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We have studied the molecular and biological consequences of overexpression of serine palmiotyltransferase (SPT) using HEK293 cells stably transfected with SPTLC1 and SPTLC2 (termed "SPT1/2 cells"). The effects of the elevated SPT activity were analyzed by liquid chromatography, electrospray ionization tandem mass spectrometry. Most sphingolipid subspecies were elevated in SPT1/2 cells, with disproportionately higher dihydrosphingolipids and ceramides with stearic acid. Sphingomyelins were lower, however, which does not appear to be due to faster degradation, but possibly by substitution by dihydrosphingomyelins. Despite large increases in potentially growth inhibitory and lethal ceramides, SPT1/2 cells grow faster than HEK293 cells. We also noted by confocal microscopy that endogenous SPT1 is not only in the endoplasmic reticulum, but also in the nucleus and focal adhesions, which was confirmed by elimination of SPT1 using SPTLC1 siRNA and co-immunoprecipitation of SPT1 with vinculin. The appearance of SPT1 in focal adhesions is lost when cells reach confluence and reappears after a scratch assay to reinitiate migration; furthermore, SPTLC1 siRNA causes cell rounding. Thus, in addition to its "traditional" role in de novo sphingolipid biosynthesis in the ER, SPT1 is present in other cellular compartments and is required for normal cell morphology and migration. It is possible that some of the previously unnoticed properties of SPT1 are due to alternative isoforms because we have found at least one splice variant that is expressed in HEK293 cells.

Sickle cell disease is a hereditary blood disorder caused by a point mutation in the gene encoding hemoglobin. This mutation causes hemoglobin molecules to polymerize during de-oxygenation of erythrocytes producing rod-shaped polymers that bend and distort the red blood cell membrane, making it more rigid and “sickled”. This sickling causes red blood cells to lose their flexibility and ability to navigate small capillaries and also enhances the production of pro-inflammatory membrane-derived microparticles, leading to chronic inflammation and many complications such as peripheral artery disease, stroke, myocardial infarction, vasculitis and even death. Sphingolipids are a class of lipids containing a backbone of sphingoid bases and are integral components of erythrocyte and microparticle membranes. Many of these lipids are known to mediate biological processes, but their expression, distribution and orientation in erythrocytes during sickle cell disease has never been explored. Sphingomyelin, the most abundant sphingolipid in the red blood cell membrane is hydrolyzed by sphingomyelinase to produce ceramide, which has been shown to alter membrane dynamics and enhance microvessel formation. Additionally, ceramide can be further metabolized to form sphingosine and sphingosine 1-phosphate, which is a bioactive ligand for 5 known G-protein coupled receptors present on most blood and vascular cells that modulates cell motility, proliferation, migration and phenotype. Prior to this work, it was not understood how sphingolipid metabolism contributes to vascular inflammation in sickle cell disease. Together, this body of work has elucidated key enzymatic and lipidomic alterations in sphingolipid metabolism (i.e. the activation of acid sphingomyelinase on red blood cells) that result in the production of sphingolipid-rich erythrocyte-derived microparticles, which enhance inflammatory cell activation. Our work has elucidated novel pharmacological targets to reduce microparticle generation and subsequent vascular inflammation in sickle cell disease.

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.

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.

