasmic portion, and then examined whether Taxol induced NFκB activation in these transfectants. Noticeable NFκB activation by Taxol was detected in Ba/F3 expressing mouse TLR4 and mouse MD-2 but not in the other transfectants. Coexpression of human TLR4 and human MD-2 did not confer Taxol responsiveness on Ba/F3 cells, suggesting that the TLR4·MD-2 complex is responsible for the species specificity with respect to Taxol responsiveness. Furthermore, Taxol-induced NFκB activation via TLR4·MD-2 was blocked by an LPS antagonist that blocks LPS-induced NFκB activation via TLR4·MD-2. These results demonstrated that coexpression of mouse TLR4 and mouse MD-2 is required for Taxol responsiveness and that the TLR4·MD-2 complex is the shared molecule in Taxol and LPS signal transduction in mice.

Efficient Trafficking of Ceramide from the Endoplasmic Reticulum to the Golgi Apparatus Requires a VAMP-associated Protein-interacting FFAT Motif of CERT

Ceramide is synthesized at the endoplasmic reticulum (ER) and transported to the Golgi apparatus by CERT for its conversion to sphingomyelin in mammalian cells. CERT has a pleck-strin homology (PH) domain for Golgi targeting and a START domain catalyzing the intermembrane transfer of ceramide. The region between the two domains contains a short peptide motif designated FFAT, which is supposed to interact with the ER-resident proteins VAP-A and VAP-B. Both VAPs were actually co-immunoprecipitated with CERT, and the CERT/VAP interaction was abolished by mutations in the FFAT motif. These mutations did not affect the Golgi targeting activity of CERT. Whereas mutations of neither the FFAT motif nor the PH domain inhibited the ceramide transfer activity of CERT in a cell-free system, they impaired the ER-to-Golgi transport of ceramide in intact and in semi-intact cells at near endogenous expression levels. By contrast, when overexpressed, both the FFAT motif and the PH domain mutants of CERT substantially supported the transport of ceramide from the ER to the site where sphingomyelin is produced. These results suggest that the Golgi-targeting PH domain and ER-interacting FFAT motif of CERT spatially restrict the random ceramide transfer activity of the START domain in cells.

A therapeutic agent with oriented carbohydrates for treatment of infections by Shiga toxin-producing Escherichia coli O157:H7

Infection with Shiga toxin (Stx)-producing Escherichia coli O157:H7, which causes diarrhea and hemorrhagic colitis in humans, often results in fatal systemic complications, such as neurological damage and hemolytic–uremic syndrome. Because Stx circulating in the blood is a major causative factor of these complications, the development of a Stx neutralizer that functions in the circulation holds promise as a viable therapy. Here we developed a series of carbosilane dendrimers, in which trisaccharides of globotriaosyl ceramide, a receptor for Stx, were variously oriented at their termini (referred to as SUPER TWIG), and identified a SUPER TWIG with six trisaccharides as a Stx neutralizer functioning in the circulation. This SUPER TWIG specifically bound to Stx with high affinity (Kd = 1.1 × 10−6 M) and inhibited the incorporation of the toxin into target cells. Intravenous administration of the SUPER TWIG along with Stx to mice substantially reduced the fatal brain damage and completely suppressed the lethal effect of Stx. Moreover, the SUPER TWIG protected mice from challenge with a fatal dose of E. coli O157:H7, even when administered after the establishment of the infection. The SUPER TWIG neutralized Stx in vivo by a mechanism in which the accumulation and immediate degradation of Stx by phagocytic macrophages present in the reticuloendothelial system were induced. Taken together, our findings indicate that this SUPER TWIG is therapeutic agent against infections by Stx-producing E. coli.

CERT mediates intermembrane transfer of various molecular species of ceramides.

Ceramide produced at the endoplasmic reticulum is transported to the Golgi apparatus for conversion to sphingomyelin. The main pathway of endoplasmic reticulum-to-Golgi transport of ceramide is mediated by CERT, a cytosolic 68-kDa protein, in a nonvesicular manner. CERT contains a domain that catalyzes the intermembrane transfer of natural C16-ceramide. In this study, we examined the ligand specificity of CERT in detail by using a cell-free assay system for intermembrane transfer of lipids. CERT did not mediate the transfer of sphingosine or sphingomyelin at all. The activity of CERT to transfer saturated and unsaturated diacylglycerols, which structurally resemble ceramide, was 5–10% of the activity toward C16-ceramide. Among four stereoisomers of C16-ceramide, CERT specifically recognized the natural d-erythro isomer. CERT efficiently transferred ceramides having C14, C16, C18, and C20 chains, but not longer acyl chains, and also mediated efficient transfer of C16-dihydroceramide and C16-phyto-ceramide. Binding assays showed that CERT also recognizes short chain fluorescent analogs of ceramide with a stoichiometry of 1:1. Moreover, (1R,3R)-N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecamide, which inhibited the CERT-dependent pathway of ceramide trafficking in intact cells, was found to be an antagonist of the CERT protein. These results indicate that CERT can mediate transfer of various types of ceramides that naturally exist and their close relatives.

