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The current CoV-SARS-2 pandemic exemplifies the profound impact viruses can have upon our lives. Virus infection is a complex process where viral proteins cooperate to exploit host cell metabolism while evading immune responses. Herpes simplex virus 1 (HSV-1) is a very prevalent human herpesvirus that causes life-long infection and is known to dramatically remodel the cellular environment upon replication. This extensive manipulation is achieved using only a limited number of virus-encoded proteins, many of which possess numerous functions and exert their effects in multiple different subcellular compartments. HSV-1 pUL21 is an example of such multi-function protein: it is known to be important for assembly of new virus particles and viral cell-to-cell spread but its exact molecular functions remained unknown. Using a high throughput interactomics screen, previous members of the group identified potential cellular binding partners of pUL21: protein phosphatase 1 (PP1) and ceramide transfer protein (CERT). This thesis presents biophysical characterization of the direct interactions between pUL21 and these partners and describes the role of pUL21 as a novel viral phosphatase adaptor that recruits PP1 to multiple substrates, including CERT, to promote their dephosphorylation. Conservational and structural analyses led to the discovery of a non-canonical linear motif in pUL21, termed TROPPO, that is critical for PP1 binding and it is absolutely conserved across α-herpesviruses. In vitro evolution experiments using HSV-1 strains with mutated TROPPO motifs revealed that the phosphatase adaptor pUL21 antagonises the activity of the virus-encoded kinase pUS3. A correct balance of kinase and phosphatase activity is shown to be essential for correct subcellular localisation of the HSV-1 nuclear egress complex and for virus replication and dissemination. Using in vitro biochemical experiments, stable expression of pUL21 in cultured cells, and infection with wild-type HSV-1 or viruses expressing pUL21 with a mutated TROPPO motif, we confirmed that pUL21 stimulates PP1-dependent dephosphorylation of CERT and the viral nuclear egress complex component pUL31, plus additional as-yet unidentified proteins. The binding interface of the pUL21:CERT complex was determined using small-angle X-ray scattering, enabling the generation of pUL21 mutants where binding to CERT, but not to other substrates, was specifically disrupted. Generation of a CERT non-binding mutant facilitated a detailed characterization of the sphingolipid-modulatory role of pUL21 using 'click chemistry'-based assays and revealed that pUL21-dependent upregulation of sphingomyelin turnover is required for the correct trafficking of maturating virions to the plasma membrane. In summary, this thesis presents structural and functional characterisation of HSV-1 pUL21, dissecting the multiple roles played by this protein during the replication of HSV-1. Furthermore, this study provides first insights into the modulation of sphingolipid homeostasis during virus infection, a critically understudied host:pathogen interaction.

Sphingolipids are a diverse class of lipids involved in fundamental cellular processes. In addition to their role in membrane architecture, sphingolipids can act as bioactive molecules, regulate membrane protein function and be presented to the immune system as lipid antigens by CD1 family molecules. The critical importance of sphingolipids for cell function is highlighted by the catastrophic diseases that result from defects in sphingolipid metabolism. Lipid loading onto CD1d is facilitated by the lysosomal lipid transfer protein Saposin B, but the lipid loading mechanism is unknown. I used a range of highly sensitive biochemical assays to probe the interaction between CD1d and SapB. Interestingly, no direct interaction was detectable. Importantly, this suggests that additional proteins may promote their interaction. In addition to their role as antigens, sphingolipids have roles in immunity by regulating the trafficking and function of immune receptors. Krabbe disease, a neurodegenerative disease caused by loss of function of the lysosomal sphingolipid hydrolase GALC, is accompanied by immune defects possibly driven by phagocytic cell dysfunction. I investigated how GALC deficiency alters the phenotype and function of the phagocytic cell line THP-1. This study was compromised by substantial off-target effects caused by genetic manipulation of THP-1 cells. Characterising these off-target changes identified perturbations in sphingolipid metabolism pathways, confirming that these cell lines are not appropriate for further studies. The lysosomal enzyme acid ceramidase (AC) hydrolyses ceramide to sphingosine, thus regulating the levels of pro-apoptotic and pro-survival lipids. This role in apoptosis means that regulation of AC activity is of interest for cancer treatment. Although the structure of AC was recently determined, it is still unclear how it accesses membrane-embedded lipid substrates. I attempted to co-crystallise AC with Saposin A lipoprotein nanodiscs to understand this process. Although this system did not prove suitable for this project, I was able to determine the crystal structure of a new saposin oligomer highlighting even greater diversity of tertiary arrangement of these important lipid transfer proteins. I next asked whether AC processes galactosylceramide to psychosine, the cytotoxic GALC substrate accumulating in Krabbe disease, contributing to Krabbe disease pathology by increasing oligodendrocyte cell death. Surprisingly, I found that GALC knockout oligodendrocytes were not more susceptible to cell death than wild-type oligodendrocytes, calling into question whether psychosine accumulation is the direct cause of oligodendrocyte cell death and demyelination in Krabbe disease. The sphingolipid-interacting adhesion protein PTPRM is lost at the surface of GALC-deficient oligodendrocytes, suggesting that specific sphingolipid-PTPRM interactions cause the mistrafficking of this adhesion protein in Krabbe disease oligodendrocytes, potentially contributing to demyelination. I probed the interaction of PTPRM with sphingolipids through X-ray crystallography studies and fluorescence-based trafficking assays.

Sphingolipids (SLs) are a diverse class of lipid molecules derived from the amino acid L-serine and long chain fatty acids (e.g. carbon chain lengths C14-C26). When combined together these building blocks form a so-called "sphingoid", also known as a long-chain base (LCB). Decades of research into these enigmatic molecules has revealed that SLs are essential components of eukaryotic cell membranes and control many critical cellular functions. The SL biosynthetic pathway begins in all organisms with the Claisen-like, decarboxylative condensation of L-Ser and long-chain fatty acid acyl-CoA thioester (CoASH) substrates (most commonly C16/palmitoyl) to form the intermediate 3-ketodihydrosphingosine (3-KDS). This first key irreversible reaction is catalysed by the pyridoxal 5'-phosphate (PLP)-dependent serine palmitoyltransferase (SPT). The 3-KDS product is then reduced by a NAD(P)H-dependent enzyme (KDS reductase, KDSR) to generate dihydrosphingosine (DHS). Acylation of this molecule with a long-chain fatty acid by a ceramide synthase (CerS) leads to the formation of ceramide (Cer). Downstream, phosphorylation with a phosphate group can occur on the serine-derived head group and lead to the formation of sphingosine-1-phosphate (S-1-P). The balance between Cer and S-1-P concentrations has been proposed to control the cell survival rate in the host system. Alternatively, sugars such as glucose can be added to give the family of glycosphingolipids (GSLs). In contrast to higher order species, research into SL biosynthesis in bacteria has lagged much further behind. However recent studies have revealed a number of important microbes that produce a range of SLs and Cers. Interestingly, SLs from the human microbiota including those from Bacteroides fragilis, a Gram-negative commensal bacterium from the human gut, and from the oral pathogen Porphyromonas gingivalis have recently been shown to play an essential role in host/microbe communication. Of note is that these bacterial SLs and Cers display many similar structural features to their mammalian homologues. However, there appears to be "chemical signatures" distinct to those of bacterial origin. For example, B. fragilis SLs have iso-Me branched acyl chains which are also similar to those found in the round-worm Caenorhabditis elegans. This suggests a PLP-dependent branched-chain amino acid transferase (BCAT/IlvE) transfers iso-Me chains from amino acid precursors such as L-Leu to branched-chain keto acids. To fully understand the role of bacterial SLs and their metabolism detailed investigations of the enzymes, pathways and metabolism are required. In this thesis, studies of different types of the three key enzymes (SPT, IlvE/BCAT and KDSR) involved in the core microbial SL biosynthetic pathway have been presented. Firstly, both recombinant B. fragilis SPT (BfSPT, encoded by gene BF2461) and P. gingivalis (PgSPT, gene PG1780) were expressed in E. coli, purified and studied with protein UV-vis spectrometry, enzyme kinetics, inhibition assays, mass spectrometry and protein crystallization screening. 3-KDS products were detected derived from a range of straight-chain CoA substrates (C14-C18) and amino acids (Gly, L-Ala and L-Ser) produced by BfSPT and PgSPT. Mutagenesis of a conserved loop (PAVAP) in SPT homologues was found to be associated with the catalytic efficiency of PgSPT. Also the presence of a Val353 residue in BfSPT was shown to be essential to allow the enzyme to interact with the C16-CoA substrate. Moreover, data suggested that the position (N- or C- terminus) of the 6His-affinity tag used to purify SPT influenced substrate inhibition by C16-CoA. A hypothetical 3D structural model of the PgSPT PLP:L-Ser external aldimine complex was built in order to explore the active site and residues involved in substrate binding and catalysis. In collaboration with Prof. Mary-Ellen Davey (Florida), the role on SL biosynthesis in P. gingivalis was also explored and found that SLs impact on the way this pathogen interacts with human cells. Secondly, a branched acid transaminase P. gingivalis IlvE (PgIlvE, gene PG1290) was expressed, purified and studied using UV-vis spectrometry to investigate substrate binding and enzyme activity. A multi-enzyme coupled assay for PgIlvE was developed in both the 'forward' and 'reverse' direction, studied with inhibitors such as L- and D- cycloserine (LCS/DCS), as well as x-ray crystallography. In collaboration with Dr. Jon Marles-Wright (University of Newcastle) the crystal structures of four different forms of PgIlvE; the PLP-bound, internal aldimine form, LCS ring-opened ring-closed form and the PMP form, were obtained. Two residues (F56 and Y188) were identified which played a role in substrate binding and activity. In the final chapter, recombinant KDSR from the yeast Saccharomyces cerevisiae (ScKDSR) was isolated from E. coli. Kinetic parameters for a soluble, truncated form of the enzyme were determined using the substrates KDS and NADPH. The product C18 DHS derived from C18 KDS was detected with MALDI-ToF-MS. Recent studies of human KDSR revealed that patients with point mutations in this enzyme suffered from skin disorders (erythrokeratoderma). To investigate the impact of these mutations, a series of ScKDSR mutant mimics (G176S, Y180F and G263E) were prepared. To date there has been no crystal structure of a KDSR determined but it is a member of the short-chain dehydrogenases/reductases (SDR) superfamily. A homology model of the 3D structure of ScKDSR with three possible NADPH docking positions were constructed, and residues involved in substrate binding and catalysis were suggested. The results in this thesis shed light on the key enzymes involved in the core biosynthetic pathway of microbial SLs and lay down a blueprint for future studies.

