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Kentaro Hanada

Publications and source records attributed to Kentaro Hanada.

At least 19 recordsLinked to original sources
PubMed2006

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.

Amino Acid Motifs↗
PubMed2006

Evidence that clustered phosphocholine head groups serve as sites for binding and assembly of an oligomeric protein pore.

High susceptibility of rabbit erythrocytes toward the pore-forming action of staphylococcal alpha-toxin correlates with the presence of saturable, high affinity binding sites. All efforts to identify a protein or glycolipid receptor have failed, and the fact that liposomes composed solely of phosphatidylcholine are efficiently permeabilized adds to the enigma. A novel concept is advanced here to explain the puzzle. We propose that low affinity binding moieties can assume the role of high affinity binding sites due to their spatial arrangement in the membrane. Evidence is presented that phosphocholine head groups of sphingomyelin, clustered in sphingomyelin-cholesterol microdomains, serve this function for alpha-toxin. Clustering is required so that oligomerization, which is prerequisite for stable attachment of the toxin to the membrane, can efficiently occur. Outside these clusters, binding to phosphocholine is too transient for toxin monomers to find each other. The principle of membrane targeting in the absence of any genuine, high affinity receptor may also underlie the assembly of other lipid-inserted oligomers including cytotoxic peptides, protein toxins, and immune effector molecules.

Animals↗
PubMed2006

Distinct mechanisms of clathrin-independent endocytosis have unique sphingolipid requirements.

Sphingolipids (SLs) play important roles in membrane structure and cell function. Here, we examine the SL requirements of various endocytic mechanisms using a mutant cell line and pharmacological inhibitors to disrupt SL biosynthesis. First, we demonstrated that in Chinese hamster ovary cells we could distinguish three distinct mechanisms of clathrin-independent endocytosis (caveolar, RhoA, and Cdc42 dependent) which differed in cargo, sensitivity to pharmacological agents, and dominant negative proteins. General depletion of SLs inhibited endocytosis by each clathrin-independent mechanism, whereas clathrin-dependent uptake was unaffected. Depletion of glycosphingolipids (GSLs; a subgroup of SLs) selectively blocked caveolar endocytosis and decreased caveolin-1 and caveolae at the plasma membrane. Caveolar endocytosis and PM caveolae could be restored in GSL-depleted cells by acute addition of exogenous GSLs. Disruption of RhoA- and Cdc42-regulated endocytosis by SL depletion was shown to be related to decreased targeting of these Rho proteins to the plasma membrane and could be partially restored by exogenous sphingomyelin but not GSLs. Both the in vivo membrane targeting and in vitro binding to artificial lipid vesicles of RhoA and Cdc42 were shown to be dependent upon sphingomyelin. These results provide the first evidence that SLs are differentially required for distinct mechanisms of clathrin-independent endocytosis.

Animals↗
PubMed2006

Ceramide response post-photodamage is absent after treatment with HA14-1.

The oxidative stress induced by photodynamic therapy using the phthalocyanine Pc 4 (PDT) can lead to apoptosis, and is accompanied by photodamage to Bcl-2 and accumulation of de novo ceramide. Similar to PDT, the oxidative stress inducer and Bcl-2 inhibitor HA14-1 triggers apoptosis. To test the specificity of the ceramide response, Jurkat cells were exposed to an equitoxic dose of HA14-1. Unlike PDT, HA14-1 did not induce accumulation of de novo ceramide, although levels of sphingomyelin, phosphatidylserine and phosphatidylethanolamine were below control values after either treatment. In contrast to PDT, (i) the transient inhibition of serine palmitoyltransferase induced by HA14-1 was associated with the initial decrease in de novo ceramide, and (ii) HA14-1-initiated inhibition of sphingomyelin synthase and glucosylceramide synthase did not result in accumulation of de novo ceramide. These results show that the ceramide response to PDT is not induced by another pro-apoptotic stimulus, and may be unique to PDT as described here.

Apoptosis↗
PubMed2006

A potent adjuvant monophosphoryl lipid A triggers various immune responses, but not secretion of IL-1beta or activation of caspase-1.

