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search.filters.applied.f.advisor: Merrill, Alfred H. × End date: 2019 × Start date: 2000 × Has files: Yes × search.filters.applied.f.isOrgUnitOfPublicationTitle: College of Sciences × search.filters.applied.f.resourcesubtype: Dissertation ×

Publication Search Results

Now showing 1 - 7 of 7
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Characterization of the role of acid ceramidase in adrenocortical steroid hormone biosynthesis

2011-11-14 , Lucki, Natasha Chrystman

Sphingolipids modulate multiple cellular functions, including steroid hormone biosynthesis. Sphingosine is an antagonist ligand for the nuclear receptor steroidogenic factor 1 (SF-1), which is the primary transcriptional regulator of most steroidogenic genes. Furthermore, sphingosine-dependent repression of SF-1 function is dependent on the expression of acid ceramidase (ASAH1), an enzyme that forms sphingosine. Based on these data, I hypothesized that ACTH/cAMP signaling regulates ASAH1 function at both transcriptional and post-transcriptional levels. In addition, because SF-1 is predominantly a nuclear protein, I postulated that ASAH1 modulates SF-1 function and, therefore, steroidogenic gene expression by controlling the nuclear concentrations of SPH. To test these hypotheses, I first examined the effect of chronic ACTH/cAMP signaling on the transcription of the ASAH1 gene. Next, the functional significance of ASAH1 expression in adrenocortical cells was probed by generating an ASAH1-knockdown cell line. I subsequently characterized the role of ASAH1 as a transcriptional nuclear receptor coregulator. Finally, I defined the role of sphingosine-1-phosphate, a bi-product of ASAH1 activity, in the acute phase of cortisol biosynthesis. Using a variety of experimental approaches, I identified cAMP response element binding protein as an essential transcriptional activator of the ASAH1 gene. Analysis of adrenocortical cells lacking ASAH1 revealed that ASAH1 is a global regulator of steroidogenic capacity. Furthermore, I identified ASAH1 as a nuclear protein and defined the molecular determinants of the interaction between ASAH1 and SF-1. Collectively, this body of work establishes the integral role of ASAH1 in the regulation of ACTH-dependent adrenocortical cortisol biosynthesis.

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Development of an assay for fatty acyl-CoAs using liquid chromatography-electrospray ionization-tandem mass spectrometry and its application to the stable isotope labeling and quantitation of sphingolipid metabolism

2009-11-16 , Haynes, Christopher Allen

Fatty acyl-Coenzyme As are metabolites of lipid anabolism and catabolism. A method was developed for their quantitation in extracts of cultured mammalian cells using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Palmitoyl-CoA (C16:0-CoA) is utilized for de novo sphingolipid biosynthesis catalyzed by serine palmitoyltransferase (SPT), which condenses palmitoyl-CoA and serine to form 3-ketosphinganine. After reduction to form sphinganine (Sa), dihydroceramide synthase (CerS) can N-acylate the Sa using a second fatty acyl-CoA molecule, forming dihydroceramide (DHCer). The CerS enzyme family utilizes different acyl chain lengths of fatty acyl-CoAs in an isoform-specific manner, resulting in DHCer with N-acyl chains ranging from C16 to C26 [and even longer] in mammalian tissues. DHCer is trans-4,5-desaturated to yield ceramide, which is further metabolized by the addition of moieties at the 1-O-position, forming sphingomyelin (SM) and ceramide monohexose (CMH). The rates of fatty acyl-CoA and sphingolipid biosynthesis were determined using stable isotope-labeling and LC-ESI-MS/MS analysis of the analyte isotopologues and isotopomers. Isotopic labeling of palmitoyl-CoA with [U-13C]-palmitate in HEK293 and RAW264.7 cells was robust and rapid (~ 60% labeling of the metabolite pool in 3 hr). Isotopic labeling of sphingolipids indicated utilization of [M + 16]-palmitoyl-CoA by SPT and CerS isoforms in both cell types. Metabolic flux modeling was applied to the data for [U-13C]-palmitate activation to [M + 16]-palmitoyl-CoA and its subsequent utilization in de novo sphingolipid biosynthesis, and this analysis indicated rapid turn-over rates for palmitoyl-CoA and ceramide in both cell types. Palmitate treatment of cultured cells alters their metabolic status and gene expression, therefore labeling of palmitoyl-CoA by treatment with [1-13C]-acetate was employed. A distribution of mass-shifted palmitoyl-CoA species (isotopologues) is observed based on the number of incorporations of [1-13C]-acetate during de novo biosynthesis, requiring computational analysis to derive two parameters: the isotopic enrichment of the precursor pool, and the fraction of palmitoyl-CoA that was biosynthesized during the experiment. Previous reports by others describe mass isotopomer distribution analysis (MIDA) and isotopomer spectral analysis (ISA) for this purpose, and both calculation approaches indicated concurrent results. In summary, the quantitation of fatty acyl-CoAs and their isotopic enrichment during stable isotope-labeling studies of lipid metabolism can provide data that significantly change the interpretation of analyte quantitation in these experiments, as demonstrated here for investigations of de novo sphingolipid biosynthesis.