Sphingolipids (SLs) are complex lipid-derived structures that are essential components of cell membranes in eukaryotes and some bacteria. SLs and their complex derivatives ceramides are known to be involved in multiple processes such as the formation of lipid rafts, cell signalling and membrane trafficking. The first step of SL biosynthesis is universal to all sphingolipid-producing organisms from bacteria to humans and is catalysed by the enzyme serine palmitoyltransferase (SPT). SPT is a member of the alpha-oxoamine synthase (AOS) family of pyridoxal- 5'-phosphate-dependent enzymes. All AOS family enzymes retain a high degree of structural homology and catalyse the decarboxylative Claisen-like condensation of amino acids with thioester substrates. The SPT enzyme catalyses the formation of the universal SL precursor, 3-ketodihydrosphingosine (KDS), by condensation of L-serine and coenzyme A-derived palmitic acid. Being the key controller in SL biosynthesis, SPT plays a big role in regulating natural and pathological processes. A lot of research interest has been recently generated by SLs isolated from bacterial members of the human microbiome and their roles in human health. Increasing evidence suggests that some of these SLs possess immunoregulatory effects and can have a direct impact on the immunity of the host. Bacteroides fragilis is a commensal gut-dwelling bacterium that belongs to a few human microbionts known to produce unique iso-branched sphingolipids (isoSLs); these have been shown to influence the human iNKT cell count. The production of SLs in B.fragilis is completely regulated by a gene product BF2461. In this work, BF2461 was expressed and purified; using a combination of UV-vis spectrometry, enzymatic assays, mass spectrometry and protein X-ray crystallography, it has been confirmed to be an SPT. The substrate specificity of the BfSPT has been assessed with a range of different chain-length substrates, including less common 15 and 17-carbon chain length coenzyme A substrates. The enzyme can produce different types of SL precursors with a preference for the 16-carbon chain substrate palmitoyl- CoA. However, at high levels of PCoA, a substrate inhibition is observed that might point to a natural control mechanism employed by the bacterium in favour of producing iso-branched SLs (isoSLs). The structure of BfSPT has been elucidated in a complex with its amino acid substrate L-serine. Search and analysis of putative SPTs from other microbiome-associated bacteria that produce isoSLs show that they share high similarity with an average amino acid conservation of 74%, suggesting they might be adapted to a particular type of substrate. In this respect, BfSPT might be the first isoSL-producing SPT to be structurally characterised, and the first one to have a direct impact on human health. Further structural data were obtained on protein complexes with L-cycloserine and L-penicillamine, some common inhibitors of the PLP-dependent enzymes. The structure obtained in the presence of L-penicillamine provides the first direct structural evidence of the inhibitory mechanism by a thiazolidine complex formation in the active site of a PLP-dependent enzyme. These findings shed light on certain aspects of the reaction and inhibition mechanisms of BfSPT as well as opening new prospects into researching this interesting target and its impact on the human microbiome.

Sphingolipids (SLs) are ubiquitous elements in the membranes of eukaryotes and are also found in some bacterial and viral species. As well as playing an integral structural role, SLs act as potent signalling molecules involved in numerous cellular pathways having been implicated in cancer, inflammatory conditions and cardiovascular disease. A central SL signalling molecule is sphingosine-1- phosphate (SIP), generated by the phosphorylation of sphingosine by two kinase isoforms sphingosine kinase 1 and 2 (SPHK1, SPHK2). The irreversible breakdown of SIP is catalysed by sphingosine-1-phosphate lyase (SIPL), a pyridoxal 5'-phosphate (PLP) dependent enzyme that catalyses the retro-aldol-like cleavage of SIP to (2E)-hexadecenal (2E-HEX) and phosphoethanolamine (PE). S1PL is a PLP Fold type I carbon-carbon bond lyase belonging to the Group II PLP-dependent decarboxylase subfamily and is the terminal enzyme in sphingolipid metabolism. In this work two putative SI PL genes (BPSS2021 and BPSS2025) were identified in Burkholderia pseudomallei K96243, an organism that has not been shown to produce sphingolipids, suggesting an auxiliary role for these enzymes in this pathogenic bacterium. Using a combination of UV-visible (UV-vis) spectroscopy, mass spectrometry (MS), X-ray crystallography and established assay methodology we validate that these genes encode enzymes with SI PL activity. There are a number of published SI PL assays that rely on two main methodologies to monitor SI PL activity; MS analysis of a derivatised product or the use of fluorescent SIP substrate derivatives. Here we describe the development of two new enzyme-coupled assays that use UV-vis to measure the activity of SIPLs providing a convenient method for enzyme characterisation. The first coupled assay uses a human bone phosphatase, PHOSPHOl, which is highly specific for the PE product of the lyase reaction. This generates inorganic phosphate (Pi) which is detected using a malachite green based dye or enzymatically via the purine nucleoside phosphorylase (PNP) degradation of 2- amino-6-mercapto-7-methylpurine riboside (MESG). The second assay employs a recombinant form of human fatty aldehyde dehydrogenase (FALDH) which uses the 2E-HEX as a substrate. Activity is monitored via the reduction of the FALDH NAD+ cofactor to NADH and the concomitant increase in absorbance at 340 nm. Using this assay we were able to kinetically characterise the two homologous B. pseudomallei SI PL isoforms (S1PL2021 and S1PL2025) and suggest that this new method could be applied to the studies of other bacterial and eukaryotic SIPLs. We also report the determination of the x-ray structure of S1PL2021, with the PLP cofactor bound, at 2.0A resolution which provides an insight into residues potentially involved in catalysis. The SI PL reaction mechanism requires a general base to deprotonate the SIP 3'-OH. This step in the enzyme's mechanism was probed via the mutagenesis of potential residues involved to give insight into this key stage in catalysis whereby the PLP-S1P intermediate is deprotonated releasing the 2E-HEX product. Based on structural interrogation of the enzyme, candidate residues are proposed as the possible base in the Burkholderia isoforms of the enzyme.