Lipid A, the membrane anchor portion of LPS, is responsible for the endotoxin activity of LPS and induces many inflammatory responses in macrophages. Monophosphoryl lipid A (MPL), a lipid A derivative lacking a phosphate residue, induces potent immune responses with low toxicity. To elucidate the mechanism underlying the low toxicity of MPL, we examined the effects of MPL on the secretion of proinflammatory cytokines by mouse peritoneal macrophages, a murine macrophage-like cell line (RAW 264.7), and a human macrophage-like cell line (THP-1). MPL enhanced the secretion of TNF-alpha, but not that of IL-1beta, whereas Escherichia coli-type lipid A (natural source-derived and chemically synthesized lipid A) enhanced the secretion of both cytokines. Although MPL enhanced the levels of IL-1beta mRNA and IL-1beta precursor protein to levels similar to those induced by lipid A, IL-1beta precursor processing in MPL-treated cells was much lower than that in E. coli-type lipid A-treated ones. Moreover, MPL, unlike E. coli-type lipid A, failed to induce activation of caspase-1, which catalyzes IL-1beta precursor processing. These results suggest that an immune response without activation of caspase-1 or secretion of IL-1beta results in the low toxicity of this adjuvant.

Adjuvants, Immunologic↗
PubMed2006

Discovery of the molecular machinery CERT for endoplasmic reticulum-to-Golgi trafficking of ceramide.

Synthesis and sorting of lipids are essential events for membrane biogenesis and its homeostasis. Ceramide is synthesised at the endoplasmic reticulum (ER), and translocated to the Golgi compartment for conversion to sphingomyelin (SM). We have recently identified a key factor (named CERT) for ceramide trafficking. In this short review, I summarise recent advances in molecular mechanisms of intracellular transport of ceramide, focusing on our genetic and biochemical approaches to this issue.

Animals↗
PubMed2006

Depletion of sphingolipids facilitates endosome to Golgi transport of ricin.

It has been previously demonstrated that depletion of cholesterol inhibits endosome to Golgi transport. Whether this inhibition is due to disruption of sphingolipid- and cholesterol-containing lipid rafts that are selected for Golgi transport or whether there is a physical requirement of cholesterol for either membrane deformations, facilitating formation of transport vesicles, or for recruitment of cytosolic constituents is not obvious. To investigate this in more detail, we have studied endosome to Golgi transport of ricin in sphingolipid-deficient cells using either a mutant cell line that does not express serine palmitoyltransferase, the first enzyme in sphingolipid biosynthesis, or a specific inhibitor, myriocin, of the same enzyme. Depletion of sphingolipids gave an increased sensitivity to ricin, and this increased sensitivity was inhibited by addition of sphingolipids. Importantly, endosome to Golgi transport of ricin, measured as sulfation of a modified ricin molecule, was increased in sphingolipid-deficient cells. No effect was seen on other pathways taken by ricin. Interestingly, cholesterol depletion inhibited endosome to Golgi transport even in cells with reduced levels of sphingolipids, suggesting that cholesterol as such is required for formation of transport vesicles. Our results indicate that the presence of sphingolipids actually limits and may function to control endosome to Golgi transport of ricin.

Animals↗
PubMed2006

Enhancement of de novo fatty acid biosynthesis in hepatic cell line Huh7 expressing hepatitis C virus core protein.