Chloroform methanol (CM) soluble extract of a cereal-based diet has been suggested to induce type 1 diabetes in an experimental animal model of type 1 diabetes. However, the individual constituent of this extract responsible for the disease induction and its disease pathogenesis mechanism remained unexplored. A previous study in our laboratory failed to show that the sphingolipid enriched fraction of CM soluble extract of wheat gluten triggers type 1 diabetes. Therefore, to study the involvement of CM soluble gliadin proteins in type 1 diabetes, we retrospectively analyzed proteins from sphingolipid enriched extract. SDS-PAGE analysis of CM soluble extract of wheat gluten and sphingolipid enriched fraction exhibited protein bands corresponding to the masses of the wheat gliadins, suggesting the presence of gliadin proteins in the CM soluble sphingolipid enriched extract. We studied the effect of five different dietary treatments on the histopathology of pancreatic tissue from BBdp rats includes insulitis scores i.e. lymphocytic infiltration in islet of Langerhans in order to test gliadin specific sphingolipid enriched extract (GSLEE) as possible a diabetogen. However, there were no significant differences in pancreatic insulitis scores and lymphoid tissue content due to addition of (GSLEE) to the hydrolyzed casein (HC) diet. We also investigated changes in jejunal histopathology and sera IFN-γ, TNF-α cytokine concentration in type 1 diabetes, induced by GSLEE. A decrease in jejunal CD4+ and γδ TCR + cell counts and inflammatory cell infiltrate were observed due to presence of CM soluble GSLEE in the HC diet, although this decrease was not statistically significant. A significant increase in sera IFN-γ cytokine concentration was found in BBdp rats fed the HC + GSLEE diet as compared to rats on HC diet. A numerical decrease in sera TNF-α concentration was also observed in BBdp rats fed the HC + GSLEE diet, when compared to BBdp rats on the HC diet. In contrast, a significant increase in serum IFN-γ concentrations in BBdp rats were observed after removing the CM soluble GSLEE from the wheat gluten based diet (WG) when compared to the WG diet alone. Removing GSLEE from WG diet resulted in insignificant increase in serum TNF-α concentration in BBdp rats when compared to WG dietary treatment group's BBdp rats. However, there were no significant differences in jejunal enteropathy parameters (i.e. lymphocytic infiltration, mucosal thickness, epithelial erosion, jejunal villi flattenings), jejunal CD4+ and jejunal γδ TCR+ cell counts; pancreatic insulitis scores, lymphoid tissue content after removing the CM soluble GSLEE from the WG diet when compared to the WG diet. Since overall findings regarding the CM soluble GSLEE's potential to induce type 1 diabetes by changing pancreatic and jejunal histopathology and elevating serum IFN-γ, TNF-α cytokine levels largely remained inconclusive, further investigations are warranted regarding immune suppression potential of the CM soluble sphingolipids in type 1 diabetes and the search of diabetogenic agents remaining in the residue after CM extraction. / Master of Science

Sphingolipids are a class of lipid molecules that function both as structural membrane components and as bioactive signaling molecules. Sphingolipids can be produced de novo or salvaged and recycled. Despite the established roles of sphingolipids such as sphingosine 1-phosphate and ceramides in regulating signaling involved in pro- and anti-tumorigenic cellular processes, the role of the de novo sphingolipid biosynthesis pathway in cancer is unclear. The main objective of this thesis study was to determine whether there is an essential role for this pathway in cancer and whether its disruption can be a cancer-specific metabolic vulnerability. Here, we find that de novo sphingolipid synthesis through the rate-limiting enzyme serine palmitoyltransferase (SPT) is not required in cancer cells due to their salvage capacity. However, upregulation of SPT in cancer cells creates a requirement to detoxify its product, 3-ketodihydrosphingosine (3KDS), via the downstream enzyme 3-ketodihydrosphingosine reductase (KDSR). We demonstrate that KDSR is essential in cancer cells both in vitro and in vivo to restrain the levels of its substrate 3KDS, the accumulation of which can disrupt ER structure and function, resulting in proteotoxic stress and cell death. Our findings also reveal that KDSR is essential specifically in cancer cells and not normal cells and that upregulation of SPT in cancer may act as a biomarker for sensitivity to targeting KDSR. Altogether, this thesis study provides new insights into the role of KDSR in the de novo sphingolipid biosynthesis pathway in both cancer and ER homeostasis and demonstrates the potential to exploit this for therapeutic purposes in a cancer-specific manner.