Background & Aims: The 'high-density lipoprotein (HDL) hypothesis', that therapeutic interventions directed at raising HDL cholesterol might translate into improved cardiovascular outcomes, has been confounded by recent reports from genetic and pharmacological studies. HDL functionality may be more important than cholesterol cargo. HDL cholesterol levels are normal or even high in Type 1 Diabetes (T1DM) but do not seem to protect against atherosclerosis as might be expected; this thesis aims to offer new insight into HDL functionality through examination of these patients. This thesis also aims to improve understanding of the qualitative changes in lipoproteins associated with diabetes and increased cardiovascular morbidity, with emphasis on atherogenic modifications of apolipoprotein B and sphingolipids, and consideration of the relationship between these changes, novel and established biomarkers, and macrovascular and microvascular diabetic complications. Materials & Methods: Patients with Type 1 (n = 91) and Type 2 (n = 40) Diabetes Mellitus and healthy volunteers (n = 104) attended for fasting blood tests, urinalysis, and examination including cardiac computed tomography, carotid doppler studies and assessments of nerve function. In vitro studies of lipoprotein modification used pooled human plasma. Results: Lipoprotein glycation represents an atherogenic modification. In vitro glycation occurs more readily in the presence of physiological concentrations of copper. HDL and copper-selective chelation with triethylenetetramine prevents glycation. Glycated apolipoprotein B, oxidized LDL and small-dense LDL levels were significantly higher in T1DM; HDL cholesterol levels were also significantly higher, but with altered apolipoprotein distribution, and significantly lower cholesterol efflux capacity and PON1 activity than in healthy controls. Significant changes were also observed in cystatin C, advanced glycation end-products, leucine-rich alpha-2-glycoprotein, lipoprotein-associated phospholipase A2, a variety of inflammatory markers, and sphingolipid and ceramide profiles. Discussion: Cardiovascular disease is the leading cause of death and disability in diabetes. Patients with diabetes show qualitative and kinetic lipoprotein abnormalities, and any cardiovascular benefit associated with intensive glucose lowering may be related to effects on lipoprotein metabolism rather than directly through altered glycaemia. The apparently relatively undisturbed lipid profile observed in many patients with diabetes hides major atherogenic changes and altered HDL functionality, which may be at least partially responsible for the persistent increased risk of cardiovascular disease in patients with diabetes. HDL-based therapy remains a largely unfulfilled promise, but there may be a role for copper-selective chelation and more aggressive low-density lipoprotein lowering in the reduction of diabetic complications.

Insulin-like growth factor-I (IGF-I) is the only known potentially modifiable risk factor for prostate cancer. Intake of dietary protein, especially from dairy products, might also be associated with risk and with circulating IGF-I, but it is not clear if amino acids play a role in these relationships. Moreover, investigations of circulating concentrations of metabolites might reveal novel risk factors for prostate cancer. This thesis investigates plasma concentrations of amino acids and other metabolites in relation to protein intake, IGF-I, and prostate cancer risk using data from the European Prospective Investigation into Cancer and Nutrition (EPIC). To characterise plasma metabolite profile in men consuming markedly different amounts and types of animal products (meat-eaters, fish-eaters, vegetarians and vegans), cross-sectional analyses of 392 men in the EPIC-Oxford sub-cohort were conducted. Of 21 amino acids, six varied significantly by diet group, and the metabolite profile of vegans was different from those of other diet groups owing to lower concentrations of several glycerophospholipids and sphingolipids. In a case-control study nested within EPIC, with a mean follow-up time of seven years, the relationship of plasma metabolites with risk of prostate cancer overall, by time to diagnosis, by tumour characteristics, and with risk of prostate cancer death, was investigated. Data from 1,077 matched sets suggested that seven metabolites, from various classes, were associated with risk of prostate cancer overall (p < 0.05). After correction for multiple testing, 12 glycerophospholipids were inversely associated with risk of advanced prostate cancer (the strongest OR1SD = 0.54; 95%CI: 0.40-0.72). In multivariate analyses, including data from 1,593 matched sets, principal component analysis (PCA) and treelet transform (TT) were used to identify patterns in metabolite profile, five of which were associated with risk of more aggressive tumour sub-types (high grade, advanced and aggressive disease) and/or prostate cancer death. There was a ≈ 50% lower risk of advanced and high grade prostate cancer in men with metabolite profiles characterised by high glycerophospholipids and sphingolipids (for advanced ORTT, top vs bottom third = 0:48; 95%CI: 0:31-0:74), with similar results for high grade and PCA). To investigate if associations between protein intake and circulating IGF-I may be mediated by plasma amino acid concentrations, cross-sectional analyses of amino acid concentrations with protein intake and IGF-I concentrations were carried out in 1,697 and 1,142 control participants, respectively, from the nested case-control study. Dairy protein intake was positively associated with concentrations of branched-chain amino acids and several other essential amino acids, while plant protein intake was strongly associated only with histidine. Serum IGF-I was positively associated with arginine and inversely with ornithine and certain amino acid ratios. In conclusion, men with different dietary habits with respect to the consumption of protein types have different amino acid and metabolite profiles, and metabolite concentrations may be associated with risk of more high-risk prostate cancer sub-types (high grade, advanced and aggressive disease) and prostate cancer death. Further large-scale studies are needed to determine if metabolites play a role in aetiology or are markers of sub-clinical prostate cancer.

Sphingolipids (SL) are essential structural components of membranes found in all eukaryotes and have also been identified in some bacteria. The first step of the SL biosynthetic pathway across all species is catalysed by serine palmitoyltransferase (SPT), a member of the alpha-oxoamine synthase (AOS) family of pyridoxal 5’- phosphate (PLP)-dependent enzymes. AOS enzymes are involved in the biosynthesis of a range of important natural products such as heme, vitamins and antibiotics where they catalyse the reaction between amino acid and acyl-thioester substrates. Substrate specificity across the family is of great importance, as human mutant SPTs shift the substrate specificity from L-serine to glycine or L-alanine that lead to production of deoxy-sphingolipids that are toxic to mammalian cells. PLP, a form of vitamin B6, is one of nature’s most versatile catalysts and is involved in over 160 enzymes that carry out diverse reactions on amine-containing substrates. This work probes the functional role of the phosphate group of PLP, usually housed in a phosphate binding cup (PBC) and investigates the need for a novel and unexpected H-bond between the hydroxyl group of the L-serine substrate and the 5’-phosphate group of PLP in SPT. In this study, the PLP cofactor was removed from SPT with amino-thiol substrates which act as mechanism-based inhibitors of SPT via production of a thiazolidine adduct. Replacement of natural PLP with the dephosphorylated form of the cofactor, pyridoxal, allowed a study on the importance of the PLP phosphate:L-serine H-bond on substrate specificity and optimal SPT activity. Furthermore, analysis of the phosphate binding cup of the ALAS:PLP:glycine external aldimine, a related AOS family member; revealed an important residue that could possibly be involved in determining substrate specificity of different members of the AOS family. PBC analysis also expanded, with a detailed and interesting study of a novel SPT:PLP:myriocin inhibitor complex. Human SPT is a heterodimeric, membrane-bound enzyme composed of two subunits (hLCB1/hLCB2) which is thought to contain a single PLP-containing active site. Mutations in human hLCB1 have been linked to the rare sphingolipid metabolic disease hereditary sensory neuropathy I (HSAN1). Recent studies identified three heterozygous missense mutations in the second human SPT subunit hLCB2 that show a significant loss in SPT activity. The three human SPT mutations V359M, G385V and I504F were mapped onto the bacterial S. paucimobilis SPT as V246M, G268V and G385F. These bacterial SPT mutant mimics reveal that the amino acid changes have varying impacts; they perturb the PLP cofactor binding, reduce the affinity for both substrates, decrease the enzyme activity, and, in the most severe case, cause the protein to be expressed in an insoluble form. SPTs and most of the other members of the AOS family utilise an acyl-CoA thioester substrate. In contrast, a sphingolipid-producing bacterium, S. wittichii, is thought to use a small type II acyl carrier protein (ACP) to deliver the acyl chain to its homodimeric SPT target. Converting the unmodified apo-ACP to the activated “substrate” acyl-ACP, has proven difficult and amino acid sequence alignment, combined with modelling studies revealed an unusual tryptophan residue that could prevent modification to the acyl-ACP form. In this study a double mutant ACP E36G/W37A has been prepared and characterised. Both wild-type and mutant S. wittichii ACP are expressed in the recombinant E. coli host in their inactive apoform. The transfer of a phosphopantethiene (4’PP) linker by a specific PPTase (also known as an acyl carrier protein synthase (AcpS)) has been successful in modifying the mutant form of ACP to its holo-form but could not transfer a palmitoyl group (C16). E.coli ACP has been successfully expressed, purified and characterised in this study. For the first time, ion mobility mass spectromerty (IM-MS) has been used on this protein to gain structural insight into the different forms of ACP. Collisional cross section (CCS) distributions have been calculated for different acylated states of the ACP concluding that the protein exists in equilibrium between two states: a compact and an extended conformation.