Hepatitis C virus (HCV) core protein plays important roles in the pathogeneses of liver steatosis as well as hepatocellular carcinomas due to HCV infection. In this study, we examined de novo fatty acid biosynthesis in hepatic cell line Huh7 cells expressing HCV core protein. The rate of metabolic labeling of cellular fatty acids with [(3)H]acetate in core-expressing (Uc39-6) cells was ca. 1.5-fold higher than that in non-expressing (Uc321) cells. The enzyme activities responsible for fatty acid biosynthesis were assayed in vitro. Cytosolic acetyl-CoA carboxylase activity in Uc39-6 cells was ca. 1.6-fold higher than that in Uc321 cells. On the other hand, cytosolic fatty acid synthase activity in Uc39-6 cells was only slightly higher than that in Uc321 cells. Immunoblot analysis of acetyl-CoA carboxylase 1 (ACC1), which is a rate-limiting enzyme for fatty acid biosynthesis, revealed a higher expression level of the protein in Uc39-6 cells than in Uc321 cells. The ACC1 mRNA content in Uc39-6 cells was 1.4-fold higher than that in Uc321 cells. These results strongly suggest that enhancement of fatty acid biosynthesis in core-expressing cells is caused by increased expression of fatty acid biosynthetic enzymes, especially ACC1. Up-regulation of de novo fatty acid biosynthesis by HCV core protein may affect cellular lipid metabolism, resulting in neutral lipid accumulation in HCV-infected cells.

Cell Line↗
PubMed2005

A role for manganese superoxide dismutase in apoptosis after photosensitization.

The oxidative stress triggered by photodynamic therapy (PDT) involves generation of cytotoxic reactive oxygen species, including superoxide radical, accumulation of de novo-generated ceramide, and induction of apoptosis. Since PDT with the photosensitizer phthalocyanine Pc 4 induces mitochondrial damage and the superoxide scavenger manganese superoxide dismutase (MnSOD) is localized to mitochondria, here we tested genetically the role of MnSOD in apoptosis and ceramide accumulation after photosensitization with Pc 4. Jurkat cells overexpressing wild-type MnSOD were protected from Pc 4-PDT-initiated apoptosis, but not from increased ceramide response to Pc 4-PDT. In Jurkat cells overexpressing mutant MnSOD, however, DEVDase activation and ceramide formation were promoted post-Pc 4-PDT. Similarly, in MnSOD-null cells, Pc 4-PDT-induced apoptosis, as well as ceramide accumulation, were enhanced compared to their normal counterparts. The data show that MnSOD affects sensitivity of cells to Pc 4-PDT-initiated apoptosis, and partly ceramide accumulation, suggesting that the processes are superoxide-mediated.

Animals↗
PubMed2005

Accumulation of diacylglycerol in the Chlamydia inclusion vacuole: possible role in the inhibition of host cell apoptosis.

Intracellular pathogens have developed strategies to survive for extended periods inside their host cells. These include avoidance of host microbicidal effectors, often by sequestration in a protected subcompartment of the host cell. In some cases, the parasites exert also an antiapoptotic effect that prolongs the life of the infected host cell. Chlamydia utilizes both strategies, but the underlying molecular mechanisms are incompletely understood. Comparatively, little is known regarding the effects that Chlamydia exerts on the metabolism and distribution of the host cell lipids. The expression of fluorescently tagged C1 domains revealed that diacylglycerol is greatly accumulated in the immediate vicinity of Chlamydia inclusion vacuoles. The concentrated diacylglycerol recruits protein kinase Cdelta (PKCdelta), a proapoptotic effector, to the immediate vicinity of the vacuole. PKCdelta normally exerts its pro-apoptotic effects at the mitochondria and in the nucleus. We speculate that Chlamydia antagonizes the pro-apoptotic effect of PKCdelta by sequestering the enzyme on the inclusion vacuole away from its conventional target sites. Accordingly, we found that the ectopic expression of a catalytic fragment of PKCdelta that cannot be recruited by the vacuole, because it lacks a functional C1 domain, overcame the anti-apoptotic effect of the bacteria. The scavenging of pro-apoptotic factors may provide a novel mechanism whereby pathogens promote their own survival by extending the life of the host cells they infect.

Acetophenones↗
PubMed2005

Diphtheria toxin translocation across cellular membranes is regulated by sphingolipids.

