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

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

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

Redox Proteomic Profile of Tirapazamine-Resistant Murine Hepatoma Cells

3-Amino-1,2,4-benzotriazine-1,4-dioxide (tirapazamine, TPZ) and other heteroaromatic N-oxides (ArN→O) exhibit tumoricidal, antibacterial, and antiprotozoal activities. Their action is attributed to the enzymatic single-electron reduction to free radicals that initiate the prooxidant processes. In order to clarify the mechanisms of aerobic mammalian cytotoxicity of ArN→O, we derived a TPZ-resistant subline of murine hepatoma MH22a cells (resistance index, 5.64). The quantitative proteomic of wild-type and TPZ-resistant cells revealed 5818 proteins, of which 237 were up- and 184 down-regulated. The expression of the antioxidant enzymes aldehyde- and alcohol dehydrogenases, carbonyl reductases, catalase, and glutathione reductase was increased 1.6–5.2 times, whereas the changes in the expression of glutathione peroxidase, superoxide dismutase, thioredoxin reductase, and peroxiredoxins were less pronounced. The expression of xenobiotics conjugating glutathione-S-transferases was increased by 1.6–2.6 times. On the other hand, the expression of NADPH:cytochrome P450 reductase was responsible for the single-electron reduction in TPZ and for the 2.1-fold decrease. These data support the fact that the main mechanism of action of TPZ under aerobic conditions is oxidative stress. The unchanged expression of intranuclear antioxidant proteins peroxiredoxin, glutaredoxin, and glutathione peroxidase, and a modest increase in the expression of DNA damage repair proteins, tend to support non-site-specific but not intranuclear oxidative stress as a main factor of TPZ aerobic cytotoxicity

Extended N-Terminal Acetyltransferase Naa50 in Filamentous Fungi Adds to Naa50 Diversity

Most eukaryotic proteins are N-terminally acetylated by a set of N$\alpha$ acetyltransferases (NATs). This ancient and ubiquitous modification plays a fundamental role in protein homeostasis, while mutations are linked to human diseases and phenotypic defects. In particular, Naa50 features species-specific differences, as it is inactive in yeast but active in higher eukaryotes. Together with NatA, it engages in NatE complex formation for cotranslational acetylation. Here, we report Naa50 homologs from the filamentous fungi $Chaetomium$ $thermophilum$ and $Neurospora$ $crassa$ with significant N- and C-terminal extensions to the conserved GNAT domain. Structural and biochemical analyses show that CtNaa50 shares the GNAT structure and substrate specificity with other homologs. However, in contrast to previously analyzed Naa50 proteins, it does not form NatE. The elongated N-terminus increases Naa50 thermostability and binds to dynein light chain protein 1, while our data suggest that conserved positive patches in the C-terminus allow for ribosome binding independent of NatA. Our study provides new insights into the many facets of Naa50 and highlights the diversification of NATs during evolution

Trifunctional lipid probes for comprehensive studies of single lipid species in living cells