Cardiovascular diseases are a major cause of death worldwide. Aneurysmal rupture in cerebral arteries or loss of endothelial integrity in the course of atherosclerosis or therapeutic angioplasty lead to exposure of vascular smooth muscle cells (SMC) to blood components such as sphingolipids. Sphingosylphosphorylcholine (SPC) and sphingosine 1-phosphate (S1P) are two naturally occurring sphingolipids, which are vasoprotective in the healthy endothelium-lined vessel, but may promote vascular disease by causing functional changes of SMC. Vascular inflammation is an important factor in various pathologies. SPC can activate pro-inflammatory signalling pathways in rat cerebral artery. Here these observations are extended by showing that SPC elicits monocyte chemoattractant protein-1 production in rat cerebral artery SMC ex vivo. Thus, in addition to being a vasoconstrictor, SPC may promote the development of life-threatening prolonged cerebral vasospasm following subarachnoid haemorrhage by supporting vascular inflammation. It is also demonstrated that SPC prevents tumour necrosis factor-a (TNF)-stimulated adhesion of macrophages to rat aortic SMC in vitro by interfering with adhesive properties of SMC, but not macrophages. While this effect appeared to be mediated by the S1P receptor S1P2, S1P itself did not reduce macrophage adhesion. The anti-adhesive action of SPC also depended on lipid rafts. However, SPC did neither prevent TNF-induced nuclear factor kB activation nor cell adhesion molecule expression in SMC. SPC-induced cyclooxygenase 2 expression in aortic SMC was dispensable for its anti-adhesive effect. In contrast, the inhibitory effect of SPC on TNFinduced expression of inducible nitric oxide synthase is probably involved in its anti-adhesive effect because it was mimicked by respective pharmacological blockade. The results also demonstrate that nitric oxide promotes leukocyte adhesion to vascular SMC, while it has the opposite effect on endothelial cells. These findings may help understand cardiovascular diseases and define novel treatment approaches.

Endolysosomal trafficking determines the ultimate destination of molecules that enter cells and therefore has great impact on multiple cellular processes. Our lab has established that bioactive lipids called sphingolipids can rewire endolysosomal trafficking to result in accumulation of endocytosed material in pre-lysosomal compartments. In this thesis research, the impact of disrupted endolysosomal trafficking caused by sphingolipids on the activity of oligonucleotide therapeutics will be discussed. Therapeutic oligonucleotides include large RNA molecules such as mRNA vaccines and those that target RNA such as single-stranded antisense oligonucleotides (ASOs) or the double-stranded small interfering RNAs (siRNAs). ASOs base pair with a target RNA to elicit RNaseH-dependent degradation, inhibition of translation, or changes to splicing. siRNAs can produce prolonged target RNA silencing after being loaded into Ago2 to form the RNA-induced silencing complex (RISC). Given that all of these oligonucleotide therapeutic platforms require cytoplasmic entry for activity, inefficient endosomal escape remains the primary barrier to the broad application of oligonucleotide therapeutics. Liver uptake after systemic administration is sufficiently robust that a therapeutic effect can be achieved but targeting extrahepatic tissues remains challenging. Prior attempts to improve oligonucleotide activity using small molecules that increase the leakiness of endosomes have failed due to unacceptable toxicity. This thesis will discuss the development and characterization of a well-tolerated and orally bioavailable synthetic sphingolipid analog called SH-BC-893 that increases the activity of antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) up to 200-fold in vitro without permeabilizing endosomes. SH-BC-893 treatment trapped endocytosed oligonucleotides within extra-lysosomal compartments thought to be more permeable due to frequent membrane fission and fusion events. Simultaneous disruption of ARF6-dependent endocytic recycling and PIKfyve-dependent lysosomal fusion was necessary and sufficient for SH-BC-893 to increase non-lysosomal oligonucleotide levels and enhance activity. In mice, oral administration of SH-BC-893 increased ASO potency in the liver by 15-fold. More importantly, SH-BC-893 enabled target RNA knockdown in the CNS and lungs of mice treated subcutaneously with cholesterol-functionalized duplexed oligonucleotides or unmodified ASOs, respectively. Together, these results establish the feasibility of using a small molecule that disrupts multiple endolysosomal trafficking steps to improve the activity of oligonucleotide therapeutics in extrahepatic tissues.

Sphingolipids are ubiquitous membrane lipids in eukaryotic cells. Ceramides, the backbone of sphingolipids, are synthesized by ceramide synthases (CerSs), which form amide bonds between long-chain bases (LCBs) and the acyl groups of acyl-CoAs. Class I CerS works predominantly towards C16-CoA, whereas Class II CerS works towards C24-CoA. Plant sphingolipids generally have 16-carbon and 24-carbon as the major acyl groups. However, the sphingolipids of Oryza sativa and Zea mays have 20-carbon and 24-carbon as the predominant acyl groups. This dissertation aims to understand the substrate preference of CerSs in monocots such as rice. Phylogenetic analysis shows the Class I CerSs from grasses form an evolutionarily distinct clade. The CerS I from the rice was expressed in Arabidopsis CerS I mutants, resulting in a GlcCer profile with C20 as the major acyl group. A 16-residue segment was found to determine the C20 acyl chain specificity of the rice CerS I. Plant LCBs generally have two double bonds (d18:2, Δ4, Δ8). However, Arabidopsis leaves have little d18:2. Overexpression of Δ4 desaturase led to an increasing amount of d18:2 but with a corresponding decrease of d18:1 (Δ8). Co-overexpression of both Δ4 desaturase and Class I CerS resulted in increased d18:2 without a reduction in d18:1 (Δ8). The co-overexpression lines showed delayed bolting and yellowish leaves. Sphingolipidome analysis revealed the formation of excessive amounts of ceramides and GlcCers. The impaired phenotype was recovered by overexpressing the GlcCer synthase to channel the excessive ceramides into GlcCers. The core structure of plant GIPCs is composed of an inositol phosphoceramide (IPC) linked to a GlcA. The additional sugar headgroups of GIPCs have not been fully studied in Zea mays. Here, four different GIPC species were identified in Zea mays using mass spectrometry, and the signal intensity/mole factorials for each class were developed. Complex GIPC synthesis is closely related to phosphate and nitrogen metabolism. The content changes of GIPCs were determined from maize grown under nitrogen and phosphate deplete conditions. Advisor: Edgar B. Cahoon

Sphingolipids are a diverse group of lipids recognized as important components of cellular membranes and regulators of processes during development and in response to environmental stresses. Serine palmitoyltransferase (SPT) catalyzes the first step in sphingolipid biosynthesis and is a primary regulatory point for homeostasis. ORM proteins have been identified as negative regulators of SPT activity, however the mechanistic details of the regulation are only beginning to be understood. In this work, we show that ORM1 and ORM2 are essential for life cycle completion in Arabidopsis thaliana. Furthermore, the study of a structural ORM1 variant provided information about a transmembrane domain involved in the interaction with SPT. We also provide insights into the physiological effects caused by mutations in SPT that induce the production of deoxysphingolipids. Our research demonstrates that plants expressing these mutations showed early senescence and reduced sensitivity to the cell death induced by Fumonisin B1. These findings suggest functional roles of deoxysphingolipids that have not been explored in plants. Finally, we describe a labeling approach to build a sphingolipid kinetic model to study the metabolic flux of sphingolipids during pathogen infection. This study considers a more comprehensive view of the sphingolipid metabolic network that changes dynamically when perturbed. Advisor: Edgar Cahoon