Diphtheria toxin is translocated across cellular membranes when receptor-bound toxin is exposed to low pH. To study the role of sphingolipids for toxin translocation, both a mutant cell line lacking the first enzyme in de novo sphingolipid synthesis, serine palmitoyltransferase, and a specific inhibitor of the same enzyme, myriocin, were used. The serine palmitoyltransferase-deficient cell line (LY-B) was found to be 10-15 times more sensitive to diphtheria toxin than the genetically complemented cell line (LY-B/cLCB1) and the wild-type cell line (CHO-K1), both when toxin translocation directly across the plasma membrane was induced by exposing cells with surface-bound toxin to low pH, and when the toxin followed its normal route via acidified endosomes into the cytosol. Toxin binding was similar in these three cell lines. Furthermore, inhibition of serine palmitoyltransferase activity by addition of myriocin sensitized the two control cell lines (LY-B/cLCB1 and CHO-K1) to diphtheria toxin, whereas, as expected, no effect was observed in cells lacking serine palmitoyltransferase (LY-B). In conclusion, diphtheria toxin translocation is facilitated by depletion of membrane sphingolipids.

Acyltransferases↗
PubMed2005

Sphingolipids in infectious diseases.

Sphingolipids are ubiquitous constituents of membrane lipids in eukaryotes. Sphingolipid metabolites modulate various cellular events including proliferation, differentiation, and apoptosis. In addition, sphingolipids, along with cholesterol, form detergent-resistant membrane microdomains, so called 'lipid-rafts', which are implicated in signal transduction and membrane trafficking. Sphingolipids are also relevant to infectious diseases. Various types of pathogens exploit the sphingolipids of host cells as membrane receptors. Sphingolipid metabolites regulate pathogen infection and host defense: for instance, a specific glycosphingolipid acts as an endogenous ligand for activation of natural killer T cells. Lipid-rafts of host cells serve as platforms also for infection signaling and entry of intracellular parasites. Moreover, some post-infectious autoimmune diseases result from production of antibodies cross-reacting with mammalian sphingolipids. Differences in the pathways of sphingolipid metabolism between mammals and non-mammals are good clues for rational development of new anti-infectious disease drugs. This review summarizes recent advances in sphingolipid biology related to infectious diseases.

Bacterial Toxins↗
PubMed2004

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 C(16)-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 C(16)-ceramide. Among four stereoisomers of C(16)-ceramide, CERT specifically recognized the natural d-erythro isomer. CERT efficiently transferred ceramides having C(14), C(16), C(18), and C(20) chains, but not longer acyl chains, and also mediated efficient transfer of C(16)-dihydroceramide and C(16)-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.

Biological Transport↗
PubMed2004

Differential effects of modification of membrane cholesterol and sphingolipids on the conformation, function, and trafficking of the G protein-coupled cholecystokinin receptor.

The lipid microenvironment of receptors can influence their conformation, function, and regulation. Cholecystokinin (CCK)-stimulated signaling is abnormal in some forms of hyperlipidemia, suggesting the possibility of unique sensitivity to its lipid environment. Here we examined the influence of cholesterol and sphingolipids on CCK receptors in model Chinese hamster ovary cell systems having lipid levels modified. Cholesterol was modulated chemically or metabolically, and sphingolipids were modulated using a temperature-sensitive cell line (SPB-1). Receptor conformation was probed with a fluorescent full agonist ligand, Alexa 488-conjugated Gly-[Nle(28,31)]CCK-(26-33), shown previously to decrease in anisotropy and lifetime when occupying a receptor in the active conformation (Harikumar, K. G., Pinon, D. L., Wessels, W. S., Prendergast, F. G., and Miller, L. J. (2002) J. Biol. Chem. 277, 18552-18560). Anisotropy and lifetime of this probe were increased and prolonged with cholesterol enrichment, and decreased and shortened with depletion of cholesterol or sphingolipids. The increase in these parameters with cholesterol enrichment may reflect change in CCK receptor conformation toward its inactive, uncoupled state. Indeed, cholesterol enrichment resulted in nonproductive agonist ligand binding, with affinity of binding higher than normal and calcium signaling in response to this reduced. In cholesterol- and sphingolipid-depleted states, the receptor moved into conformations that were less than optimal. With cholesterol depletion, both ligand binding and signaling were decreased, yet internalization and trafficking were unperturbed. With sphingolipid depletion, ligand binding and signaling were normal, but internalization and trafficking were markedly inhibited. Of note, normal transferrin receptor trafficking through the same clathrin-dependent pathway was maintained under these conditions. Thus, lipid microenvironment of the CCK receptor is particularly important, with different lipids having distinct effects.

