Sphingolipids are important signaling molecules regulating cell growth, cell death and differentiation, thus making them important molecules in determining the fate of a cell and in the pathogenesis of chronic illnesses. The sphingolipid signaling pathway can be initiated by reactive oxygen species (ROS) and inflammatory molecules, both of which are believed to be upregulated in a state of obesity. The hypothesis tested in this dissertation is that due to the inflammatory state of obese animals, the sphingolipid pathway is altered, shifting the balance of pro- and anti-apoptotic proteins and contributing to the pathogenesis of diseases associated with an obese state. The specific aims were to compare, 1) key sphingolipid signaling enzymes; 2) levels of sphingolipid signaling molecules and 3) pro and anti-apoptotic protein levels, in hepatic and colonic tissues procured from lean and obese animals. Obese animals are susceptible to various diseases, including colon cancer and hepatic steatosis. To assess the effect of obesity on sphingolipid signaling, and to provide insight as to the pathogenesis of diseases in a state of obesity, liver and colon tissues from Zucker obese female rats (fa/fa) were compared to tissues from their lean counterparts (Fa/fa or Fa/Fa Zucker rats). Enzyme analyses included an assay of sphingomyelinase (SMase) activity and quantification of ceramidase and sphingosine kinase-1 (SK1) protein expression by western blot. Also, sphingomyelin (SM), ceramide, ceramide-1 phosphate (C1P), sphingosine and sphingosine-1-phosphate (S1P) levels were determined by high-performance liquid chromatography (HPLC) -tandem mass spectroscopy (MS). Representative apoptotic proteins, Bax and Bcl-2 were quantified by western blot. Obese liver demonstrates hepatic steatosis in the Zucker animal model. Among the major differences noted between obese and lean liver were significantly upregulated ceramidase, and downregulated SK1 and C1P levels (P<0.05), as well as a difference in ceramide and SM species composition. Bax was overexpressed while Bcl-2 level was lower in obese compared to lean liver (P<0.05). Taken together, the results indicate a shift toward higher apoptotic signaling in obese liver tissue and correspond with the diseased state of the steatotic liver. Analysis of the sphingolipid pathway in colon revealed upregulation of ceramidase and downregulation of SK1 (P<0.05), similar to liver tissue. C1P levels were lower (P<0.05) but no changes were observed for ceramide, SM or sphingosine levels. A trend toward higher SMase activity in obese colon was observed. Bax was overexpressed in obese colon tissue (P< 0.05), while Bcl-2 results were inconclusive. The liver expressed lower level of molecules associated with sphingolipid signaling than the colons. This study is first to demonstrate tissue-specific differences in the sphingolipid signaling pathway, regardless of genotype. Nevertheless, overall the genotype of Zucker model was found to be a factor altering the expression levels of various sphingolipid enzymes and metabolites in both colon and liver. The findings of the present research provide incentive to further understand the role and modulation of sphingolipid signaling pathway in causation and prevention of chronic diseases prevalent in obese state.

Acquired Immunodeficiency Syndrome (AIDS) was first reported in the United States in 1981 and has since become a major worldwide epidemic. The typical course of HIV disease begins with a primary infection followed by a relatively long latency phase and finally ends in the advance phase also called AIDS. There are two aspects considered the most important in HIV pathogenesis, namely viral replication and CD4 + T cell depletion. During the latency phase, tumor necrosis factor (TNF ) has been shown to play a predominant role in HIV-1 replication and disease progression. Since ethanol is also an important risk factor and has been implicated in HIV-1 replication, we investigate the effects of ethanol on TNF inducible signaling associated with HIV-1 replication in human CD4 + T cells. We demonstrate that clinically relevant ethanol concentrations significantly potentiate TNF inducible NF B. Although ethanol effectively collaborated with TNF , by itself it does not have a direct effect on NF activation. The ethanol dependent potentiation of TNF inducible NF B nuclear translocation is observed to involve the enhanced degradation of I B . Additionally, the ethanol mediated potentiation of TNF inducible NF B activation is abrogated by the known antioxidant pyrrolidinedithiocarbamate (PDTC), suggesting an important mechanistic role for reactive oxygen species (ROS) in this process. In correspondence with its effect on NF B, ethanol is also able to significantly enhance HIV-1 long terminal repeat (HIV-1-LTR) dependent transcription induced by TNF . Apoptosis has been proposed as a critical mechanism for CD4 + T cell depletion in HIV pathogenesis. Ceramide, a sphingolipid metabolite, is a common apoptotic transducer involved in CD4 + T cell apoptosis. In the current study, we show that ceramide potently induces CD4 + T cell apoptosis through activating caspase 3, which may further increase Fas Ligand expression to amplify the apoptotic signaling. Interestingly, the apoptotic effect of ceramide is completely blocked by pretreatment with zinc and the underneath mechanism is suggested to be a direct inhibition of caspase 3 activity by zinc. Survival factors are equally important in the regulation of apoptotic process. We demonstrate that PI3-kinase/Akt pathway is indispensable for the survival of CD4 + T cells. Further, Akt kinase is significantly inactivated and downregulated in oxidative stress induced CD4 + T cell apoptosis. N-acetyl-cysteine (NAC) can rescue CD4 + T cell from H2O2 induced caspase 3 activation and apoptosis, while depletion of glutathione (GSH) exacerbate it. Overall, this work identifies several mechanisms underlying CD4 + T cell apoptosis and provides molecular basis for the role of ethanol as a cofactor that can adversely affect HIV-1 infection and pathogenesis.