Sphingosine 1-phosphate (S1P) is a signalling lipid that mediates biological processes through five G-protein coupled receptors (S1PR1-5). S1P synthesis occurs through the phosphorylation of sphingosine by sphingosine kinase 1 (SphK1) or SphK2. S1P levels and SphK activity are reduced in multiple neurodegenerative paradigms including Alzheimer’s disease (AD). Pharmacological targeting of S1PRs is neuroprotective in animal models of these diseases. This thesis investigates the cellular and molecular mechanisms behind S1P-mediated neuroprotection. S1P up-regulates four neurotrophic genes (BDNF, PDGFB, HBEGF, and LIF) in primary astrocytes, but not neurons. Induction of these genes is mainly driven by S1PR2 signalling, with minor contributions from S1PR1. Phosphoproteomic analysis showed time-dependent activation of canonical pathways such as the mitogen-activated protein kinase and RhoA pathways, and phosphorylation of cJUN and Yes associated protein (YAP). Immediate early genes (IEGs) were also induced with S1P. Transcription start sites of these neurotrophic factors show predicted binding sites for cJUN and YAP, and the IEGs. While distinct phosphosites were regulated by S1PR1 and S1PR2 signalling, a large subset was regulated by both. However, the RhoA-YAP pathway was exclusively activated by S1PR2. The clinical drug Fingolimod (FTY720) is a sphingosine analogue that is phosphorylated in vivo by SphK2 to form the S1P structural analogue FTY720-P. Previous reports detailing the neuroprotective properties of S1PRs used FTY720-P to investigate the neurotrophic response. Unlike S1P, FTY720-P does not activate the S1PR2-RhoA-YAP pathway in astrocytes. Accordingly, S1P was a much more potent inducer of IEGs and neurotrophic gene expression than FTY720-P. Additionally, S1P but not FTY720-P significantly attenuated excitotoxic neuronal cell death in vitro when co-cultured with glia. Neuroprotection was ablated upon incubation with a LIF neutralising antibody, but not with antagonists to BDNF or HBEGF signalling. I have therefore established a novel neuroprotective pathway in which S1P stimulates secretion of the growth factor LIF by astrocytes, which protects neurons against excitotoxic cell death. This pathway requires dual S1PR1 and S1PR2 signalling in astrocytes, thus explaining the inefficacy of FTY720-P. Current S1PR therapies do not target S1PR2 and targeting this receptor may be a novel therapeutic option for AD and other neurodegenerative conditions.

Mammalian ATP-binding cassette subfamily F member 3 (ABCF3) is a class 2 ABC protein that has previously been identified as a partner of the mouse flavivirus resistance protein 2′,5′-oligoadenylate synthetase 1B (OAS1B). The functions and natural substrates of ABCF3 are currently not known. In Chapter 1 of this study, we show that purified ABCF3 is an active ATPase. Binding analyses with a fluorescent ATP analog TNP-ATP suggested unequal contributions by the two nucleotide-binding domains. We further showed that ABCF3 activity is increased by lipids, including sphingosine, sphingomyelin, platelet-activating factor, and lysophosphatidylcholine. However, cholesterol inhibited ABCF3 activity, whereas alkyl ether lipids either inhibited or resulted in a biphasic response, suggesting small changes in lipid structure differentially affect ABCF3 activity. Point mutations in the two nucleotide-binding domains of ABCF3 affected basal and sphingosine-stimulated ATPase activity differently, further supporting different roles for the two catalytic pockets. We propose a model in which pocket 1 is the site of basal catalysis, whereas pocket 2 engages in ligand-stimulated ATP hydrolysis. Co-localization of the ABCF3–OAS1B complex to the virus-remodeled endoplasmic reticulum membrane has been shown before. We show that co-expression of ABCF3 and OAS1B in bacteria alleviated growth inhibition caused by expression of OAS1B alone, and significantly enhanced OAS1B levels, indirectly showing interaction between these two proteins in bacterial cells. As viral RNA synthesis requires large amounts of ATP, we conclude that lipid-stimulated ATP hydrolysis may contribute to the reduction in viral RNA production characteristic of the flavivirus resistance phenotype. Chapter 2 of this dissertation provides a comprehensive review of the major known antibiotic resistance mechanisms, including the function of ABC proteins, found in producer soil bacteria and discusses different horizontal gene transfer mechanisms that may play a role in the dissemination of resistance genes from producer and non-producer environmental bacteria to pathogenic bacteria in clinical settings. Many bacterial and eukaryotic ABC proteins are polyspecific in nature and are capable of transporting structurally diverse compounds, including drugs and lipids. These proteins are responsible for intrinsic or acquired multidrug resistance, which can also spread to pathogenic organisms through the horizontal transfer mechanisms discussed in this review.

The signalling lipid ceramide has been implicated in the pathogenesis of obesity-related insulin resistance. Ceramide is synthesized by a family of six ceramide synthases in mammals (CerS1-6), each of which preferentially utilises fatty acids of particular acyl chain length (C14-C28). In skeletal muscle (SkM), C18 ceramide, which is synthesized almost exclusively by ceramide synthase 1 (CerS1), has been suggested to mediate systemic insulin resistance in obese humans and rodent models. Therefore, targeted reduction of SkM C18 ceramide may improve detrimental metabolic outcomes induced by a high fat diet (HFD). This thesis describes the characterisation of a new, selective inhibitor of CerS1, P053. P053 inhibits CerS1 with an IC50 of 0.5 micromolar and selectively reduced C18 ceramide in cultured cells. Lipidomic profiling revealed that P053 administration specifically reduced C18 ceramide levels in SkM of mice fed with standard chow or HFD for 4-6 weeks. The reduction of SkM C18 ceramide was coupled to a compensatory increase in C24 ceramides, which are synthesized by CerS2, and loss of triacylglycerols. No other complex lipid species were affected by P053 treatment. At the physiological level, P053 impeded fat deposition induced by a HFD. Despite its effect on adiposity, P053 treatment had no effect on glucose tolerance and insulin sensitivity, assessed by glucose tolerance test and hyperinsulinemic-euglycemic clamps. Unexpectedly, prolonged CerS1 inhibition with P053 improved mitochondrial oxidative capacity in SkM. This effect was associated with significant increases in mitochondrial respiratory chain complex levels, increased activity of TCA cycle and β-oxidation enzymes, and increased mitochondrial respiratory capacity in SkM. Enhanced mitochondrial metabolism may therefore underlie the reduced lipid accretion in mice treated with P053. A significant positive correlation between SkM C18 ceramide and body adiposity was identified. In contrast, SkM C24 ceramides were inversely correlated with adiposity. Thus, P053 may reduce adiposity both directly by reducing SkM C18 ceramide and indirectly through the associated increase in C24 ceramides. In conclusion, we have generated the first selective CerS1 inhibitor. Results from this thesis revealed a potential new role for CerS1 as an endogenous inhibitor of mitochondrial oxidative function in SkM and regulator of whole-body adiposity.

Sphingolipids are major structural components of the plasma membrane and endomembrane system. Research suggests that sphingolipids are involved with the formation of lipid microdomains, also known as lipid rafts, which may help to organize proteins within the membrane and may be important for membrane trafficking. Aside from their structural roles in membranes, sphingolipids and their metabolic products have been implicated in several cellular signaling responses like programmed cell death (PCD). Because of this, maintenance of sphingolipid homeostasis is critical for eukaryotic cell growth and development. Serine palmitoyltransferase (SPT) catalyzes the first step in sphingolipid biosynthesis and is the primary regulatory point for sphingolipid homeostasis. We have characterized two sets of Arabidopsis proteins that physically interact with and impact Arabidopsis SPT activity. The first set, the ssSPTs (Arabidopsis thaliana), have been shown to be essential and redundant. Modulation of AtssSPT expression was shown to alter SPT activity, LCB accumulation and sensitivity to the mycotoxin, Fumonisin B1 (FB1), in a way that is consistent with the AtssSPTs being activators of SPT. Alternatively, modulating expression of the other set of proteins, the AtORMs, was shown to alter SPT activity, LCB accumulation and sensitivity to FB1, consistent with them acting as SPT inhibitors. Both the AtssSPTs and the AtORMs appear to be limiting as transgenic up/down regulation of these genes leads to predictable changes to SPT activity. Interestingly, we also see changes in ceramide synthase activity with modulation of AtORM expression, suggesting a more complex regulatory role for these proteins and pointing towards coordinate regulation of SPT with downstream enzymes. Our research also demonstrates that SPT substrate specificity can be altered through point mutations in AtssSPT and AtLCB1, leading to the production of aberrant long-chain bases (LCBs). Alteration of SPT substrate specificity may function as another regulatory control point by altering SPT products through changes in the composition of SPT subunits. We also discuss a system utilizing a point mutation in AtLCB1 that can be used as a tool to better measure SPT activity in planta. Collectively our data point towards a complex and nuanced regulatory scheme for maintaining sphingolipid homeostasis in Arabidopsis thaliana. Advisor: Edgar Cahoon