Autophagy is the process of “cell self-eating” which has been implicated both in cell survival and cell death. Sphingosine kinase 1 (SphK1) regulates the intracellular balance between ceramide and sphingosine, bioactive lipids associated with cell death, and sphingosine-1-phosphate (S1P), whose actions are associated with survival and proliferation. Previous studies have implicated upregulation of SphK1 in the induction of autophagy. In this study, SK1-I, a SphK1 specific competitive inhibitor, induced autophagy in a concentration and time dependent manner in HCT116 colorectal carcinoma cells. This autophagic response was observed to be more intense in wild type p53 expressing HCT116 cells than in p53 null cells and ultimately led to non-apoptotic death in wild type and apoptotic death in p53 null cells. In agreement, cell death in wild type cells was not accompanied by cleavage of polyADP ribose polymerase, a hallmark of apoptosis. Knockdown of Beclin 1 demonstrated that it and its binding partners do not have a significant role in the induction of autophagy in response to SK1-I treatment. Similarly, mTORC1 signaling was not observed. In contrast, SK1-I markedly decreased Akt phosphorylation. However, this might not be the sole factor important for SK1-I induced autophagy, as pharmacological inhibition of Akt only led to a comparatively weak autophagic response. Indeed, phosphorylation of the endoplasmic reticulum (ER) stress marker eIF2 α, was greatly reduced, suggesting that an ER mediated mechanism also contributes to SK1-I induced autophagy. Thus, SK1-I induced autophagy was likely triggered by ER stress signaling and led to non-apoptotic cell death in the more highly autophagic wild type 53 expressing cells. These results suggest that an isotype specific SphK1 inhibitor might be a useful adjunct for the treatment of cancer or other diseases in which enhancement of cytotoxicity or autophagy is desirable.

Alcoholic liver disease (ALD) is one of the most common liver diseases worldwide characterized by the accumulation of lipids within the liver, inflammation and the possibility of progressing to cirrhosis and liver failure. More importantly, there are currently no effective treatments for ALD and liver transplantation remains the only therapeutic option for end-stage liver disease. Previous studies have shown that ALD is a result of a combination of endoplasmic reticulum (ER) stress, lipid metabolism dysregulation and inflammation. It has been previously reported that alcohol disrupts gut microbiota homeostasis and causes increased endotoxins that contribute to the pathology of ALD. However, the detailed mechanism(s) underlying ALD and disease progression is poorly understood. We have discovered that sphingosine kinase 2 (SphK2) deficient (SphK2-/-) mice on an alcohol diet exhibit increased steatosis and inflammation compared to wild type mice. Sphingosine 1-phosphate receptor 2 (S1PR2) and SphK2 have been previously shown to play a key role in nutrient metabolism and signaling. However, their roles in alcohol-induced liver injury have not been characterized. The overall objective of this study is to determine the molecular mechanism(s) by which disruption of S1PR2-mediated SphK2 signaling contributes to ALD. The effects of alcohol on mouse primary hepatocytes and cultured RAW264.7 macrophages were examined. The acute on chronic alcohol mouse model from NIAAA that recapitulates the drinking pattern of human ALD patients was used to study the effects of SphK2 deficiency in ALD. In addition, 60-day chronic alcohol mouse model was used to determine whether a more severe form of ALD was present in SphK2-/- mice. The results indicated that SphK2-/- mice on an alcohol diet exhibited an increased amount of hepatic steatosis compared to wild type mice. Genes regulating lipid metabolism were also dysregulated in SphK2-/- mice. SphK2-/- mice also had increased inflammation and liver injury as shown by an upregulation of inflammatory markers and increased levels of liver enzymes. Moreover, SphK2 protein expression levels were downregulated in the human livers of alcoholic cirrhotic and hepatocellular carcinoma (HCC) patients. These findings contribute to a greater understanding of the pathophysiology of ALD and could provide information on the development of novel therapeutics against ALD.

