Development of a new inhibitor of glucosylceramide synthase.
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Analogs of the potent inhibitor of glucosylceramide (GlcCer) synthase, D-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (P4), based on substitutions in the palmitoyl group were made by means of a stereo-selective synthetic method in order to elucidate the role of the hydrophobic portion in both the inhibitory action toward the enzyme and the biological effects. While P4 strongly inhibited GlcCer synthase with an IC(50) of 0.5 microM in vitro, it also inhibited cell growth by 50% at the concentration of 7 microM. The shorter N-acyl chain analogs including decanoyl, octanoyl, and hexanoyl groups showed similar IC(50) values for GlcCer synthase (around 2 microM) but the hexanoyl analog exhibited only a slight inhibitory effect on cell growth, showing the dissociation between GlcCer depletion and cell growth. Several compounds which exhibit similar hydrophobicity to the hexanoyl analog of P4 were subsequently designed. We found that D-threo-1-phenyl-2-benzyloxycarbonylamino-3-pyrrolidino-1-pr opanol (PBPP) was a most potent inhibitor, showing an IC50 of 0.3 microM. In cultured cells, PBPP was able to deplete glycosphingolipids without affecting cell growth or the ceramide level. (+info)
Phosphatidylinositol 4,5-bisphosphate induces actin-based movement of raft-enriched vesicles through WASP-Arp2/3.
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BACKGROUND: Phosphatidylinositol 4,5-bisphosphate (PIP(2)) has been implicated in the regulation of the actin cytoskeleton and vesicle trafficking. It stimulates de novo actin polymerization by activating the pathway involving the Wiskott-Aldrich syndrome protein (WASP) and the actin-related protein complex Arp2/3. Other studies show that actin polymerizes from cholesterol-sphingolipid-rich membrane microdomains called 'rafts', in a manner dependent on tyrosine phosphorylation. Although actin has been implicated in vesicle trafficking, and rafts are sites of active phosphoinositide and tyrosine kinase signaling that mediate apically directed vesicle trafficking, it is not known whether phosphoinositide regulation of actin dynamics occurs in rafts, or if it is linked to vesicle movements. RESULTS: Overexpression of type I phosphatidylinositol phosphate 5-kinase (PIP5KI), which synthesizes PIP(2), promoted actin polymerization from membrane-bound vesicles to form motile actin comets. Pervanadate (PV), a tyrosine phosphatase inhibitor, induced comets even in the absence of PIP5KI overexpression. PV increased PIP(2) levels, suggesting that it induces comets by changing PIP(2) homeostasis and by increasing tyrosine phosphorylation. Platelet-derived growth factor (PDGF) enhanced PV-induced comet formation, and these stimuli together potentiated the PIP5KI effect. The vesicles at the heads of comets were enriched in PIP5KIs and tyrosine phosphoproteins. WASP-Arp2/3 involvement was established using dominant-negative WASP constructs. Endocytic and exocytic markers identified vesicles enriched in lipid rafts as preferential sites of comet generation. Extraction of cholesterol with methyl-beta-cyclodextrin reduced comets, establishing that rafts promote comet formation. CONCLUSIONS: Sphingolipid-cholesterol rafts are preferred platforms for membrane-linked actin polymerization. This is mediated by in situ PIP(2) synthesis and tyrosine kinase signaling through the WASP-Arp2/3 pathway. Actin comets may provide a novel mechanism for raft-dependent vesicle transport and apical membrane trafficking. (+info)
The polar-lipid composition of the sphingolipid-producing bacterium Flectobacillus major.