Alzheimer’s disease (AD) is a debilitating neurodegenerative disease and a significant burden on families and society. A major hallmark of AD is lipid occlusions within neural cell types, indicating dysfunctional lipid metabolism. Sphingolipids (SL), a subgroup of lipids, play a vast array of functions in the CNS, including as essential components of myelin, a hydrophobic barrier insulating axons and facilitating action potential propagation. SL dysregulation has been studied in post mortem AD affected brain tissues compared to controls, however, SL changes in pre-clinical stages of AD has not been investigated. Applying liquid chromatography tandem mass spectrometry, this thesis describes the sphingolipid profile of six AD affected brain regions infiltrated to varying degrees by neurofibrillary tangle (NFT) pathology, indicated by Braak staging I-VI. A pronounced loss in myelin lipids galactosylceramide (GalCer) and sulfatide from frontal and temporal grey matter (GM) and hippocampus was observed, leading to investigation of de novo synthesis of the precursor ceramide. Activity of the enzyme ceramide synthase 2 (CERS2), essential for very long chain ceramide synthesis and myelin lipid production, was significantly reduced in multiple brain regions at a stage of disease preceding characteristic NFT pathology. Lipid analysis also showed a marked decline of the neuroprotective signalling lipid sphingosine 1-phosphate (S1P) in hippocampus and temporal GM that tracked closely with NFT pathology. S1P loss was attributed to loss of activity in sphingosine kinases 1 and 2, required for S1P synthesis. The major genetic risk factor of AD is expression of the E4 isoform of the apolipoprotein E (ApoE) lipid transporter, which aids in brain lipid delivery and clearance. Despite no correlation between APOE genotype and ceramide levels, a positive correlation between age and C16 ceramide, recently associated with systemic insulin resistance, was observed. Overall we hypothesise that CERS2 activity loss destabilises myelin, triggering neurological decline and sensitising neurons to AD induced neurotoxicity. The effect of insulin resistance in the AD affected brain including cognitive function is garnering interest in AD research, warranting further investigation into the study of C16 ceramide levels with age. Pharmacological restoration of deficient S1P signalling could also open therapeutic perspectives for AD.

Sphingolipids are essential components of eukaryote membranes. The ceramide backbone of complex sphingolipids is composed of an 18 carbon Long Chain Base (LCB) bound to a 16-26 carbon fatty acid (FA) through an amide linkage. Ceramides are synthesized de novo from a free LCB and fatty acyl coA by ceramide synthase (sphingosine N-acyl transferase, EC 2.3.1.24) which can be inhibited by the fungal mycotoxin Fumonisin B1. Arabidopsis thaliana contains three ceramide synthases denoted LOH1, LOH2, and LOH3 that have previously been hypothesized to have unique substrate preferences that control the final sphingolipid composition, different susceptibilities to Fumonisin, and different influences plant growth/development. This dissertation works to answers to these questions as well as identify novel complex sphingolipid biosynthetic pathways. Through the use of in vitro assays it was found that LOH1 and LOH3 prefer LCBs witth hydroxyls at the C1, C2, and C4 positions (trihydroxy) and C20-24 saturated FA while LOH2 prefers LCBs with hydroxyls at the C1 and C2 positions (dihydroxy) and C16 saturated fatty acids. None of the isoforms were able to use ω9 desaturated acyl CoAs which are abundant in the final sphingolipid profile. Surprisingly LOH2 showed the highest level of activity with C4 unsaturated LCBs which are not commonly found in leaf. Each isoform was also overexpressed in planta to determine the effects ceramide composition has on plant growth. Overexpression of LOH1 or LOH3 led to an increase in biomass while overexpression of LOH2 resulted in a dwarf phenotype. Both the in vitro assays and in planta overexpression found LOH1 to the most susceptible to FB1 inhibition. In addition to ceramide synthesis a novel Δ8 LCB desaturase from castor bean was identified which required the presence of a Δ4 double bond for activity. The presence of Δ4,8 unsaturated LCBs was found to result in increased glucoscylceramide levels as revealed by LCB feeding experiments and pollen sphingolipid profiling. Therefore, it is hypothesized that the presence of a Δ4 unsaturation targets LCBs through a LOH2-like ceramide synthase for subsequent Δ8 desaturation and glucosylceramide synthesis. Advisor: Edgar B. Cahoon

The correct functioning of the cell, and hence a multicellular organism, is dependent on proteins and lipids reaching their proper destination at the correct time. Intracellular trafficking is the predominant regulated process by which these materials are transported between various organelles. In route, material is transferred via vesicle and membrane tubule carriers. Previous work from our lab and others have demonstrated that the formation of these carriers are dependent on localized changes in membrane curvature, which is generated by phospholipid modifying enzymes such as lysophospholipid acyltransferases and phospholipid lipases. While our understanding about these carrier intermediates has greatly advanced over the years, many questions remain unanswered. One such question is how cargoes are marked to traffic via a vesicle or a membrane tubule. Aiming to uncover kinase regulators of Golgi membrane tubule formation, our lab conducted a kinome-directed RNAi screen in mammalian cells. Through this screen ceramide kinase, or CERK, was identified as having a positive role in Golgi membrane tubulation. CERK phosphorylates the sphingolipid ceramide to produce ceramide-1-phosphate (C1P). The studies to date indicate CERK and/or C1P may have roles in eicosanoid production and phagolysosome function. However it is unclear whether this is a direct effect of CERK/C1P or is secondary to more fundamental changes in intracellular functions and pathways as indicated by our RNAi screen. The role of CERK in intracellular endolysosomal trafficking and membrane   tubulation at the Golgi complex and early endosomes were investigated. By utilizing overexpression and knockdown experiments in conjunction with microscopy-based trafficking assays in mammalian cells, I discovered that CERK activity positively affects membrane tubulation at the Golgi complex and early endosomes. Furthermore, CERK influences endolysosomal morphology without affecting compartment identity, through both catalytic and non-catalytic means. Finally, CERK negatively influences the endocytic trafficking of cholera toxin. Evidence from my studies adds to our understanding of CERK function and will aid in directing future research into the specific biological roles of CERK. Ultimately, further studies are warranted to refine our knowledge regarding CERK function in intracellular trafficking and membrane tubulation.

Eukaryotic plasma membranes are highly complex structures, both with respect to molecular composition and spatial organization. This composition and organization is essential for cellular function and must be maintained during dramatic shifts in extracellular environment that challenge membrane homeostasis. In the model eukaryote Saccharomyces cerevisiae, signaling by TOR Complex 2 (TORC2) and its effector kinases Ypk1 and Ypk2 is modulated in response to various membrane stresses and cells deficient in TORC2-Ypk signaling have membrane defects, suggesting that this signaling pathway is important for maintaining membrane homeostasis. To understand the molecular mechanisms by which TORC2-Ypk1 signaling regulates membrane homeostasis, I devised a three-tiered genome-wide screen to identify substrates of Ypk kinases. This screen identified 12 novel Ypk substrates in a variety of processes linking TORC2-Ypk signaling to membrane homeostasis including: lipid metabolism, glycerol metabolism, peroxisome function and autophagy. I then performed detailed studies of the regulation of identified substrates Lac1 and Lag1, components of the ceramide synthase complex, a lipid metabolism enzyme. In response to various membrane stresses, TORC2-Ypk1 activates ceramide synthase. This activation is essential in allowing cells to survive these stresses. Interestingly, it appears that activation of ceramide synthase promotes stress survival not by increasing ceramide levels, but rather by preventing the accumulation of ceramide precursors. Lastly, I characterized the TORC2-Ypk regulation of identified substrates Fps1, Gpt2 and Smp1, proteins involved in glycerol metabolism and the cellular response to hyperosmotic shock. I show that TORC2-Ypk is a novel hyperosmotic shock responsive pathway and that TORC2-Ypk coordinately regulates glycerol metabolism substrates to promote accumulation of glycerol, balance osmolarity and promote cellular survival. Thus, this work has identified at least some of the molecular mechanisms by which TORC2-Ypk signaling restores membrane homeostasis in the face of environmental stress.