Das Ceramid Transfer Protein (CERT) ist einer der geschwindigkeitsbestimmenden Proteine in der de novo Biosynthese von Sphingomyelin. Es ist verantwortlich für den nicht-vesikulären Transfer von Ceramid vom Endoplasmatischen Retikulum zum Golgi-Apparat. Die Inhibition von CERT wird als potenzielle Behandlung für Krankheiten wie Infektionen, Krebs oder Gain-of-Function-Mutationen des CERT Gens diskutiert. Kürzlich, wurde Lomitapide als potenter Inhibitor des CERT-vermittelten intermembran-Transfers identifiziert. Im ersten Teil dieser Arbeit wird die Synthese von Lomitapide Derivaten mit verbesserter Wirksamkeit und Selektivität präsentiert. Die synthetisierten Analoga wurden in vitro mithilfe eines liposomalen Transferassays auf ihre Inhibition der CERT-Transferaktivität getestet. Zusätzlich konnte durch die Messung des Ceramid-Sphingomyelin-Verhältnisses nach Inhibitor Behandlung die Aktivität in cellulo bestätigt werden. Die Selektivität gegenüber dem Mikrosomalen Triglycerid Transfer Protein (MTP) wurde durch Messung der MTP-vermittelten Sekretion von apoB ermittelt. Unter den synthetisierten Analoga zeigten einige verbesserte CERT-Transfer Inhibition und niedrigere Inhibition der apoB Sekretion, sogar bei fünffacher Konzentration verglichen mit Lomitapide. Obwohl die Bewertung der biologischen Aktivität noch im Gange ist, wurde eine vorläufige Struktur-Aktivitäts-Beziehung etabliert. Es wurden strukturelle Bestandteile identifiziert, die wichtig für die CERT-Inhibition sind und andere welche variabel sind, um die Wirksamkeit und Selektivität in Zukunft noch weiter zu steigern. Der zweite Teil dieser Arbeit beschreibt die Synthese von Lomitapide-basierten proteolysis targeting chimeras (PROTACs) für CERT. PROTACs haben sich im letzten Jahrzehnt zu einem vielversprechenden therapeutischen Ansatz entwickelt und mehrere potenzielle Wirkstoffe hervorgebracht. PROTACs sind heterobifunktionale Moleküle, die sich den zellulären Weg der Proteinzersetzung zunutze machen, indem sie das gewünschte Protein zur Zersetzung markieren. Es wurde eine erste Serie von CERT PROTACs mit vielversprechender Abbauwirkung synthetisiert, welche eine bevorzugte Zersetzung von CERT aber nicht CERTL andeuten. CERTL ist eine längere Spleiß-Variante, welche vornehmlich im Herz, Gehirn und den Skelettmuskeln exprimiert wird. Eine zweite Serie von PROTACs mit variierter Linker Kettenlänge wurde synthetisiert. Untersuchung des Einflusses auf die apoB Sekretion aus HepG2 Zellen zeigte sogar bei 50-facher Konzentration einen niedrigeren Einfluss auf diese als Lomitapide. / The ceramide transfer protein (CERT) is one of the rate-limiting proteins in the de novo biosynthesis of sphingomyelin, facilitating the non-vesicular transfer of ceramide from the Endoplasmic Reticulum to the Golgi-apparatus. Inhibition of CERT has been proposed as a potential treatment for pathogenesis like infectious diseases, cancer, or disease-causing gain-of-function mutations within the CERT gene. Recently Lomitapide has been identified as a potent inhibitor of CERT-mediated intermembrane transfer. In the first part of this thesis, the synthesis of Lomitapide derivatives with improved potency and selectivity is presented. The synthesized analogs were tested in vitro for their inhibition of CERT-transfer using a liposomal transfer assay. Additionally, the activity could be confirmed in cellulo by monitoring the ceramide-sphingomyelin-ratio after inhibitor treatment. Selectivity against the microsomal triglyceride transfer protein (MTP) has been determined by monitoring the MTP-mediated cellular secretion of apoB. Among the synthesized analogs, several showed improved CERT-transfer inhibition and lower inhibition of apoB secretion even at five-fold higher concentrations compared to Lomitapide. Although the biological evaluation is still underway, a preliminary structure-activity-relationship has been established and identified structural motifs important for CERT inhibition and modifiable moieties to increase potency and selectivity even further in the future. The second part of the thesis describes the synthesis of Lomitapide-based proteolysis targeting chimeras (PROTACs) for CERT. PROTACs have evolved in the last decade as a promising therapeutic technique and resulted in the development of several drugs which are currently in clinical trials. PROTACs are heterobifunctional small molecules that mediate the degradation of the target protein by hijacking the cellular proteasomal pathway. A first series of synthesized CERT PROTACs showed promising preliminary results for CERT degrader activity and indicated a preferred degradation of CERT over CERTL, a longer splicing variant expressed in the heart, brain, and skeletal muscles. Motivated by this a second generation of PROTACs with varying linker chain lengths was synthesized. Investigation of their inhibition of apoB secretion from HepG2 cells revealed lower activity on secretion than Lomitapide even at 50-fold concentrations for a set of CERT PROTACs.