<p>Endozepine is an alternative name for the testis-specific isoform of the acyl-CoA binding protein (t-ACBP). Acyl-CoA binding proteins form a highly conserved family of proteins, which bind long chain fatty acid esters with nanomolar affinity. They are also known to be endogenous ligands to the --amino butyric acid (GABA) receptor in the central nervous system and to play a role in a wide variety of cellular functions such as vesicular trafficking, fatty acid biosynthesis and gene regulation. A role for endozepine in apoptosis was suggested through promoter gene trapping studies using CHO22 cells in which 90 % reduction in the expression of endozepine correlated with delayed mitochondrial permeabilization, a reduced activation of caspase-3 (an activator of apoptosis) and a consequent resistance to C2-ceramide induced apoptosis.</p> <p>Transduction studies using Tat-GFP-ELP fusion protein showed that endozepine restored the sensitivity of mutant CHO22 cells to C2-ceramide induced apoptosis. In this thesis, we have investigated two hypotheses for the involvement of endozepine in ceramide-induced apoptosis. The first hypothesis is that endozepine contributes to apoptosis through the transport of palmitoyl-CoA, a substrate required for the de novo synthesis of ceramide. The second hypothesis is that endozepine interacts directly with ceramide leading to interaction with peripheral benzodiazepine receptor and a subsequent opening of the mitochondria permeability transition pore, leading to apoptosis.</p>

Magister Scientiae - MSc / Endozepine is an alternative name for the testis-specific isoform of the acyl-CoA binding protein (t-ACBP). Acyl-CoA binding proteins form a highly conserved family of proteins, which bind long chain fatty acid esters with nanomolar affinity. They are also known to be endogenous ligands to the --amino butyric acid (GABA) receptor in the central nervous system and to play a role in a wide variety of cellular functions such as vesicular trafficking, fatty acid biosynthesis and gene regulation. A role for endozepine in apoptosis was suggested through promoter gene trapping studies using CHO22 cells in which 90 % reduction in the expression of endozepine correlated with delayed mitochondrial permeabilization, a reduced activation of caspase-3 (an activator of apoptosis) and a consequent resistance to C2-ceramide induced apoptosis. Transduction studies using Tat-GFP-ELP fusion protein showed that endozepine restored the sensitivity of mutant CHO22 cells to C2-ceramide induced apoptosis. In this thesis, we have investigated two hypotheses for the involvement of endozepine in ceramide-induced apoptosis. The first hypothesis is that endozepine contributes to apoptosis through the transport of palmitoyl-CoA, a substrate required for the de novo synthesis of ceramide. The second hypothesis is that endozepine interacts directly with ceramide leading to interaction with peripheral benzodiazepine receptor and a subsequent opening of the mitochondria permeability transition pore, leading to apoptosis. / South Africa

The resolution of fluorescence light microscopy was long believed to be limited by the diffraction limit of light of around 200-250 nm described in 1873 by Ernst Abbe. Within the last decade, several approaches, such as structured illumination microscopy (SIM), stimulated emission depletion STED and (direct) stochastic optical reconstruction microscopy (d)STORM have been established to bypass the diffraction limit. However, such super-resolution techniques enabling a resolution <100 nm require specialized and expensive setups as well as expert knowledge in order to avoid artifacts. They are therefore limited to specialized laboratories. Recently, Boyden and colleagues introduced an alternate approach, termed expansion microscopy (ExM). The latter offers the possibility to perform superresolution microscopy on conventional confocal microscopes by embedding the sample into a swellable hydrogel that is isotropically expanded. Since its introduction in 2015, expansion microscopy has developed rapidly