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KEGG PATHWAY: Metabolic pathways - Reference pathway

Functional Organization of the Golgi Apparatus in Glycosphingolipid Biosynthesis ..

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After treatment, cells were washed three times in PBS, pH 7.4. Lipids were extracted directly from the culture dishes using hexane:2-propanol (32, v/v) and the extracted lipids were dried under a nitrogen stream. Lipids were re-dissolved in hexane:2-propanol and analyzed on high performance thin-layer chromatography (HPTLC) silica plates (Whatman, UK) using a developing solvent containing chloroform:methanol:acetone:acetic acid:water (102:42:1). Identification of the different lipid species was done using lipid standards run in parallel with the samples. Visualization of the lipid migration was done using an iodine chamber, or by spraying with orcinol (0.2% orcinol in a 50% H2SO4 solution) and heating the plate on 120°C for 5 minutes. The lipid spots were scraped, mixed with Optiphase ‘Hi phase’ scintillation liquid (PerkinElmer-Wallac, Turku, Finland) in scintillation vials and the radioactivity was measured using a liquid scintillation counter, 1216 Rackbeta (PerkinElmer-Wallac, Turku, Finland). For the lipid mass quantification experiments, the band intensities of scanned HPTLC silica plates were analyzed and quantified using ImageJ software . After lipid extraction, the cellular proteins were extracted with 0.1 M NaOH and the protein content was analyzed with the Lowry method . Total counts per minute (cpm) obtained for the various experiments were normalized to total cellular protein. The results are displayed as the relative 3H-sphinganine or 3H-palmitic acid incorporation into various lipid species, or total lipid mass normalized to control cells.

The requirement for FAPP2 makes the whole glycosphingolipid synthetic pathway sensitive to …

The fungal toxin BFA inhibits vesicular transfer of proteins and lipids to the plasma membrane by inducing retrograde protein transport from the Golgi apparatus to the ER. This effect in turn results in the fusion of these organelles, creating a Golgi-ER complex and leaving the trans-Golgi network fused with late endosomes . Previously, it has been demonstrated that BFA treatment increases the incorporation of radiolabeled precursors into GlcCer, GalCer, LacCer and the gangliosides GM3 and GD3 , . Monensin is a monovalent cationophore, which is also known to interfere with vesicular transport through the Golgi apparatus, affecting distal Golgi function because of swelling of its cisternae . Monensin has been shown to inhibit the synthesis of sphingomyelin (SM) while increasing levels of radiolabeled precursor incorporation into GlcCer, GalCer and ceramide in cells .

Important dates in the history of lipids ..

In this report we present direct evidence that Egghead and Brainiac do function in vivo in the glycosphingolipid pathway and ..

Also, the synthesis and processing of GlcCer are more sophisticated than suspected (): (1) The GCS is concentrated in the trans-Golgi, not in the cis-Golgi, and its activity was inhibited more than twofold upon CERT knockdown. (2) In contrast to short-chain GlcCer, natural GlcCer did not flop efficiently across the Golgi membrane and was not a substrate for the multidrug transporter ABCB1. (3) Instead, newly synthesized GlcCer reached the outside of the plasma membrane by a nonvesicular transport pathway and was translocated via a mechanism that was inhibited by concanamycin A, an inhibitor of the vacuolar ATPase, suggesting the involvement of a proton gradient. (4) Finally, most GlcCer reached the LCS in the Golgi lumen via the ER, with a role for the trans-Golgi glycolipid-binding protein FAPP2 in shuttling GlcCer to the ER. This suggests that GlcCer may play a role in transport and sorting events at the ER. In addition, FAPP2 appears to regulate complex GSL synthesis. The finding that multiple pathways remove GlcCer from the cytosolic surface of the trans-Golgi suggests the possibility that GlcCer exerts a physiological function at that location.

Our results indicate that GSL synthesis is critical for embryonic development and for differentiation of certain tissues. Earlier studies have suggested an important role for cell-surface carbohydrate interactions in preimplantation embryogenesis, although the nature of these structures, whether glycolipid or glycoprotein, could not be ascertained. The LeX structure, found on both glycoproteins and GSLs, was shown to be important for embryo compaction at the morula stage (). Elimination of O-acetylated sialic acids, found on some gangliosides in addition to glycoproteins, aborted development at the two-cell stage (). Our results indicate that embryos defective in GSL synthesis are able to proceed through preimplantation development. We cannot rule out, however, the possibility that maternally derived Ugcg mRNA or that residual GSLs may suffice for these very early stages of embryogenesis. It also is not known whether galactose-based GSLs (Fig. A) are expressed at this stage and whether they might substitute for the absence of the glucose-based GSLs.