Duchenne Muscular Dystrophy (DMD) is characterized by progressive muscle degeneration and a chronic inflammatory response. Sphingolipid metabolites are associated with the generation or perpetuation of low-grade chronic inflammation critical in atherosclerosis, obesity and cancer. Dietary sphingolipids, however, can suppress intestinal inflammation. We hypothesized that dietary sphingomyelin (SM) from bovine milk can modulate the inflammatory signature and improve muscle function in mdx mice, a model of DMD. C57BL10 (WT) and mdx mice were fed AIN 76A diet ± 0.1% SM for 7 weeks starting at age 4 weeks (n=10/group: WT, WT + S, mdx, mdx + S). At ages 5, 7, and 9 weeks, ankle flexor torque was determined in vivo. Mice were euthanized at 11 wks. Serum creatine kinase and extensor digitorum longus (EDL) contractile properties in vitro were determined; Tibialis Anterior (TA) inflammatory markers were profiled by qRT-PCR; TA sections were stained with H&E and immunohistochemistry for p-Akt was performed. At age 9 weeks, in vivo ankle flexor torque at stimulation frequencies 50-150 Hz was greater in mdx+S vs. mdx (P=0.0160) and WT (P

Glioblastoma (GBM), a genetically heterogeneous disease, has a significant burden on our society. Currently, the standard treatment for newly diagnosed GBM patients consists of surgery followed by concomitant radiotherapy and temozolomide chemotherapy, resulting in a median survival of 12-15 months. Targeted therapies are being developed to inhibit oncogenes based upon GBM molecular profiling, though have not been as successful as expected. Our understanding of DNA and RNA alterations in GBM has grown considerably over the past few years. However, our understanding of the lipid biology, specifically sphingolipids, in GBM is lagging and may prove useful in the arsenal of targeted therapies. The sphingolipid pathway contains lipid signalling molecules, which modulate cellular survival through the balance of ceramide, a pro-apoptotic metabolite, and Sphingosine-1-Phosphate (S1P), a pro-survival metabolite. Herein, I characterise for the first time the sphingolipid profile of normal grey matter (NGM), diffuse astrocytomas (AII), anaplastic astrocytomas (AIII), and GBM using liquid chromatography tandem mass spectrometry. The lipid profile is supported by an enzyme expression profile favouring ceramide catabolism and S1P formation, including upregulation of acid ceramidase (ASAH1) and sphingosine kinase 1 (SPHK1), and a down regulation of S1P phosphatase 2 (SGPP2). Significantly, C18 ceramide was reduced 5-fold in GBM compared to NGM, while S1P was increased in GBM by approximately 9-fold compared to NGM. Based on the sphingolipid profiles, ASAH1 and SPHK1 were assessed for functional relevance in vitro. Using gene silencing and pharmacological inhibition, I found SPHK1 to be critical for U87MG-induced angiogenesis through S1P paracrine signalling, which was independent of VEGF levels. EGFR mutations were associated with increased C16 and C22 ceramide levels. For the first time, I measured sphingolipid metabolites in plasma extracted from GBM patients and healthy controls. Elevated levels of S1P were found in GBM plasma and together with tumour S1P levels, were associated with a poor survival outcome. In contrast low S1P levels in tissue combined with MGMT methylation was associated with a good survival outcome. Overall, the data presented in this thesis reaffirm the importance of sphingolipid metabolism in GBM biology, reflected by a shift in the ceramide-S1P balance, favouring the pro-angiogenic S1P. Additionally, sphingolipid interactions with altered genetic pathways and potential biomarker capacity are novel findings that require further validation, with the hope of informing and monitoring therapeutic responses for GBM patients.

Lipids of the stratum corneum (SC), the outer layer of the epidermis of birds and mammals, provide a barrier to water vapor diffusion through the skin. The SC of birds consists of flat dead cells, called corneocytes, and two lipid compartments: an intercellular matrix and a monolayer of covalently bound lipids (CBL) attached to the outer surface of corneocytes. We previously found two classes of sphingolipids, ceramides and cerebrosides, covalently bound to corneocytes in the SC of house sparrows and that these were associated with cutaneous water loss (CWL). In this study, we collected adult and nestling house sparrows from Ohio and nestlings from Saudi Arabia, acclimated them to either a high or low humidity environment, and measured their rates of CWL. We also collected natural populations of nestlings from Ohio and Saudi Arabia from 2 days after hatching until they fledged, and measured their CWL rates. We then evaluated the composition of the CBL of the SC using thin layer chromatography. We found that CBL development differed between habitats, but that lipid density generally increased with age. CBL profile did not exhibit phenotypic plasticity with acclimation and was mostly the same between habitats. CWL appears to be functionally related with the interactions of CBL classes as a whole, and this may be associated with age. Finally, we found that house sparrows have a very diverse range of CBLs in their SC, including free fatty acids and cholesteryl esters, and we propose a new model for CBL organization.

Sphingolipids have been implicated as potential atherogenic lipids. Inhibition of hepatic serine palmitoyl transferase (SPT) reduces plasma sphingomyelin (SM) levels leading to a reduction of atherosclerosis in apolipoprotein-E gene knockout (apoE-/-) mice. In my thesis I have investigated the possibility that the reduced atherosclerosis resulting from SPT inhibition is associated with decreases in plasma glycosphingolipids (GSL). Furthermore, I examined whether SPT inhibition can lead to regression of atherosclerotic lesions. This thesis shows the SPT inhibitor myriocin inhibits atherosclerosis in apoE-/- mice fed a high fat diet. Lesion inhibition was most pronounced at the aortic arch and distal sites of the thoracic and abdominal aorta. There was also a trend towards a reduction in lesion area at the aortic root. Myriocin treatment resulted in significant reductions in both plasma SM and GSL concentrations. Moreover, it was demonstrated that myriocin significantly inhibited the progression of established atherosclerosis. Although the inhibition of lesion progression was observed mainly in the distal regions of the aorta, regression of lesion size was not detected. In addition, this thesis demonstrated for the first time that selective inhibition of GSL synthesis by the GSL synthesis inhibitor, d-threo-1- ethylendioxyphenyl-2-palmitoylamino-3-pyrrolidino-propanol (EtDO-P4) had no significant impact on lesion area in apoE-deficient mice. Thus, despite the previously observed positive correlations between plasma and aortic GSL concentrations and the development of atherosclerosis, this thesis indicates that inhibition of GSL synthesis does not inhibit atherosclerosis in vivo. In other studies we assessed the possibility that myriocin may also be acting to increase hepatic apoA-I production via the inhibition of ERK phosphorylation. To address this, HepG2 cells and primary mouse hepatocytes were treated with myriocin. This significantly increased apoA-I mRNA, and protein levels. It also increased apoA-I secrection, and decreased ERK phosphorylation. These in vitro data indicate that ERK phosphorylation plays a role in modulating apoA-I expression, and that myriocin’s mechanism of action is linked to this pathway. Overall, this thesis has expanded the current literature regarding the role of sphingolipid synthesis inhibition and atherosclerosis.

Cellular accumulation of Sphingosine 1-phosphate (S1P) stimulates cellular motility, invasion, proliferation, adhesion, and angiogenesis. S1P tranduces these cellular effects by signaling through one of a family of high affinity G-protein coupled receptors that include EDG-1/S1P1, EDG-5/S1P2, EDG-3/S1P3, EDG-6/S1P4, and EDG-8/S1P5. Upon receptor stimulation, unique and preferential G-protein signaling pathways are activated to varying degrees to elicit these cellular responses. Enzymatic production of S1P is a result of the activity of sphingosine kinase (SK). Expression of this protein has been shown to correlate to poor glioblastoma multiforme (GBM) patient prognosis. This deadly neoplasm is diagnosed through histologic criteria that include significant neovascularization, high mitotic index, and diffuse infiltrative appearance. Although progress has been made in the treatment of many cancers in recent years, the dismal patient prognosis for GBM patients has not. Mo lecular based therapies targeting the infiltrative nature of this tumor could ultimately improve patient outcome. This work focuses on GBM malignant behavior as influenced by S1P receptor subtype signaling. All the individual receptor subtypes were found to have unique influences over in vitro GBM proliferative, migratory, adhesive, and invasive responses. The S1P2 receptor subtype displayed novel S1P-induced pro-invasive, but anti-migratory effects, which were mediated through increased adhesion to the extracellular matrix. Invasiveness was promoted through CCN1, uPA, and uPAR, which have all been correlated to GBM tumorigenesis. S1P induced expression of CCN1 through S1P1-2 promoted invasion that was limited with antibody in a spheroid invasion assay. Enhanced activation of SK with receptor subtype S1P1 overexpression resulted in endogenous upregulation of uPA and uPAR expression as well as uPA activity. Additionally, antibody targeting uPA also limited total invasiv e distance of spheroids. The invasive mediators uPA and uPAR were shut down with the inhibition of SK and spheroid invasiveness was eliminated regardless of receptor subtype overexpression. The results of this work better delineate the complex S1P receptor subtype signaling pathways in GBM cells as well as their contributions to malignant behavior. The results on GBM invasiveness have illustrated the key role that SK plays and validated uPA and CCN1 as viable targets for molecular based approaches to therapy.