offering protocols for 4x, 10x and 20x expansion of proteins and RNA in cells, tissues and human clinical specimens. Mitochondria are double membrane-bound organelles and crucial to the cell by performing numerous tasks, from ATP production through oxidative phosphorylation, production of many important metabolites, cell signaling to the regulation of apoptosis. The inner mitochondrial membrane is strongly folded forming so-called cristae. Besides being the location of the oxidative phosphorylation and therefore energy conversion and ATP production, cristae have been of great interest because changes in morphology have been linked to a plethora of diseases from cancer, diabetes, neurodegenerative diseases, to aging and infection. However, cristae imaging remains challenging as the distance between two individual cristae is often below 100 nm. Within this work, we demonstrate that the mitochondrial creatine kinase MtCK linked to fluorescent protein GFP (MtCK-GFP) can be used as a cristae marker. Upon fourfold expansion, we illustrate that our novel marker enables visualization of cristae morphology and localization of mitochondrial proteins relative to cristae without the need for specialized setups. Furthermore, we show the applicability of expansion microscopy for several bacterial pathogens, such as Chlamydia trachomatis, Simkania negevensis, Neisseria gonorrhoeae and Staphylococcus aureus. Due to differences in bacterial cell walls, we reveal important aspects for the digestion of pathogens for isotropic expansion. We further show that expansion of the intracellular pathogens C. trachomatis and S. negevensis, enables the differentiation between the two distinct developmental forms, catabolic active reticulate bodies (RB) and infectious elementary bodies (EB), on a conventional confocal microscope. We demonstrate the possibility to precisely locate chlamydial effector proteins, such as CPAF or Cdu1, within and outside the chlamydial inclusion. Moreover, we show that expansion microscopy enables the investigation of bacteria, herein S. aureus, within LAMP1 and LC3-II vesicles. With the introduction of the unnatural α-NH2-ω-N3-C6-ceramide, we further present the first approach for the expansion of lipids that may also be suitable for far inaccessible molecule classes like carbohydrates. The efficient accumulation and high labeling density of our functionalized α-NH2-ω-N3-C6-ceramide in both cells and bacteria enables in combination with tenfold expansion nanoscale resolution (10-20 nm) of the interaction of proteins with the plasma membrane, membrane of organelles and bacteria. Ceramide is the central molecule of the sphingolipid metabolism, an important constituent of cellular membranes and regulates many important cellular processes such as differentiation, proliferation and apoptosis. Many studies report about the importance of sphingolipids during infection of various pathogens. While the transport of ceramide to Chlamydia has been reported earlier, one of the unanswered questions remaining was if ceramide forms parts of the outer or inner bacterial membrane. Expansion of α-NH2-ω-N3-C6-ceramide enabled the visualization of ceramide in the inner and outer membrane of C. trachomatis and their distance was determined to be 27.6 ± 7.7 nm. / Aufgrund der Beugungseigenschaften des Lichtes wurde bereits 1873 durch Ernst Abbe für die Lichtmikroskopie eine theoretische Auflösungsgrenze von 200-250 nm definiert. Durch die Einführung verschiedener hochauflösender Mikroskopiemethoden, wie beispielsweise SIM-Mikroskopie (structured illumination microscopy), STED-Mikroskopie (stimulated emission depletion) und (d)STORM-Mikroskopie ((direct) stochastic optical reconstruction microscopy), konnte im letzten Jahrzehnt jedoch die Auflösung auf unter 100 nm verbessert werden. Allerdings benötigen solche Hochauflösungstechniken sowohl spezialisierte und kostenintensive Geräte als auch Expertenwissen zur Vermeidung von Artefakten, sodass diese nur in wenigen Laboren angewendet werden können. Ein alternativer Ansatz, die sogenannte