Le rôle des lipides comme des acteurs moléculaires de la voie sécrétoire des plantes n'est pas encore complètement élucidé. Le Glucosylceramide (GlcCer) est synthétisé par la Glucosylceramide synthase (GCS) chez les plantes et constitue un des sphingolipides complexes clé dans les membranes, mais on connaît peu les exigences cellulaires pour le GlcCer. Cette étude repose sur le blocage de la biosynthèse de GlcCer, par l'utilisation d'inhibiteurs chez le tabac et l’arabidopsis, et la production de mutants d'Arabidopsis, pour déterminer l'impact de la biosynthèse du GlcCer sur la dynamique endomembranaire des cellules végétales. Dans une approche qui inclue la biochimie des lipides, l'imagerie de cellules vivantes, des études ultrastructurales par microscopie electronique à transmission et des études sur le développement de la plante entière, nous avons acquis une meilleure compréhension de l'impact de la synthèse du GlcCer dans les cellules végétales qui peut être résumée comme suit: (1) Sur la base d’un analyse théorique et de la microscopie de cellules vivantes on a déterminé que la GCS est situé dans le Réticulum Endoplasmique (au début de la voie sécrétoire) dans les cellules végétales, (2) PDMP est un inhibiteur spécifique de GCS chez les plantes, (3) La perturbation de la biosynthèse du GlcCer par le PDMP et par un approche génétique ont montré que le GlcCer est important pour le trafic normale des protéines et pour la dynamique des endomembranes, notamment dans le maintien de la structure de l’appareil de Golgi, (4) La régulation du trafic des protéines mediée par la synthèse du GlcCer pourrait être critique dans l’etablissement et le maintien de la polarité cellulaire, tel qu’il est suggéré par des changements dans la localisation des marqueurs polaires chez l’Arabidopsis traitée avec PDMP, et (5) Un bloc dans la synthèse du GlcCer peut conduire à des défauts importants dans le développement des plantes, comme le montrent des mutants d'Arabidopsis avec une croissance anormale des racines primaires et incapables à se développer jusqu’aux étapes reproductives. Les interactions potentielles entre GlcCer et les machineries de transport sont discutés, ainsi que les mécanismes cellulaires qui sont potentiellement déclenchés dans les cellules végétales pour compenser une perturbation de la biosynthèse du GlcCer. / The role of lipids as molecular actors in protein secretion is not well understood in plants. Glucosylceramide (GlcCer) is synthesized by Glucosylceramide Synthase (GCS) in plants and constitutes a key complex sphingolipid in membranes, but little is known about the plant cellular requirements for GlcCer. This study relied upon the block of GlcCer biosynthesis, by the use of inhibitors in tobacco and Arabidopsis, and the production of Arabidopsis mutants, to determine the impact of GlcCer biosynthesis on plant cell endomembrane dynamics. In a comprehensive approach that included lipid biochemistry, live cell imaging, ultrastructural studies by Transmission Electron Microscopy, and whole plant developmental studies, we have gained a better understanding of the impact of GlcCer in plant cells that can be summarised as follows: (1) Based on theoretical analysis and live-cell microscopy the GCS is located to the Endoplasmic Reticulum (at the beginning of the secretory pathway) in plant cells, (2) PDMP is a specific inhibitor of GCS in plants, (3) Disruption of GlcCer biosynthesis using PDMP and genetic approaches showed that GlcCer is important for normal protein trafficking and endomembrane dynamics, notably in the maintenance of Golgi structure, (4) The regulation of protein trafficking by the synthesis of GlcCer could be critical in the establishment and maintenance of cell polarity, as suggested by defects in the localisation of polar markers in Arabidopsis treated with PDMP, and (5) A block in GlcCer synthesis may be conducive to severe defects in plant development, as Arabidopsis mutants showed abnormal primary root growth and inability to develop to reproductive stages. Potential interactions between GlcCer and the transport machineries are discussed, as well as cellular mechanisms that may be set off following a disruption of GlcCer biosynthesis in plant cells.