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Polar lipids comprise about 90% of the total chloroform-methanol extractable lipids of the Gram-negative, fresh-water, ring-forming bacterium Flectobacillus major FM and consist of at least 10 constituents. These are aminophosphosphingolipids, 2-N-(2'-D-hydroxy-13'-methyltetradecanoyl)-15-methyl-4(E)-hexad ecasph ingenyl-1-phosphoethanolamine (36.8% of the total polar lipids) and its 2'-deoxy derivative (3.7%); sulfonic-acid analogues of ceramide, 2-D-(2'-D-hydroxy-13'-methyltetradecanoyl)amino-3-D-hydroxy-15-met hyl hexadecane-1-sulfonic acid (18.1%) and its 2'-deoxy derivative (3. 5%); a lipoamino acid, N-[3-D-(15'-methylhexadecanoyloxy)-15-methylhexadecanoyl]-gl ycine (3. 7%); a lipodipeptide, N- inverted question markN'-[3"-D-(15"'-methylhexadecanoyloxy)-15"-methylhexadecanoyl ]glycy l inverted question mark-L-serine (7.8%); 1,2-diacyl-sn-glycero-3-phosphoethanolamine (7. 7%), 1,2-diacyl-3-alpha-D-galactopyranosyl-sn-glycerol (2.9%); ceramide phospho-myo-inositol (4.9%), and a previously described unusual glycosphingolipid, 7-deoxy-7-amino-D-manno-heptulosonopyranosyl (1-hydroxycarbonyl-6-deoxy-6-amino-alpha-D-mannopyranosyl) ceramide (10.9%); the last two lipids contain only 15-methyl-4(E)-hexadecasphingenine as a long-chain base. The sole structural type of amide-bound fatty acids in the sphingolipids, including the sulfonic-acid analogues, is iso-15:0, either non-hydroxylated or hydroxylated at 2-C, whereas 15-methylhexadecanoic acid is the major ester-bound fatty acid in the remaining lipids. (+info)
Sphingolipid-enriched membrane domains from rat cerebellar granule cells differentiated in culture. A compositional study.
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Sphingolipid-enriched membrane domains, characterized by a particular protein and lipid composition, have been detected in a variety of cells. However, limited data are available concerning these domains in neuronal cells. We analyzed the lipid and protein composition of a sphingolipid-enriched membrane fraction prepared from primary rat cerebellar granule cells differentiated in culture. Although the protein content of this fraction was only 1.4% of total cellular protein, 60% of the gangliosides, 67% of the sphingomyelin, 50% of the ceramide, and 40% of the cholesterol were located in this fraction. The protein pattern of the sphingolipid-enriched domain fraction was dramatically different from that associated with the cell homogenate. This fraction contained 25% of the tyrosine-phosphorylated proteins and was enriched in two proteins with apparent molecular masses of 135 and 15 kDa. 12% of cellular glycerophospholipids were located in the fraction, with phosphatidylcholine having the highest enrichment. The molar ratio between proteins, glycerophospholipids, cholesterol, sphingomyelin, ceramide and gangliosides in cerebellar granule cells was 1.6:41.6:6. 1:1.3:0.3:1 in the cell homogenate and 0.04:8.3:4.0:1.4:0.2:1 in the sphingolipid-enriched membrane fraction. These data indicate that selected proteins segregate with sphingolipids in specialized domains in the membrane of cultured neurons. (+info)
Syringomycin E inhibition of Saccharomyces cerevisiae: requirement for biosynthesis of sphingolipids with very-long-chain fatty acids and mannose- and phosphoinositol-containing head groups.
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Syringomycin E is an antifungal cyclic lipodepsinonapeptide that inhibits the growth of Saccharomyces cerevisiae by interaction with the plasma membrane. A screen conducted to find the yeast genes necessary for its fungicidal action identified two novel syringomycin E response genes, SYR3 and SYR4. A syr3 mutant allele was complemented by ELO2 and ELO3. These genes encode enzymes that catalyze the elongation of sphingolipid very long chain fatty acids. Tetrad analysis showed that SYR3 was ELO2. Strains with deletions of SYR3/ELO2 and ELO3 were resistant to syringomycin E, and lipid analyses of both mutants revealed shortened fatty acid chains and lower levels of sphingolipids. SYR4 was identified by Tn5 inactivation of genomic library plasmids that complemented a syr4 mutant allele. SYR4 was found to be identical to IPT1, which encodes the terminal sphingolipid biosynthetic enzyme, mannosyl-diinositolphosphoryl-ceramide synthase. Deletion Deltasyr4/ipt1 strains were viable, were resistant to syringomycin E, did not produce mannosyl-diinositolphosphoryl-ceramide, and accumulated mannosyl-inositolphosphoryl-ceramide. Accumulation of mannosyl-inositolphosphoryl-ceramide was not responsible for resistance since a temperature-sensitive secretory pathway mutant (sec14-3(ts)) accumulated this sphingolipid and was sensitive to syringomycin E. Finally, Deltacsg1/sur1 and Deltacsg2 strains defective in the transfer of mannose to inositolphosphoryl-ceramide were resistant to syringomycin E. These findings show that syringomycin E growth inhibition of yeast is promoted by the production of sphingolipids with fully elongated fatty acid chains and the mannosyl and terminal phosphorylinositol moieties of the polar head group. (+info)
Embryonic striatal neurons from niemann-pick type C mice exhibit defects in cholesterol metabolism and neurotrophin responsiveness.