Human cytomegalovirus (HCMV) is a beta-herpes virus which can cause serious disease and even death in congenitally-infected infants and in immunocompromised individuals or immunosuppressed transplant recipients. Sphingolipids are structural components of cell membranes that can act as critical mediators of cell signaling. An unexplored area of research is whether HCMV modulates sphingolipids and their signaling pathways. Our data show that HCMV infection results in increased accumulation and activity of sphingosine kinase (SphK) within different cell types and occurs during early times of infection. Measuring the levels of transcripts encoding key enzymes of the sphingolipid metabolic pathway during HCMV infection revealed a temporal regulation of both synthetic and degradative enzymes of this pathway. Using mass spectrometry, we were able to generate a sphingolipidomic profile of HCMV-infected cells, which suggests an enhancement of de novo sphingolipid synthesis at early times of infection. This was followed by a decrease in the levels of several sphingolipids at 48 hrs, correlating with the upregulation of degradative enzymes. Then by knocking down SphK1 expression with siRNA, we showed that this enzyme may function within HCMV infected cells to sustain levels of the immediate early (IE) transactivator, IE1. Later, we show evidence suggesting that de novo sphingolipid biosynthesis is necessary for the production of optimal levels of infectious virus progeny, but has an intermediate product which suppresses the levels of IE1 protein accumulation. Through exogenous addition of dhSph, an intermediate of de novo sphingolipid synthesis, we show that this lipid can inhibit HCMV protein accumulation. dhS1P treatment, however, results in increased accumulation of HCMV proteins, although only at early times during infection. Moreover, pretreatment of cells with dhS1P or S1P prior to infection results in reduced accumulation of HCMV gene products, thus indicating a time-dependent need for increased dhS1P levels by HCMV. Finally, our results, using a drug inhibitor to suppress SphK activity during infection with HCMV, suggest that the stimulation of SphK activity may act to promote virus replication. These studies indicate that HCMV modulates sphingolipid metabolism to temporally regulate the levels of dhSph and dhS1P in order to optimize viral protein accumulation and growth.

Prostate cancer is the second leading cause of cancer death in men, and diet is thought to play a role in the development of this disease. The “Westernized” diet, rich in red meat and dairy products, is associated with increased prostate cancer risk. Despite the negative perception of dairy products, milk contains several components, such as whey proteins and sphingomyelin (SM), which may play a role in prevention and/or treatment of prostate cancer. Whey proteins are a cysteine-rich protein source, and consumption of these proteins can elevate plasma concentrations of the antioxidant glutathione (GSH). Elevation of prostate GSH may reduce inflammation that is associated with cancer development. SM is a phospholipid that can induce apoptosis in cancer cells, and consumption of SM is associated with reduced incidence of colon cancer in mice. It is hypothesized that SM may also limit the proliferation of prostate cancer cells. The present studies were designed to examine the role of whey proteins and SM in prostate cells to determine if there is a potential role for these dairy components in prostate cancer prevention and treatment. First, the effects of oxidative stress in non-cancerous prostate cells was examined. Next, we examined the elevation of GSH in non-cancerous prostate cells by hydrolyzed whey proteins. Finally, the antiproliferative effects of SM in human prostate cancer cells were studied. The results of the present study revealed that bovine pituitary extract, a supplement used in the growth medium of the non-cancerous prostate cells, provides significant protection against oxidant-induced cell damage and may represent a confounding variable when studying oxidative stress in cell lines requiring this supplement. Hydrolyzed whey proteins elevated GSH concentrations in non-cancerous prostate cells by 64% and this GSH elevation protected the cells against oxidant-induced cell death. Exogenous SM significantly reduced prostate cancer cell proliferation by 17% after 24 h incubation, and this reduction was, in part, due to apoptosis. Taken together, results from these studies suggest a potential role for whey proteins and SM in reducing the risk for development of prostate cancer.

Cardiovascular disease, including stroke and atherosclerosis, remains a major cause of morbidity and mortality in the United States due to consumption of a high-fat “Western” diet. A major indication of these disease states is excess lipid deposition, particularly cholesterol, in the arterial walls. It has been well documented that high levels of circulating high-density lipoprotein (HDL) and its main protein component apolipoprotein AI (apoA-I) are associated with lowered risk of cardiovascular disease. The protective effects of HDL are thought to be mediated by a process called reverse cholesterol transport - in which HDL takes up excess cholesterol from peripheral tissues and transports it to the liver for excretion in the bile. It is now widely accepted that the interaction of apoA-I with the cell membrane protein ATP-binding cassette transporter AI (ABCAI) is critical for the formation of nascent HDL particles. Since sphingomyelin maintains a preferential interaction with cholesterol in membranes, the breakdown of sphingomyelin may regulate the availability of cellular cholesterol utilized by ABCAI. Furthermore, the catabolite of sphingomyelin, ceramide, is a potent signaling molecule and may play an important role in ABCAI regulation or function. The following study examines the potential contribution of sphingolipids in ABCAI-mediated cholesterol removal from the cell. It was discovered that treatment with C2-ceramide enhances cholesterol release to apoA-I. This effect appeared to becaused by an increase in cellular ABCAI content with enrichment at the cell surface. These findings may lead to new ways to increase cellular ABCAI and further promote cholesterol removal from regions of excess cholesterol such as the atherosclerotic lesion.

This study was designed to examine if a sphingolipid-enriched lipid fraction from wheat gluten could affect the incidence of type I diabetes in BioBreeding diabetes prone (BBdp) rats. Wheat gluten was extracted with a chloroform-methanol (CM) mixture to isolate most of the lipids. Isolated lipids were subjected to silica gel column chromatography and saponification to remove most of neutral lipids and phospholipids, leaving behind a lipid fraction enriched in sphingolipids. This sphingolipid-enriched lipid fraction was used in a BBdp rat feeding study. BBdp rats were fed with one of five diets from weaning at 23 days of age until 125 days of age: a hydrolyzed casein based diet (HC), a NTP-2000 standard rodent diet (NTP-2000), a wheat gluten based diet (WG), a sphingolipid-free wheat gluten based diet (WGSLF), and a hydrolyzed casein plus sphingolipid-enriched lipid fraction diet (HC+SL). The yield of sphingolipid-enriched lipid fraction was about 0.62% of wheat gluten. The content of glycosylceramide in sphingolipid-enriched lipid fraction was increased more than five fold compared to that in total isolated lipids. Rats fed the NTP-2000 diet had the highest incidence of diabetes; while rats on the HC diet had the lowest diabetes incidence. There was no significant difference with regard to the onset age of diabetes among rats in the five diet groups. The addition of sphingolipid-enriched fraction to the HC diet caused a significant increase in the incidence of diabetes in BBdp rats in the first 80 days of the study. However, the ultimate diabetes incidence at day 125 was not changed. The removal of lipids from wheat gluten did not change the diabetes incidence in BBdp rats at any stages of the feeding study. These findings suggest that the sphingolipid-enriched fraction from wheat gluten acted as a possible promoter but not as a trigger of the development of type I diabetes in BBdp rats. There must be something that remains in wheat gluten after chloroform-methanol extraction that serves as a trigger for type I diabetes in these rodents. Type I diabetes in this animal model for the human disease seems to be caused by multiple factors, most likely, by the interaction of sphingolipids and some other unknown substances in wheat gluten.