10.1021/jm049486a - American Chemical Society

Expression of ceramide glucosyltransferases, which are essential for glycosphingolipid synthesis, is only required in a small su

Apart from being the precursor for the complex GSLs and playing a role in membrane organization as part of lipid rafts on the lumenal surface of cellular membranes, GlcCer may exert specific functions at one other location that is unusual for GSLs, the cytosolic surface of the Golgi and the ER membrane. GlcCer is synthesized on the cytosolic surface of the Golgi, from where it does not simply disappear by translocation across the Golgi membrane. From independent work, we have suggested a function for GlcCer on the cytosolic surface of the Golgi in sorting membrane proteins to the melanosomes (), which we have now identified as a stimulation of the vacuolar proton pump (unpublished data). Relevant issues are the sidedness of GlcCer in the ER and the location and mechanism by which it flops to the lumenal side of the membrane. In addition, the function of the recently discovered GlcCer degrading ER and cytosolic β-glucosidases (; ; ), the absence of which results in raised GlcCer levels, remains to be resolved. The fact that the GCS occurs in most eukaryotes, even in 50% of all fungi (), suggests that eukaryotes use GlcCer for basic functions that we are about to unravel, but it is likely that evolution has overlaid the basic principles by many levels of regulation. This may explain why these basic principles of GlcCer action have remained obscure to date.

The finding that GlcCer reached the ER lumen as measured via an assay involving enzymatic modification suggested the involvement of one of the two known cytosolic GlcCer-binding proteins. Because MEB4 cells have low levels of GLTP (not depicted), we knocked down FAPP2, a protein with a GLTP domain that is recruited to the trans-Golgi and TGN by PI4P (Fig. S1 A; ). The FAPP2 knockdown dramatically reduced GlcCer transport to the ER lumen (). FAPP2 may transport GlcCer as a monomer via its GlcCer-binding domain, which is analogous to ceramide transfer by CERT. Presently, we cannot exclude the possibility that FAPP2 is involved in the generation of retrograde vesicles that, on their cytosolic surface, are enriched in GlcCer. FAPP2 knockdown inhibited GM3 synthesis by 50% (), suggesting that under normal conditions, at least half of the GlcCer used for GM3 synthesis passes through the ER. It would be delivered to the cytosolic ER surface by FAPP2, flip across the ER, and would be concentrated into ER–Golgi transport vesicles. This lateral concentration in the ER membrane may be very similar to the proposed formation of lipid rafts in the Golgi (). Localization of the Drosophila GCS to the ER () has the same implications. Also, GalCer, which is synthesized on the lumenal surface of the ER of some cell types, may contribute to such a mechanism. The occurrence of lipid rafts in the ER (defined by detergent insolubility) has been reported for yeast () and mammalian cells, where they were GlcCer dependent (). They were suggested to play a role in protein sorting and toxin retrotranslocation across the ER membrane, respectively. The effects of FAPP2 knockdown on sphingolipid-mediated events at the TGN and apical plasma membrane of MDCK cells (, ) may be caused by the inhibition by FAPP2 of the GlcCer flux through the ER or by its inhibitory effect on complex GSL synthesis ().

The requirement for FAPP2 makes the whole glycosphingolipid synthetic pathway sensitive to regulation by ..
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of glycosphingolipid synthesis ameliorates ..

To this date, it is not known how GLTP is regulated in the cell, and if GLTP is involved in intracellular glycolipid synthesis and trafficking. Based on a point mutational data study, GLTP has been indirectly suggested to be involved in the intracellular sensoring and/or trafficking of GlcCer . Further support for this suggestion comes, not only from the seemingly strong evolutionary connection between GlcCer and GLTP as mentioned above, but also from the fact that GlcCer is synthesized on the outer side of the Golgi membrane, leaving the lipid accessible for interaction with the cytosolic GLTP. Previously, we have shown that overexpression of GLTP in HeLa cells, and in this study human skin fibroblast cells (HSFs), leads to increased GlcCer levels while siRNA-mediated GLTP depletion does not alter GlcCer levels significantly . This indicates that GLTP does not like FAPP2 have a direct role in the synthesis of higher glycosphingolipids, because depletion of GLTP does not affect their synthesis . We have also demonstrated in vitro that GLTP interacts with the vesicle-associated protein-associated protein VAP-A, an ER protein that interacts with the FFAT-like motif (two phenylalanines in an acidic tract) in GLTP and other cytosolic lipid-binding proteins . In a recent screening study, weaker FFAT-like VAP association motifs were found, both in FAPP2 as well as in different GLTPs that might suggest a possibility for different strengths of ER targeting .

Stemming the tide: glycosphingolipid synthesis inhibitors as ..

Ceramides are synthesized in the ER by various ceramide synthases (). Some cell types express the galactosylceramide (GalCer) synthase (GalCS), which acts on the lumenal side of the ER (, ). However, all other synthetic enzymes of ceramide-containing lipids are located in the Golgi, with the exception of SMS2 at the plasma membrane (). Newly synthesized ceramides are transported from the ER to the Golgi, where they are converted by the GlcCer synthase (GCS) and SMS1. An important question is how cells regulate the ceramide supply to these enzymes. A first clue has come from the finding that the synthesis of SM but not GlcCer depended on ceramide transport to the trans-Golgi by ceramide transport protein (CERT; ), a pathway that is regulated via phosphoinositides, sterols, and CERT phosphorylation (). However, both SMS1 and GCS have been localized biochemically to the cis-medial Golgi, whereas GCS has also been assigned to pre-Golgi and trans-Golgi membranes (; , ; ; ). The current agreement is that GlcCer is synthesized on a cytosolic surface and translocates across the Golgi membrane for higher GSL synthesis in the late Golgi (, ).

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