Gaucher disease is one of the lysosomal storage disorders belonging to inherited defects of catabolism of sphingolipids. These defects are caused by mutation in genes of sphingolipid hydrolases or their protein activators. Subsequent storage of non-degraded sphingolipids leads to severe clinical phenotypes in patients. Gaucher disease is caused by deficiency of lysosomal β-glucocerebrosidase (GBA1) activity. Non-degraded glycosphingolipids are glucosylceramide (GlcCer) and glucosylpsychosine (lyso-GlcCer). Accumulation of these glycosphingolipids is related to Gaucher cells which are derived from macrophages and are abundant in spleen, liver or lung. The objective of the diploma thesis is pathobiochemistry of above mentioned glycosphingolipids. One of the topics of this work was optimization of mass spectrometry method for determination of activity of lysosomal β-glucocerebrosidase, using a natural substrate. The method was successfully optimized and can be effectively used for diagnostic purpose, instead of methods utilizing artificial substrates. In the next step, we performed analysis of pH profiles of lysosomal β-glucocerebrosidase activity focusing to search for non-lysosomal enzyme, which is able to degrade glucosylceramide. Evaluation of pH profiles did not confirm the existence of such an...

The molecular mechanisms underlying the association between mutations in GBA1 and risk of developing the ‘synucleinopathy’ disorders Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) remain elusive. To better understand the precise molecular cascade that connects GBA1 mutations with α-synuclein dysregulation, a modified lipid extraction and HPTLC protocol was optimized to detect changes in levels of neutral sphingolipids (SLs) from neural cells and tissue expressing wild-type (WT) GBA1, mutant GBA1, or both. We demonstrate that mutant GBA1 does not confer a dominant-negative effect on WT GBA1-mediated activity; however, bona fide loss-of-enzymatic function mutation events led to the accumulation of lipid substrates in neural cells and tissue, and enhance α- synuclein/ubiquitin reactivity in brain tissue of mutant gba1 mice. Our HPLC-MS/MS data are consistent with other studies demonstrating that heterozygous GBA1 mutations do not lead to lipid accumulation, but may alter α-synuclein degradation through a yet-to-be defined novel gain-of-toxic function event.

The molecular mechanisms underlying the association between mutations in GBA1 and risk of developing the ‘synucleinopathy’ disorders Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) remain elusive. To better understand the precise molecular cascade that connects GBA1 mutations with α-synuclein dysregulation, a modified lipid extraction and HPTLC protocol was optimized to detect changes in levels of neutral sphingolipids (SLs) from neural cells and tissue expressing wild-type (WT) GBA1, mutant GBA1, or both. We demonstrate that mutant GBA1 does not confer a dominant-negative effect on WT GBA1-mediated activity; however, bona fide loss-of-enzymatic function mutation events led to the accumulation of lipid substrates in neural cells and tissue, and enhance α- synuclein/ubiquitin reactivity in brain tissue of mutant gba1 mice. Our HPLC-MS/MS data are consistent with other studies demonstrating that heterozygous GBA1 mutations do not lead to lipid accumulation, but may alter α-synuclein degradation through a yet-to-be defined novel gain-of-toxic function event.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23940号 / 農博第2489号 / 新制||農||1090(附属図書館) / 学位論文||R4||N5375(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 / DGAM

Sphingolipids are found in essentially all animals, plants and fungi, and some prokaryotic organisms and viruses. Sphingolipids function as structural components of membranes, lipoproteins, and as cell signaling modulators and mediators. To complicate matters further, sphingolipids often vary in type in different regions of tissues, and even in single cells, the subcellular localization of sphingolipids and their metabolic enzymes, transport proteins and targets may influence their functions. It is important to study sphingolipids spatial distribution within living organisms to understand how sphingolipids are involved in complex biochemical processes. As part of this thesis, procedures were optimized for the use of matrix assisted laser desorption/ionization (MALDI) tissue mass spectrometry (TIMS) to visualize the location of several types of lipids including sulfatides (ST), gangliosides and phosphoglycerolipids in brains from a mouse model for Tay-Sachs/Sandhoff disease. MALDI-TIMS was next applied to human ovarian carcinoma tissue to detect sulfatide location and established that ST are associated specifically with the regions of the ovarian tissue that bear the carcinoma. Electrospray ionization tandem mass spectrometry (ESI-MS-MS) was also used to confirm that ST and galactosylceramide (GalCer) are elevated in ovarian cancer. Gene expression data using tumor cells collected using laser capture microdissection revealed greater expression of mRNAs for GalCer synthase, GalCer sulfotransferase (Gal3ST1) and other enzymes of ST biosynthesis in epithelial ovarian carcinoma cells. This is a unique combination of two complementary, profiling technologies--mass spectrometry (metabolomic approach) with analysis of gene expression to study complex cancer pathology. The next study focused on the subcellular location of sphingolipids. In comparison with wild type Hek293 cells, a Hek293 cell line stably overexpressing serine palmitoyltransferase (SPT1/2 cells) was found to have elevated amounts of all subspecies of ceramide (Cer), but produces disproportionately higher amounts of C18-Cer and GalCer. Since Cer is known to inhibit protein ER/Golgi trafficking, these studies found that the higher production of Cer caused impairment of ER/Golgi trafficking of Ceramide synthase 1 (CerS1), thus increased C18-Cer. In addition, since GalCer is only synthesized in the lumen of the ER, this impairement of ER/Golgi trafficking also gave GalCer synthase access to its substrate and increased GalCer biosynthesis. These studies illustrate the complexity of sphingolipid biology and the usefulness of multiple tools to understand sphingolipid complex biological processes.