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Niemann-Pick type C (NP-C) disease is a progressive and fatal neuropathological disorder previously characterized by abnormal cholesterol metabolism in peripheral tissues. Although a defective gene has been identified in both humans and the npc(nih) mouse model of NP-C disease, how this leads to abnormal neuronal function is unclear. Here we show that whereas embryonic striatal neurons from npc(nih) mice can take up low density lipoprotein-derived cholesterol, its subsequent hydrolysis and esterification are significantly reduced. Given the importance of cholesterol to a variety of signal transduction mechanisms, we assessed the effect of this abnormality on the ability of these neurons to respond to brain-derived neurotrophic factor (BDNF). In contrast to its effects on wild type neurons, BDNF failed to induce autophosphorylation of the TrkB receptor and to increase neurite outgrowth in npc(nih) neurons, despite expression of TrkB on the cell surface. The results suggest that abnormal cholesterol metabolism occurs in neurons in the brain during NP-C disease, even at embryonic stages of development prior to the onset of phenotypic symptoms. Moreover, this defect is associated with a lack of TrkB function and BDNF responsiveness, which may contribute to the loss of neuronal function observed in NP-C disease. (+info)
A longevity assurance gene homolog of tomato mediates resistance to Alternaria alternata f. sp. lycopersici toxins and fumonisin B1.
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The phytopathogenic fungus Alternaria alternata f. sp. lycopersici (AAL) produces toxins that are essential for pathogenicity of the fungus on tomato (Lycopersicon esculentum). AAL toxins and fumonisins of the unrelated fungus Fusarium moniliforme are sphinganine-analog mycotoxins (SAMs), which cause inhibition of sphingolipid biosynthesis in vitro and are toxic for some plant species and mammalian cell lines. Sphingolipids can be determinants in the proliferation or death of cells. We investigated the tomato Alternaria stem canker (Asc) locus, which mediates resistance to SAM-induced apoptosis. Until now, mycotoxin resistance of plants has been associated with detoxification and altered affinity or absence of the toxin targets. Here we show that SAM resistance of tomato is determined by Asc-1, a gene homologous to the yeast longevity assurance gene LAG1 and that susceptibility is associated with a mutant Asc-1. Because both sphingolipid synthesis and LAG1 facilitate endocytosis of glycosylphosphatidylinositol-anchored proteins in yeast, we propose a role for Asc-1 in a salvage mechanism of sphingolipid-depleted plant cells. (+info)
Role of a new mammalian gene family in the biosynthesis of very long chain fatty acids and sphingolipids.
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Whereas the physiological significance of microsomal fatty acid elongation is generally appreciated, its molecular nature is poorly understood. Here, we describe tissue-specific regulation of a novel mouse gene family encoding components implicated in the synthesis of very long chain fatty acids. The Ssc1 gene appears to be ubiquitously expressed, whereas Ssc2 and Cig30 show a restricted expression pattern. Their translation products are all integral membrane proteins with five putative transmembrane domains. By complementing the homologous yeast mutants, we found that Ssc1 could rescue normal sphingolipid synthesis in the sur4/elo3 mutant lacking the ability to synthesize cerotic acid (C(26:0)). Similarly, Cig30 reverted the phenotype of the fen1/elo2 mutant that has reduced levels of fatty acids in the C(20)-C(24) range. Further, we show that Ssc1 mRNA levels were markedly decreased in the brains of myelin-deficient mouse mutants known to have very low fatty acid chain elongation activity. Conversely, the dramatic induction of Cig30 expression during brown fat recruitment coincided with elevated elongation activity. Our results strongly implicate this new mammalian gene family in tissue-specific synthesis of very long chain fatty acids and sphingolipids. (+info)