Cytosolic Glucosylceramide regulates endolysosomal function in Niemann-Pick type C disease

A new paradigm for Niemann-Pick C disease is presented where lysosomal storage leads to a deficit in cytoplasmic glucosylceramide (GlcCer) where it performs important functions. Previously it had been reported that Gaucher cells have defective endolysosomal pH. GlcCer also accumulates in Niemann-Pick C disease and also shows this defect. Niemann-Pick C cells were found to have reduced cytoplasmic glucosylceramide (GlcCer) transport. Inhibiting cytoplasmic glucocerebrosidase (GBA2), increased GlcCer, decreased endolysosomal pH in normal cells, reversed increases in endolysosomal pH and restored disrupted BODIPY-LacCer trafficking and increased expression of vATPase a subunit in Niemann-Pick C fibroblasts. The results are consistent with a model where both endolysosomal pH and Golgi targeting of BODIPY-LacCer are dependent on adequate levels of cytosolic GlcCer which are reduced in NPC disease. This work consequently suggests GBA2 and vATPase as new therapeutic targets in Niemann-Pick C and related neurodegenerative diseases. The work was in collaboration with colleagues in the Netherlands and Leicester University. The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link.Niemann-Pick type C disease (NPCD) is a neurodegenerative disease associated with increases in cellular cholesterol and glycolipids and most commonly caused by defective NPC1, a late endosomal protein. Using ratiometric probes we find that NPCD cells show increased endolysosomal pH. In addition U18666A, an inhibitor of NPC1, was found to increase endolysosomal pH, and the number, size and heterogeneity of endolysosomal vesicles. NPCD fibroblasts and cells treated with U18666A also show disrupted targeting of fluorescent lipid BODIPY-LacCer to high pH vesicles. Inhibiting non-lysosomal glucocerebrosidase (GBA2) reversed increases in endolysosomal pH and restored disrupted BODIPY-LacCer trafficking in NPCD fibroblasts. GBA2 KO cells also show decreased endolysosomal pH. NPCD fibroblasts also show increased expression of a key subunit of the lysosomal proton pump vATPase on GBA2 inhibition. The results are consistent with a model where both endolysosomal pH and Golgi targeting of BODIPY-LacCer are dependent on adequate levels of cytosolic-facing GlcCer, which are reduced in NPC disease

Zur spezifischen Interaktion von Sphingolipiden mit Membranproteinen

Ziel der vorliegenden Arbeit war die Etablierung einer Methode zur Untersuchung von Protein-Sphingolipid-Interaktionen. In vitro generierte COPI-Vesikel weisen eine im Vergleich zur Donormembran höhere Konzentration der SM-Spezies SM 18:0 auf (Brugger et al. 2000). Die Anreicherung der SM-Spezie könnte auf eine spezifische Protein-Lipid-Interaktion zurückzuführen sein. Bei den Proteinen der p24-Familie handelt es sich um Transmembranproteine, welche ein Teil der Budding-Maschinerie zur Bildung von COPI-Vesikeln sind und in diesen angereichert vorliegen (Stamnes et al. 1995; Sohn et al. 1996). Sie stellen somit potentielle Kandidaten der Lipidsortierung dar. Die zu etablierende Methode sollte Hinweise liefern, ob den Proteinen der p24-Familie eine Funktion bei der durch COPI-Vesikel vermittelten Lipidsortierung zukommt. Im Rahmen der vorliegenden Arbeit konnte ein radioaktives und durch UV-Licht aktivierbares Sphingosin-Derivat - [³H]-D-erythro-photoSph - synthetisiert werden. Markierungsexperimente von Zellen mit dieser Verbindung zeigten, dass dieses wie sein natürliches Analogon Sphingosin von der Zelle aufgenommen und zu photoaktivierbaren Sphingolipiden verstoffwechselt wird. Die radioaktive Markierung des Ceramid Transporters - einem Sphingolipid bindenden Protein - zeigte, dass sich [³H]-D-erythro-photoSph zum Nachweis von Protein-Sphingolipid-Interaktionen eignet. Photoaktivierbares Cholesterin und die photoaktivierbare Stearinsäure 10-ASA in Kombination mit [³H]-Cholin wurden bereits erfolgreich zur Analyse von Protein-Cholesterin bzw. Protein-Phosphatidylcholin-Interaktionen eingesetzt (Thiele et al. 2000). Das hier vorgestellte Sphingosin-Derivat ermöglicht nun auch die Analyse von Protein-Sphingolipid-Interaktionen. Die Methode wurde auf die p24-Proteine p23 und p24 angewandt. Auf diese Weise konnte eine spezifische Interaktion von p24 mit einem Sphingolipid gezeigt werden. Erste Untersuchungen deuten darauf hin, dass es sich um eine Interaktion mit Sphingomyelin handelt. Die in dieser Arbeit gewonnenen Erkenntnisse zeigen, dass dem p24-Protein p24 eine Rolle bei der Lipidsortierung bzw. der Membranorganisation zukommen könnte