Animals↗
PubMed2004

Ceramide sensitizes astrocytes to oxidative stress: protective role of cannabinoids.

Cannabinoids induce apoptosis on glioma cells via stimulation of ceramide synthesis de novo, whereas they do not affect viability of primary astrocytes. In the present study, we show that incubation with Delta9-tetrahydrocannabinol did not induce accumulation of ceramide on astrocytes, although incubation of these cells in a serum-free medium (with or without cannabinoids) led to stimulation of ceramide synthesis de novo and sensitization to oxidative stress. Thus treatment with H2O2 induced apoptosis of 5-day-serum-deprived astrocytes and this effect was abrogated by pharmacological blockade of ceramide synthesis de novo. The sensitizing effect of ceramide accumulation may depend on p38 mitogen-activated protein kinase activation rather than on other ceramide targets. Finally, a protective role of cannabinoids on astrocytes is shown as a long-term incubation with cannabinoids prevented H2O2-induced loss of viability in a CB1 receptor-dependent manner. In summary, our results show that whereas challenge of glioma cells with cannabinoids induces accumulation of de novo -synthesized ceramide and apoptosis, long-term treatment of astrocytes with these compounds does not stimulate this pathway and also abrogates the sensitizing effects of ceramide accumulation.

Animals↗
PubMed2004

De novo ceramide accumulation due to inhibition of its conversion to complex sphingolipids in apoptotic photosensitized cells.

The oxidative stress induced by photodynamic therapy (PDT) with the photosensitizer phthalocyanine 4 is accompanied by increases in ceramide mass. To assess the regulation of de novo sphingolipid metabolism during PDT-induced apoptosis, Jurkat human T lymphoma and Chinese hamster ovary cells were labeled with [14C]serine, a substrate of serine palmitoyltransferase (SPT), the enzyme catalyzing the initial step in the sphingolipid biosynthesis. A substantial elevation in [14C]ceramide with a concomitant decrease in [14C]sphingomyelin was detected. The labeling of [14C]ceramide was completely abrogated by the SPT inhibitor ISP-1. In addition, ISP-1 partly suppressed PDT-induced apoptosis. Pulse-chase experiments showed that the contribution of sphingomyelin degradation to PDT-initiated increase in de novo ceramide was absent or minor. PDT had no effect on either mRNA amounts of the SPT subunits LCB1 and LCB2, LCB1 protein expression, or SPT activity in Jurkat cells. Moreover in Chinese hamster ovary cells LCB1 protein underwent substantial photodestruction, and SPT activity was profoundly inhibited after treatment. We next examined whether PDT affects conversion of ceramide to complex sphingolipids. Sphingomyelin synthase, as well as glucosylceramide synthase, was inactivated by PDT in both cell lines in a dose-dependent manner. These results are the first to show that in the absence of SPT up-regulation PDT induces accumulation of de novo ceramide by inhibiting its conversion to complex sphingolipids.

Acyltransferases↗
PubMed2004

Hydrolysis of sphingosylphosphocholine by neutral sphingomyelinases.

Sphingosylphosphocholine (SPC), the N-deacylated form of sphingomyelin (SM), is a naturally occurring lipid mediator. However, little is known about the metabolism of SPC. We here report an in vitro assay system for SPC-phospholipase C (PLC). Using this assay system, we demonstrated that nSMase1 and nSMase2, human neutral sphingomyelinases (SMases), are capable of hydrolyzing SPC efficiently under detergent-free conditions. Bacterial and plasmodial neutral SMases also showed SPC-PLC activity. The substrate specificity of neutral SMases that hydrolyze SM, SPC, and monoradyl glycerophosphocholine, but not diradyl glycerophosphocholine, suggested that a hydrogen-bond donor at the C-2 or sn-2 position in the substrate is required for recognition by the enzymes.

Cell Line↗