Expansionsmikroskopie, wurde kürzlich von der Arbeitsgruppe um Ed Boyden etabliert. Hierbei wird eine Probe mit einem quellfähigen Gel vernetzt, welches daraufhin isotrop expandiert wird, sodass auch an konventionellen konfokalen Mikroskopen Hochauflösung ermöglicht wird. Seit ihrer Einführung im Jahre 2015 hat sich die Expansionsmikroskopie schnell entwickelt und bietet Protokolle für 4-fache, 10-fache oder sogar 20-fache Expansion von Proteinen als auch RNA in Zellen oder sogar komplexen Geweben. Mitochondrien besitzen zwei Membranen und sind für die Zelle von großer Bedeutung, da sie eine Vielzahl wichtiger Aufgaben übernehmen - von der ATP-Produktion durch die oxidative Phosphorylierung über die Produktion vieler wichtiger Metabolite bis hin zur Regulation zellulärer Signalwege. Die innere Mitochondrienmembran ist stark gefaltet und bildet Einstülpungen, die sogenannten Cristae, in welchen die oxidative Phosphorylierung und somit die Energieumwandlung und ATP-Synthese stattfindet. Morphologische Veränderungen der Cristae können sowohl beim Altern von Zellen, als auch bei verschiedenen Infektionen beobachtet werden und können darüber hinaus auch im Rahmen diverser Erkrankungen, wie beispielsweise Krebs, Diabetes oder neurodegenerativen Erkrankungen auftreten. Die Visualisierung der Cristae durch Fluoreszenzmikroskopie ist herausfordernd, da der Abstand zwischen einzelnen Cristae oftmals unter 100 nm beträgt. In der vorliegenden Arbeit wird gezeigt, dass die Expression der mitochondrialen Kreatinkinase gekoppelt an das Fluoreszenzprotein GFP (MtCK-GFP) als Cristaemarker genutzt werden kann. In Kombination mit vierfacher Expansion ermöglicht unser Marker die Untersuchung morphologischer Veränderungen von Cristae, sowie die Lokalisierung mitochondrialer Proteine relativ zu den Cristae. Darüber hinaus wird im Rahmen dieser Arbeit die Anwendbarkeit der Expansionsmikroskopie für mehrere bakterielle Pathogene, und zwar Chlamydia trachomatis, Simkania negevensis, Neisseria gonorrhoeae und Staphylococcus aureus, gezeigt. Hierbei verdeutlichen wir wichtige Aspekte für den vollständigen Verdau unterschiedlicher bakterieller Zellwände und somit isotropen Expansion. Die Expansion der intrazellulären Pathogene C. trachomatis und S. negevensis ermöglichte es uns an konventionellen konfokalen Mikroskopen zwischen den zwei verschiedenen Entwicklungsstadien, der katabolisch aktiven Retikulärkörperchen (RBs) und der infektiösen Elementarkörperchen (EBs), zu unterscheiden. Außerdem konnte die Möglichkeit der präzisen Lokalisierung chlamydialer Proteine wie CPAF und Cdu1 innerhalb und außerhalb der chlamydialen Inklusion gezeigt werden und Bakterien, in diesem Fall S. aureus, in LAMP1 und LC3-II Vesikeln visualisiert werden. Mit der Einführung des unnatürlichen α-NH2-ω-N3-C6-Ceramides, präsentieren wir zudem ein erstes Konzept für die Expansion von Lipiden, welches möglicherweise auch für deutlich unzugänglichere Molekülklassen wie beispielsweise Kohlehydrate geeignet ist. Die effiziente Akkumulierung unseres funktionalisierten α-NH2-ω-N3-C6-Ceramides in Zellen sowie Bakterien ermöglicht in Kombination mit zehnfacher Expansion die Untersuchung der Interaktion von Proteinen mit der Zellmembran, Membranen von Organellen und Bakterien mit einer räumlichen Auflösung von 10-20 nm. Ceramid ist das zentrale Molekül des Sphingolipidstoffwechsels, ein wichtiger Baustein zellulärer Membrane und reguliert viele essentielle Prozesse wie die Zelldifferenzierung, die Proliferation als auch die Apoptose. Viele Studien berichten von der Bedeutung der Sphingolipide während der Infektion verschiedener Pathogene. So wurde beispielsweise zuvor berichtet, dass Ceramide aktiv zu Chlamydien transportiert und in deren Membranen eingebaut werden. Hierbei verblieb allerdings die Frage, ob Ceramide in der äußeren oder inneren bakteriellen Membran lokalisiert sind. Die Expansion unseres α-NH2-ω-N3-C6-Ceramides ermöglichte es uns Ceramide in der inneren und äußeren Membran von C. trachomatis zu visualisieren und den Abstand zwischen beiden Membranen auf 27.6 ± 7.7 nm zu bestimmen.