Optical manipulation of sphingolipid biosynthesis using photoswitchable ceramides

Ceramides are central intermediates of sphingolipid metabolism that also function as potent messengers in stress signaling and apoptosis. Progress in understanding how ceramides execute their biological roles is hampered by a lack of methods to manipulate their cellular levels and metabolic fate with appropriate spatiotemporal precision. Here, we report on clickable, azobenzene-containing ceramides, caCers, as photoswitchable metabolic substrates to exert optical control over sphingolipid production in cells. Combining atomic force microscopy on model bilayers with metabolic tracing studies in cells, we demonstrate that light-induced alterations in the lateral packing of caCers lead to marked differences in their metabolic conversion by sphingomyelin synthase and glucosylceramide synthase. These changes in metabolic rates are instant and reversible over several cycles of photoswitching. Our findings disclose new opportunities to probe the causal roles of ceramides and their metabolic derivatives in a wide array of sphingolipid-dependent cellular processes with the spatiotemporal precision of light

Stroma Transcriptomic and Proteomic Profile of Prostate Cancer Metastasis Xenograft Models Reveals Prognostic Value of Stroma Signatures.

Resistance acquisition to androgen deprivation treatment and metastasis progression are a major clinical issue associated with prostate cancer (PCa). The role of stroma during disease progression is insufficiently defined. Using transcriptomic and proteomic analyses on differentially aggressive patient-derived xenografts (PDXs), we investigated whether PCa tumors predispose their microenvironment (stroma) to a metastatic gene expression pattern. RNA sequencing was performed on the PCa PDXs BM18 (castration-sensitive) and LAPC9 (castration-resistant), representing different disease stages. Using organism-specific reference databases, the human-specific transcriptome (tumor) was identified and separated from the mouse-specific transcriptome (stroma). To identify proteomic changes in the tumor (human) versus the stroma (mouse), we performed human/mouse cell separation and subjected protein lysates to quantitative Tandem Mass Tag labeling and mass spectrometry. Tenascin C (TNC) was among the most abundant stromal genes, modulated by androgen levels in vivo and highly expressed in castration-resistant LAPC9 PDX. The tissue microarray of primary PCa samples (n = 210) showed that TNC is a negative prognostic marker of the clinical progression to recurrence or metastasis. Stroma markers of osteoblastic PCa bone metastases seven-up signature were induced in the stroma by the host organism in metastatic xenografts, indicating conserved mechanisms of tumor cells to induce a stromal premetastatic signature. A 50-gene list stroma signature was identified based on androgen-dependent responses, which shows a linear association with the Gleason score, metastasis progression and progression-free survival. Our data show that metastatic PCa PDXs, which differ in androgen sensitivity, trigger differential stroma responses, which show the metastasis risk stratification and prognostic biomarker potential

Sialyllactose in Viral Membrane Gangliosides Is a Novel Molecular Recognition Pattern for Mature Dendritic Cell Capture of HIV-1

An accessible sialyllactose moiety on viral membrane gangliosides is shown to be essential for HIV-1 uptake into mature dendritic cells, thereby promoting viral transfer and infection of bystander CD4+ T lymphocytes

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Protein-lipid interactions: paparazzi hunting for snap-shots

Photoactivatable groups meeting the criterion of minimal perturbance allow the investigation of interactions in biological samples. Here, we review the application of photoactivatable groups in lipids enabling the study of protein-lipid interactions in (biological) membranes. The chemistry of various photoactivatable groups is summarized and the specificity of the interactions detected is discussed. The recent introduction of ‘click chemistry’ in photocrosslinking of membrane proteins by photoactivatable lipids opens new possibilities for the analysis of crosslinked products and will help to close the gap between proteomics and lipidomics

Sphingolipid Asymmetry and Transmembrane Translocation in Mammalian Cells

Sphingolipids are a typical feature of eukaryotic cells, and indeed, they have been found to fulfi ll a number of intra - and intercellular functions that are specifi c for eukaryotes. Membrane sphingolipids are organized in specialized membrane domains that are involved in the sorting of membrane proteins and lipids along the cellular vesicular transport pathways. In addition, the domains have been invoked in various types of signaling events, like the formation of the T - cell receptor complex and the formation of cell – cell signaling domains. On the other hand, individual sphingolipids act as lipid second messengers, the clearest examples being sphingosine - 1 - phosphate and ceramide. Sphingolipids act at discrete locations, and they are synthesized and degraded at defi ned locations. These are not always on the same side of the membrane, which necessitates transmembrane transport. The sites of transmembrane translocation, the molecular mechanism, and its possible regulation are the topic of the present chapter (Fig. 4.1 )