Glycolipids play important roles in the biology of prokaryotes and eukaryotes, including humans, and although theyare found on the cell-membrane surface of all eukaryotic cells, not much is known about their biosynthesis. The aim ofthis project was to characterize two enzymes: glucosylceramide synthase (GCS) which is involved in the biosynthesisof glycolipids such as gangliosides that are abundant in the membranes of nerve cells; and dolicholphosphate mannosesynthase (DPMS), involved in the synthesis precursor for protein glycosylation. Both GCS and DPMS have been shown play a role in cancer as well as in congenital disorders of glycosylation, and are therefore interesting targets tostudy from a therapeutic perspective.With the goal to identify a suitable expression system for GCS, the genes coding for GCS from lancelet (Branchiostoma floridae) and human (Homo sapiens) were cloned and tested for expression in Escherichia coliBL21(DE3)T1 and C41(DE3) using different vectors. Cloning into three different vectors was successful and initial expression testing was performed. SDS-PAGE analysis confirmed initial expression of proteins. Although the correctsize of the protein could be confirmed by Western blot, no fluorescence of the GFP-fusion protein could be detected.DPMS from Thermococcus onnurineus (ToDP) was expressed in E. coli C41(DE3) and purified by immobilized metal ion affinity chromatography and gel filtration. Crystallization optimization was performed for ToDP produced from the vector pNIC28-Bsa4 and plate-like crystals were obtained. X-ray intensity data analysis indicated that thesecrystals contained lipid rather than protein. Crystallization screening for ToDP produced from the vector pNIC-CTHO construct was successful. Crystallization screening using the commercially available MemGold-HT96 crystallization kit resulted in initial crystallization that yielded protein crystals that diffracted to 10 °A resolution. / Glykolipider är viktiga biologiska byggstenar hos prokaryoter och eukaryoter, även människor. Trots att glykolipider finns på cellmembran ytan hos alla eukaryota celler är inte mycket känt kring syntesen av glykolipider. Målet med detta projekt var att karaktärisera två enzym: glukosylceramidsyntas (GCS) som är involverat i biosyntesen av glykolipider som gangliosider vilka förekommer i cellmembranet hos människors nervceller; och dolikolfosfatmannossyntas (DPMS) som är involverat i syntesen av substrat för proteinglykosylering. Både GCS och DPMS harvisat sig spela en roll i cancer och medfödda glykosyleringssjukdomar och är därför intressanta enzym att studera ur ett medicinskt perspektiv.Med målet att identifiera ett lämpligt expressionssystem för GCS, klonades gener från lansett (Branschiostomafloridae) och människa (Homo sapiens) och testades för expression i Escherichia coli BL21(DE3)T1 och C41(DE3)med olika vektorer. Kloning av tre olika vektorer lyckades och expressionstester utfördes. Analys med SDS-PAGE bekräftade expression av protein. Trots att korrekt storlek av proteinet kunde bekräftas med Western blot, detekterades ingen fluorescens från GFP-fusionsproteinet. DPMS från Thermococcus onnurineus (ToDP) i två olika konstrukt uttrycktes i E. coli C41(DE3) och renades med immobiliserad metalljonaffinitetskromatografi och gelfiltrering. Kristalliseringsoptimering utfördes för ToDP uttryckt i vektorn pNIC28-Bsa4 och skivliknande kristaller erhölls. Diffraktionsdata indikerade dock att kristallerna innehöll lipider och inte protein. Kristallisering av ToDP uttryckt i vektorn pNIC-CTHO lyckades och initiala kristallingsförhållanden hittades genom att använda det kommersiellt tillgängliga kristalliseringskitet MemGold-HT96. Diffraktionsdata visade på upplösning ner till 10 Å.