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The regulation of cellular trafficking of the human lysophosphatidic acid receptor 1: identification of the molecular determinants required for receptor trafficking

2007-05-16 , Urs, Nikhil Mahabir

The following thesis research was undertaken to gain a better understanding of the mechanisms that regulate the cellular trafficking and signaling of the endothelial differentiation gene (EDG) family of G-protein coupled receptors, LPA1, LPA2, and LPA3. This thesis will specifically focus on the regulation of the trafficking of the LPA1 Lysophosphatidic acid receptor, which is the most widely expressed and has been shown to be a major regulator of migration of cells expressing it. The initial studies undertaken in this project were aimed at understanding the endocytic pathway followed by the LPA1 receptor. Lysophosphatidic acid (LPA), an abundant serum phospholipid, stimulates heterotrimeric G protein signaling by activating three closely related receptors, termed LPA1, LPA2 and LPA3. In the first part of the project we show that in addition to promoting LPA1 signaling, membrane cholesterol is essential for the association of LPA1 with β-arrestin, which leads to signal attenuation and clathrin dependent endocytosis of LPA1. The second phase of the project was aimed at elucidating the different structural motifs required for the trafficking and signaling of the LPA1 receptor and helping us gain a more mechanistic view of the processes involved in its regulation. In the second part of the project we show that agonist-independent internalization of the LPA1 receptor is clathrin adaptor, AP-2 dependent and PKC-dependent and that it requires a distal dileucine motif, whereas agonist-dependent internalization of the LPA1 receptor is β-arrestin and clathrin-dependent and requires a cluster of serine residues in the tail region, which is upstream of the dileucine motif. These studies collectively vastly enhance our understanding of mechanisms that regulate LPA1 trafficking and signaling. These studies can also be applied to other G-protein coupled receptors making the task easier for other scientists to understand this vast family of receptors.

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Application of bioinformatics in studies of sphingolipid biosynthesis

2010-05-17 , Momin, Amin Altaf

The studies in this dissertation demonstrate that the gene expression pathway maps are useful tools to notice alteration in different branches of sphingolipid biosynthesis pathway based on microarray and other transcriptomic analysis. To facilitate the integrative analysis of gene expression and sphingolipid amounts, updated pathway maps were prepared using an open access visualization tool, Pathvisio v1.1. The datasets were formatted using Perl scripts and visualized with the aid of color coded pathway diagrams. Comparative analysis of transcriptomics and sphingolipid alterations from experimental studies and published literature revealed 72.8 % correlation between mRNA and sphingolipid differences (p-value < 0.0001 by the Fisher's exact test).The high correlation between gene expression differences and sphingolipid alterations highlights the application of this tool to evaluate molecular changes associate with sphingolipid alterations as well as predict differences in specific metabolites that can be experimentally verified using sensitive approaches such as mass spectrometry. In addition, bioinformatics sequence analysis was used to identify transcripts for sphingolipid biosynthesis enzyme 3-ketosphinganine reductase, and homology modeling studies helped in the evaluation of a cell line defective in sphingolipid metabolism due to mutation in the enzyme serine palmitoyltransferase, the first enzyme of de novo biosynthesis pathway. Hence, the combination of different bioinformatics approaches, including protein and DNA sequence analysis, structure modeling and pathway diagrams can provide valuable inputs for biochemical and molecular studies of sphingolipid metabolism.

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Characterization of ceramide synthases (Cers) in mammalian cells

2009-05-13 , Park, Hyejung

This thesis describes the characterization of ceramide (Cer) biosynthesis by mammalian cells. The possibility that Cer undergo developmental changes was explored using mouse embryonic stem cells versus embryoid bodies by analysis of the Cer subspecies by liquid chromatography, electrospray ionization-tandem mass spectrometry (LC ESI-MS/MS) and of the transcript levels for enzymes involved in Cer biosynthesis by qRT-PCR. Cer of embroid bodies had higher proportions of very-long-chain fatty acids, which correlated with the relative expression of mRNA for the respective Cer synthases (CerS) and fatty acyl-CoA elongases, as well as changes in the fatty acyl-CoA's of the cells. Therefore, it is clear that Cer subspecies change during embryogenesis, possibly for functionally important reasons. One CerS isoform, CerS2, was studied further because it has the broadest tissue distribution and a remarkable fatty acyl-CoA specificity, utilizing longer acyl-chain CoAs (C20-C26) in vitro. The fatty acid chain selectivity was refined by analysis of the Cer from livers from CerS2 null mice, which displayed very little Cer with fatty acyl chains with 24 + 2 carbons. Another interesting structural variation was discovered in studies of cells treated with fumonisin B1 (FB1), which inhibits CerS. Under these conditions, cells in culture and animals accumulate substantial amounts of a novel sphingoid base that was identified as 1-deoxysphinganine. This compound arises from utilization of L-alanine instead of L-serine by serine palmitoyltransferase (SPT) based on the inability of LYB cells, which lack SPT, to make 1-deoxysphinganine. In the absence of FB1, 1-deoxysphinganine is primarily acylated to 1-deoxydihydroceramides. These are an underappreciated category of bioactive sphingoid bases and "ceramides" that might play important roles in cell regulation and disease. In summary, cells contain a wide variety of Cer subspecies that are determined by changes in expression of CerS, enzymes that produce co-substrates (such as fatty acyl-CoAs), and the types of amino acids utilized by SPT, the initial enzyme of de novo sphingolipid biosynthesis. One can envision how these changes might impact membranes structure as well as signaling by this family of highly bioactive compounds.

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Regulation of ceramide and its metabolites: biosynthesis and; in situ sphingolipid analysis

2010-01-19 , Liu, Ying

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

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Study of serine palmitoyltransferase and de novo synthesis of sphingolipids

2009-04-06 , Wei, Jia

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.

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