Indiana University-Purdue University Indianapolis (IUPUI) / Key regulatory decisions of protein synthesis and autophagy are controlled by the lysosomal nutrient sensing complex (LYNUS). To engage protein synthesis signaling, LYNUS requires cellular availability of amino acids, adenosine triphosphate (ATP), growth factors, and docking at the lysosomal membrane. The molecular determinants of LYNUS signaling and docking are not completely elucidated and may involve regulators of the lipid membrane structure and function of the lysosome. Since ceramides are both bioactive second messengers and determinants of lipid membrane stiffness, we investigated the role of the ceramide-producing lysosomal acid sphingomyelinase (ASM) in the homeostatic function of mammalian target of rapamycin (mTOR) signaling and autophagy. Using ASM inhibition with either imipramine or siRNA against SMPD1, in primary human lung cells or Smpd1+/- mice, we demonstrated that ASM is an endogenous inhibitor of autophagy. ASM was necessary for physiological mTOR signaling and maintenance of sphingosine levels. Whereas overstimulation of ASM has been shown to trigger autophagy with impaired flux, inhibition of ASM activity during homeostatic, non-stressed conditions triggered autophagy with degradative potential, associated with enhanced transcription factor EB (TFEB), a master regulator of autophagy and lysosomal biogenesis genes, translocation to the nucleus and decreased sphingosine levels. These findings suggest LYNUS signaling and autophagy are partially regulated by ASM.

No description available.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21804号 / 農博第2317号 / 新制||農||1065(附属図書館) / 学位論文||H31||N5176(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 菅原 達也, 教授 佐藤 健司, 教授 松井 徹 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Background: Ceramide synthesis and metabolism is a promising target in cancer drug development. γ-tocotrienol (GT3), a member of the vitamin E family, orchestrates multiple effects that ensure the induction of apoptosis in both, wild-type and RAS-mutated pancreatic cancer cells. Here, we investigated whether these effects involve changes in ceramide synthesis and transport. Methods: The effects of GT3 on the synthesis of ceramide via the de novo pathway, and the hydrolysis of sphingomyelin were analyzed by the expression levels of the enzymes serine palmitoyl transferase, ceramide synthase-6, and dihydroceramide desaturase, and acid sphingomyelinase in wild-type RAS BxPC3, and RAS-mutated MIA PaCa-2 and Panc 1 pancreatic cancer cells. Quantitative changes in ceramides, dihydroceramides, and sphingomyelin at the cell membrane were detected by LCMS. Modulation of ceramide transport by GT3 was studied by immunochemistry of CERT and ARV-1, and the subsequent effects at the cell membrane was analyzed via immunofluorescence of ceramide, caveolin, and DR5. Results: GT3 favors the upregulation of ceramide by stimulating synthesis at the ER and the plasma membrane. Additionally, the conversion of newly synthesized ceramide to sphingomyelin and glucosylceramide at the Golgi is prevented by the inhibition of CERT. Modulation ARV1 and previously observed inhibition of the HMG-CoA pathway, contribute to changes in membrane structure and signaling functions, allows the clustering of DR5, effectively initiating apoptosis. Conclusions: Our results suggest that GT3 targets ceramide synthesis and transport, and that the upregulation of ceramide and modulation of transporters CERT and ARV1 are important contributors to the apoptotic properties demonstrated by GT3 in pancreatic cancer cells.

Acid Sphingomyelinase (ASMase) activity has been proposed to mediate LPS signaling in various cell types. This study shows that in macrophages, ASMase is a negative regulator of LPS-induced TNFα secretion. ASMasedeficient (asm-/-) mice and isolated peritoneal macrophages produce several fold more TNFα than their wild-type (asm+/+) counterparts when stimulated with LPS. The mechanism for these differences however is not transcriptional but post-translational. The TNFα converting enzyme (TACE) catalyzes the maturation of the 26kD precursor (proTNFα) to the active 17kD form (sTNFα). In mouse peritoneal macrophages, the activity of TACE rather than the rate of TNFα mRNA synthesis was the rate-limiting factor regulating TNFα production. Substantial portion of the translated proTNFα was not processed to sTNFα; instead it was rapidly internalized and degraded in the lysosomes. TACE activity was 2 to 3 fold higher in asm-/- macrophages as compared to asm+/+ macrophages and was suppressed when cells were treated with exogenous ceramide and SMase. In asm-/- but not in asm+/+macrophages, indirect immunofluorescence experiments revealed distinct TNFα-positive structures in close vicinity of the plasma membrane. Asm-/- cells also had higher number of EEA1-positive early endosomes. Co-localization experiments that involved inhibitors of TACE and/or lysosomal proteolysis suggest that in asm-/-cells a significant portion of proTNFα is sequestered within the early endosomes, and instead of undergoing lysosomal proteolysis it is recycled to the plasma membrane and processed to sTNFα.