Glycosphingolipid (GSL) metabolism is a complex process involving proteins and enzymes at distinct locations within the cell. Mammalian GSLs are typically based on glucose or galactose, forming glucosylceramide (GlcCer) and galactosylceramide (GalCer). Most GSLs are derived from GlcCer, which is synthesized on the cytosolic leaflet of the Golgi, while all subsequent GSLs are synthesized on the lumenal side. We have utilized both pharamacological and genetic manipulation approaches to selectively regulate GSL metabolism and better understand its mechanistic details. We have developed analogues of GlcCer and GalCer by substituting the fatty acid moiety with an adamanatane frame. The resulting adamantylGSLs are more water-soluble than their natural counterparts. These analogues selectively interfere with GSL metabolism at particular points within the metabolic pathway. At 40 µM, adaGlcCer prevents synthesis of all GSLs downstream of GlcCer, while also elevating GlcCer levels, by inhibiting lactosylceramide (LacCer) synthase and glucocerebrosidase, respectively. AdaGalCer specifically reduces synthesis of globotriaosylceramide (Gb3) and downstream globo-series GSLs. AdaGalCer also increases Gaucher disease N370S glucocerebrosidase expression, lysosomal localization and activity. AdaGSLs, therefore, have potential as novel therapeutic agents in diseases characterized by GSL anomalies and as tools to study the effects of GSL modulation. Two predominant theories have been developed to explain how GlcCer accesses the Golgi lumen: one involving direct translocation from the cytosolic-to-lumenal leaflet of the Golgi by the ABC transporter P-glycoprotein (P-gp, ABCB1, MDR1), and the other involving retrograde transport of GlcCer by FAPP2 to the ER, followed by entry into the vesicular transport system for Golgi lumenal access. To examine the in vivo involvement of P-gp in GSL metabolism, we generated a knockout model by crossbreeding the Fabry disease mouse with the P-gp knockout mouse. HPLC analyses of tissue Gb3 levels revealed a tissue-specific reduction in MDR1/Fabry mice. TLC analyses, however, did not show such reduction. In addition, we performed a gene knockdown study using siRNA against P-gp and FAPP2. Results show these siRNA to have distinct effects on GSL levels that are cell-type specific. These results give rise to the prospect of unique therapeutic approaches by targeting P-gp or FAPP2 for synthesis inhibition of particular GSL pathways.

Glycosphingolipid (GSL) metabolism is a complex process involving proteins and enzymes at distinct locations within the cell. Mammalian GSLs are typically based on glucose or galactose, forming glucosylceramide (GlcCer) and galactosylceramide (GalCer). Most GSLs are derived from GlcCer, which is synthesized on the cytosolic leaflet of the Golgi, while all subsequent GSLs are synthesized on the lumenal side. We have utilized both pharamacological and genetic manipulation approaches to selectively regulate GSL metabolism and better understand its mechanistic details. We have developed analogues of GlcCer and GalCer by substituting the fatty acid moiety with an adamanatane frame. The resulting adamantylGSLs are more water-soluble than their natural counterparts. These analogues selectively interfere with GSL metabolism at particular points within the metabolic pathway. At 40 µM, adaGlcCer prevents synthesis of all GSLs downstream of GlcCer, while also elevating GlcCer levels, by inhibiting lactosylceramide (LacCer) synthase and glucocerebrosidase, respectively. AdaGalCer specifically reduces synthesis of globotriaosylceramide (Gb3) and downstream globo-series GSLs. AdaGalCer also increases Gaucher disease N370S glucocerebrosidase expression, lysosomal localization and activity. AdaGSLs, therefore, have potential as novel therapeutic agents in diseases characterized by GSL anomalies and as tools to study the effects of GSL modulation. Two predominant theories have been developed to explain how GlcCer accesses the Golgi lumen: one involving direct translocation from the cytosolic-to-lumenal leaflet of the Golgi by the ABC transporter P-glycoprotein (P-gp, ABCB1, MDR1), and the other involving retrograde transport of GlcCer by FAPP2 to the ER, followed by entry into the vesicular transport system for Golgi lumenal access. To examine the in vivo involvement of P-gp in GSL metabolism, we generated a knockout model by crossbreeding the Fabry disease mouse with the P-gp knockout mouse. HPLC analyses of tissue Gb3 levels revealed a tissue-specific reduction in MDR1/Fabry mice. TLC analyses, however, did not show such reduction. In addition, we performed a gene knockdown study using siRNA against P-gp and FAPP2. Results show these siRNA to have distinct effects on GSL levels that are cell-type specific. These results give rise to the prospect of unique therapeutic approaches by targeting P-gp or FAPP2 for synthesis inhibition of particular GSL pathways.