Critical Role of Virion-Associated Cholesterol and Sphingolipid in Hepatitis C Virus Infection

ABSTRACT In this study, we establish that cholesterol and sphingolipid associated with hepatitis C virus (HCV) particles are important for virion maturation and infectivity. In a recently developed culture system enabling study of the complete life cycle of HCV, mature virions were enriched with cholesterol as assessed by the molar ratio of cholesterol to phospholipid in virion and cell membranes. Depletion of cholesterol from the virus or hydrolysis of virion-associated sphingomyelin almost completely abolished HCV infectivity. Supplementation of cholesterol-depleted virus with exogenous cholesterol enhanced infectivity to a level equivalent to that of the untreated control. Cholesterol-depleted or sphingomyelin-hydrolyzed virus had markedly defective internalization, but no influence on cell attachment was observed. Significant portions of HCV structural proteins partitioned into cellular detergent-resistant, lipid-raft-like membranes. Combined with the observation that inhibitors of the sphingolipid biosynthetic pathway block virion production, but not RNA accumulation, in a JFH-1 isolate, our findings suggest that alteration of the lipid composition of HCV particles might be a useful approach in the design of anti-HCV therapy.

Regulatory Roles for MD-2 and TLR4 in Ligand-Induced Receptor Clustering

LPS, a principal membrane component in Gram-negative bacteria, is recognized by a receptor complex consisting of TLR4 and MD-2. MD-2 is an extracellular molecule that is associated with the extracellular domain of TLR4 and has a critical role in LPS recognition. MD-2 directly interacts with LPS, and the region from Phe119 to Lys132 (Arg132 in mice) has been shown to be important for interaction between LPS and TLR4/MD-2. With mouse MD-2 mutants, we show in this study that Gly59 was found to be a novel critical amino acid for LPS binding outside the region 119–132. LPS signaling is thought to be triggered by ligand-induced TLR4 clustering, which is also regulated by MD-2. Little is known, however, about a region or an amino acid in the MD-2 molecule that regulates ligand-induced receptor clustering. MD-2 mutants substituting alanine for Phe126 or Gly129 impaired LPS-induced TLR4 clustering, but not LPS binding to TLR4/MD-2, demonstrating that ligand-induced receptor clustering is differentially regulated by MD-2 from ligand binding. We further show that dissociation of ligand-induced receptor clustering and of ligand-receptor interaction occurs in a manner dependent on TLR4 signaling and requires endosomal acidification. These results support a principal role for MD-2 in LPS recognition.

Structural basis for specific lipid recognition by CERT responsible for nonvesicular trafficking of ceramide

In mammalian cells, ceramide is synthesized in the endoplasmic reticulum and transferred to the Golgi apparatus for conversion to sphingomyelin. Ceramide transport occurs in a nonvesicular manner and is mediated by CERT, a cytosolic 68-kDa protein with a C-terminal steroidogenic acute regulatory protein-related lipid transfer (START) domain. The CERT START domain efficiently transfers natural d-erythro-C16-ceramide, but not lipids with longer (C20) amide-acyl chains. The molecular mechanisms of ceramide specificity, both stereo-specific recognition and length limit, are not well understood. Here we report the crystal structures of the CERT START domain in its apo-form and in complex with ceramides having different acyl chain lengths. In these complex structures, one ceramide molecule is buried in a long amphiphilic cavity. At the far end of the cavity, the amide and hydroxyl groups of ceramide form a hydrogen bond network with specific amino acid residues that play key roles in stereo-specific ceramide recognition. At the head of the ceramide molecule, there is no extra space to accommodate additional bulky groups. The two aliphatic chains of ceramide are surrounded by the hydrophobic wall of the cavity, whose size and shape dictate the length limit for cognate ceramides. Furthermore, local high-crystallographic B-factors suggest that the α-3 and the Ω1 loop might work as a gate to incorporate the ceramide into the cavity. Thus, the structures demonstrate the structural basis for the mechanism by which CERT can distinguish ceramide from other lipid types yet still recognize multiple species of ceramides.

E6AP Ubiquitin Ligase Mediates Ubiquitylation and Degradation of Hepatitis C Virus Core Protein

ABSTRACT Hepatitis C virus (HCV) core protein is a major component of viral nucleocapsid and a multifunctional protein involved in viral pathogenesis and hepatocarcinogenesis. We previously showed that the HCV core protein is degraded through the ubiquitin-proteasome pathway. However, the molecular machinery for core ubiquitylation is unknown. Using tandem affinity purification, we identified the ubiquitin ligase E6AP as an HCV core-binding protein. E6AP was found to bind to the core protein in vitro and in vivo and promote its degradation in hepatic and nonhepatic cells. Knockdown of endogenous E6AP by RNA interference increased the HCV core protein level. In vitro and in vivo ubiquitylation assays showed that E6AP promotes ubiquitylation of the core protein. Exogenous expression of E6AP decreased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected Huh-7 cells. Furthermore, knockdown of endogenous E6AP by RNA interference increased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected cells. Taken together, our results provide evidence that E6AP mediates ubiquitylation and degradation of HCV core protein. We propose that the E6AP-mediated ubiquitin-proteasome pathway may affect the production of HCV particles through controlling the amounts of viral nucleocapsid protein.