Lipid-mediated signaling events regulate many cellular processes. Investigations of the complex underlying mechanisms are difficult because several different methods need to be used under varying conditions. Here we introduce multifunctional lipid derivatives to study lipid metabolism, lipid−protein interactions, and intracellular lipid localization with a single tool per target lipid. The probes are equipped with two photoreactive groups to allow photoliberation (uncaging) and photo–cross-linking in a sequential manner, as well as a click-handle for subsequent functionalization. We demonstrate the versatility of the design for the signaling lipids sphingosine and diacylglycerol; uncaging of the probe for these two species triggered calcium signaling and intracellular protein translocation events, respectively. We performed proteomic screens to map the lipid-interacting proteome for both lipids. Finally, we visualized a sphingosine transport deficiency in patient-derived Niemann−Pick disease type C fibroblasts by fluorescence as well as correlative light and electron microscopy, pointing toward the diagnostic potential of such tools. We envision that this type of probe will become important for analyzing and ultimately understanding lipid signaling events in a comprehensive manner. The roles of lipids in cells go far beyond providing the structural backbone of cellular membranes. Certain lipid species are powerful signaling molecules. Examples include the roles of sphingosine (Sph) and the diacylglycerol (DAG) variant, stearoyl-arachidonylglycerol (SAG) in intracellular calcium signaling (1, 2). The study of such signaling lipids is often complicated by the fact that they are under tight metabolic control and that they occur only in very low concentrations. Overexpression of metabolic enzymes for manipulation of signaling lipid levels is a slow process compared with the rapid turnover of those lipids and may therefore produce not only the target lipid but also multiple downstream metabolites. Chemical dimerizer and optogenetic approaches are options to manipulate lipid contents more rapidly, but they depend on cytosolic lipid-metabolizing enzymes. In the past, many applications therefore focused on phosphoinositides (3, 4). A more general way to rapidly increase lipid concentration is the use of caged lipids. These are equipped with a photocleavable protecting group (caging group), which blocks biological activity and renders them resistant to metabolic turnover before the active lipid is released using a flash of light (2, 5⇓–7). The sudden increase in target lipid concentration facilitates analysis of downstream lipid signaling events as well as lipid metabolism within living cells in pulse−chase experiments. To correctly interpret such signaling events, underlying processes such as lipid−protein interactions, intracellular lipid localization, and kinetics of lipid metabolism need to be considered. To date, lipid metabolism is typically monitored using isotope-labeled or alkyne-modified lipids (8⇓–10). Fluorescent lipids, lipid-binding antibodies, or lipid biosensors are mainly used to study lipid localization (11, 12). Most assays for studying lipid−protein interactions rely on reconstituted membranes/liposomes and are therefore largely restricted to soluble proteins (13⇓⇓–16). The plethora of methods used to investigate these different processes makes it difficult to compare or validate their respective results. A promising approach to integrate the study of lipid metabolism, lipid localization, and lipid−protein interactions has emerged in recent years; bifunctional lipids feature a small diazirine group to allow photo–cross-linking with interacting proteins in the intact cellular environment and a terminal alkyne for subsequent functionalization (17). Biotinylation of cross-linked lipid−protein conjugates enables their enrichment and identification of lipid-interacting proteins. To date, bifunctional lipids are one of the few methods to screen for lipid−protein interactions in living cells (18⇓⇓–21). Alternatively, bifunctional lipids can be used to visualize lipid localization by click reaction with a fluorophore (1, 18, 20). The application of the bifunctional lipid principle to signaling lipids, however, is handicapped by their tight metabolic control. Any precursor is rapidly incorporated into downstream lipids, complicating the interpretation of resulting data. The ability to liberate a single, well-defined signaling lipid species within cells and to immediately capture its interacting partners, investigate downstream signaling, and study its subcellular localization would enable much-needed insight into the regulation of lipid-dependent signaling. Here, we present “trifunctional” lipids as tools, combining the advantages of caged and bifunctional lipids in a single molecule to allow for a wide range of studies in living cells with tight temporal control. Applied to Sph and DAG, we show that trifunctional lipids enable (i) acute alteration of signaling lipid concentration, (ii) measurement of lipid metabolism on a population-wide as well as on a single-cell level, (iii) screening for lipid−protein interactions, and (iv) direct visualization of lipid localization by light and correlative light and electron microscopy (CLEM) in comparable experimental settings

Intracellular sphingosine releases calcium from lysosomes

To elucidate new functions of sphingosine (Sph), we demonstrate that the spontaneous elevation of intracellular Sph levels via caged Sph leads to a significant and transient calcium release from acidic stores that is independent of sphingosine 1-phosphate, extracellular and ER calcium levels. This photo-induced Sph-driven calcium release requires the two-pore channel 1 (TPC1) residing on endosomes and lysosomes. Further, uncaging of Sph leads to the translocation of the autophagy-relevant transcription factor EB (TFEB) to the nucleus specifically after lysosomal calcium release. We confirm that Sph accumulates in late endosomes and lysosomes of cells derived from Niemann-Pick disease type C (NPC) patients and demonstrate a greatly reduced calcium release upon Sph uncaging. We conclude that sphingosine is a positive regulator of calcium release from acidic stores and that understanding the interplay between Sph homeostasis, calcium signaling and autophagy will be crucial in developing new therapies for lipid storage disorders such as NPC