Ceramid, ein zentrales Signalmolekül des Sphingolipidstoffwechsels, ist neben der de novo Synthese über die enzymatische Spaltung von Sphingomyelin und Glucosylceramid zugänglich. Genetische Mutationen, die eine Fehlfaltung der verantwortlichen Enzyme saure Sphingomyelinase (aSMase) und Glucocerebrosidase (GCase) begünstigen, könnten somit zu einer Dysregulation des gesamten Sphingolipidstoffwechsels und den damit verbundenen Signaltransduktionsprozessen führen. Niedermolekulare Inhibitoren können in Zellstudien einen Einblick in diese Prozesse geben und den Defekt eines Enzyms simulieren oder eine etwaige Überaktivität derselben Enzyme verhindern. Für derartige Studien ist die Möglichkeit einer zeitaufgelösten Inhibition von Vorteil. Für diese Methode müssten photolabile Schutzgruppen in eine bereits bekannte Inhibitorstruktur integriert werden. Im Fall der aSMase würden sich hierfür myo-Inosit-bisphosphat-Derivate anbieten, die starke, kompetitive Inhibitoren des Enzyms darstellen. Auf dieser Grundlage werden in der vorliegenden Arbeit die Synthese sowie die in vitro und in cellulo Wirkung des ersten zellpermeablen, photoaktivierbaren Inhibitors für die aSMase präsentiert. Kompetitive Inhibitoren können ebenso als sogenannte pharmakologische Chaperone fungieren, welche Proteine durch Herabsetzung der freien Energie des jeweiligen Faltungszustandes stabilisieren. Dies ist besonders bei von Mutationen betroffenen lysosomalen Enzymen von Interesse, um diese vor einem proteasomalen Abbau zu bewahren und einen geregelten Transport in die Lysosomen zu gewährleisten. So wurden in der vorliegenden Arbeit verschiedene myo-Inositderivate als potenzielle pharmakologische Chaperone für die aSMase und GCase synthetisiert. Um eine Verdrängung der Verbindungen vom aktiven Zentrum des Enzyms durch das natürliche Substrat zu beschleunigen, wurde eine Orthoesterfunktion in die Seitenkette der Inhibitorstruktur integriert, die im sauren Milieu der Lysosomen gespalten werden kann. / Ceramide, a central signaling molecule in sphingolipid metabolism, is in addition to the novo synthesis accessible via the enzymatic cleavage of sphingomyelin and glucosylceramide. Genetic mutations that promote misfolding of the responsible enzymes acid sphingomyelinase (aSMase) and glucocerebrosidase (GCase) could thus lead to a dysregulation of the entire sphingolipid metabolism and the associated signal transduction processes. Small molecule inhibitors can provide insight into these processes in cell studies and simulate the defect of an enzyme or prevent eventual overactivity of the same enzyme. For such studies, the possibility of a time-resolved inhibition would be advantageous. For this method, photolabile protecting groups would have to be integrated into the structure of a known inhibitor. In the case of aSMase, myo-inositol-diphosphate derivatives, which represent strong, competitive inhibitors of the enzyme, would be suitable for this purpose. On this basis, the synthesis as well as the in vitro and in cellulo effects of the first cell-permeable photocaged inhibitor for acid sphingomyelinase are presented in this work. Competitive inhibitors can also act as so-called pharmacological chaperones, which stabilize proteins by reducing the free energy of the respective folding state. This is of particular interest in the case of lysosomal enzymes affected by mutations, in order to protect them from proteasomal degradation and to ensure regulated transport into the lysosomes. In the present work, various myo-inositol derivatives were synthesized as potential pharmacological chaperones for aSMase and GCase. To accelerate displacement of the compounds from the enzyme's active site by the natural substrate, an orthoester function was integrated into the side chain of the inhibitor structure, which can be cleaved in the acidic environment of the lysosome.