Browsing by Subject "Lipid Metabolism"
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Item Alternative Processing of SREBP in Site 2 protease and Scap mutants during larval development in Drosophila(2009-01-14) Matthews, Krista Ann; Rawson, Robert B.Lipid metabolism is regulated by the membrane-bound transcription factor, sterol regulatory element binding protein (SREBP). SREBP requires release of the amino terminus from the membrane to activate transcription of genes involved in cholesterol and fatty acid synthesis. In response to low sterol levels, Scap escorts SREBP from the ER to the Golgi where it is cleaved by Site-1 and Site-2 proteases. The SREBP pathway is conserved in Drosophila despite these organisms being cholesterol auxotrophs. dSREBP is essential for activating genes involved in the uptake and synthesis of fatty acids which are required for rapid growth during larval development. I have demonstrated that processing of SREBP in Drosophila does not require the S2P or Scap, in contrast to the mammalian system. Flies lacking dS2P are viable and still process dSREBP. dS2P homozygotes were subviable, only emerging at 40% of the expected ratio. This phenotype can be rescued completely by supplementation with fatty acids. dSREBP activity was detected in the fat body of dS2P mutant larvae and to a lesser extent in the oenoctyes. Additionally, SREBP target genes were expressed at higher levels in dS2P homozygotes compared to dSREBP mutants, though less than wild type. dS2P mutants were viable due to alternative cleavage of dSREBP within the juxtamembrane region by the effector caspase, Drice. Flies lacking both dS2P and Drice, or the initiator caspase Dronc, exhibited an early larval lethality that could be rescued by lipid supplementation. Caspase cleavage was dependant upon the aspartic acid at residue 386 in dSREBP. dScap was not essential for larval growth or dSREBP processing in Drosophila. dScap mutants were relatively healthy, emerging at 70% of the expected numbers. dSREBP was actively cleaved in midgut and oenocytes, but significantly reduced in fat body. Levels of dSREBP mRNA and precursor were reduced in larvae lacking dScap, thus demonstrating that Drosophila SREBP is subject to feed-forward activation of its own transcription. Addition of soy lipids suppress dSREBP processing in dScap mutants, but whether this regulation is translational or post-translational is unknown. Furthermore, flies lacking both dScap and dS2P are viable, but survive less well than either single mutant alone. Membrane-bound intermediate dSREBP accumulates in double mutants, suggesting that dSREBP is processed normally by dS1P and dS2P in dScap single mutants. Thus, dScap mutants escape the larval lethality seen in dSREBP mutants due to alternative processing of dSREBP, but through different mechanism than that seen in dS2P mutants.Item CDNA Cloning and Characterization of Enzymes That Synthesize Bile Acids, Vitamin D and Waxes(2006-05-15) Cheng, Jeffrey Binyan; Russell, DavidCountless enzymes are required for the synthesis of the diverse array of lipids found in nature. The identification and characterization of five different lipid metabolizing enzymes are reported here. The 3beta -hydroxy-delta 5-C27-steroid oxidoreductase (C27 3beta-HSD) enzyme catalyzes a step in bile acid synthesis. Subjects with mutations in the encoding gene fail to synthesize bile acids and develop liver disease. Fifteen patients were screened and twelve different mutations were identified in the C27 3beta -HSD gene. Vitamin D is required for normal bone metabolism and maintenance of serum calcium levels. The conversion of vitamin D into an active ligand requires 25-hydroxylation. I report here the identification by expression cloning of a cytochrome P450 (CYP2R1) with vitamin D 25-hydroxylase activity. A patient with low circulating levels of 25-hydroxyvitamin D and classic symptoms of vitamin D deficiency was identified. Molecular analysis of this individual revealed homozygosity for a transition mutation in the CYP2R1 gene causing the substitution of a proline for a leucine in the protein and eliminating vitamin D 25-hydroxylase enzyme activity. These data identify CYP2R1 as a biologically relevant vitamin D 25-hydroxylase and reveal the molecular basis of a human genetic disease, selective 25-hydroxyvitamin D deficiency. The reduction of fatty acids to fatty alcohols, by a fatty acyl-CoA reductase enzyme, is required for the synthesis of wax monoesters and ether lipids. Using a bioinformatics approach, the first two mammalian fatty acyl-CoA reductase genes (FAR1 and FAR2) were identified. The two mouse FAR enzymes, which share 57% sequence identity at the amino acid level, have differing substrate specificities and tissue distributions implying unique physiological roles for each. Wax monoesters are synthesized by the esterification of fatty alcohols and fatty acids. A mammalian enzyme that catalyzes this reaction has not been isolated. Here, I report the identification by expression cloning of a wax synthase gene. Co-expression of cDNAs specifying FAR1 and wax synthase led to the synthesis of wax monoesters. The data suggests that wax monoester synthesis in mammals involves a two step biosynthetic pathway catalyzed by fatty acyl-CoA reductase and wax synthase enzymes.Item Examination of Abnormal Dolichol Metabolism in Infantile Batten Disease Caused by Palmitoyl Protein Thioesterase-1 (PPT1) Deficiency(2004-08-19) Cho, Steve Kyungrae; Lehrman, Mark A.The neuronal ceroid lipofuscinosis (NCLs, also known collectively as Batten disease) are a group of lysosomal storage disorders characterized by the accumulation of autofluorescent storage material in the brain. Although a number of genes underlying different forms of NCL have been cloned, the underlying mechanism for the neurodegeneration is still unknown. The most severe form of NCL (infantile NCL) is caused by mutations in the CLN1/PPT1 gene, which encodes a soluble lysosomal hydrolase (palmitoyl protein thioesterase-1) that removes fatty acids from lipid modified proteins in the lysosome. It has been postulated that abnormal dolichol metabolism might be involved in NCL pathogenesis because high levels of dolichol phosphate (Dol-P) and lipid linked oligosaccharides (LLOs) accumulate in NCL patients including infantile NCL. Here, a possible relationship between fatty acid and dolichol metabolism in the neuropathogenesis of NCL has been explored by analyzing LLOs from mouse models of Batten disease and other non-NCL lysosomal storage diseases (LSDs), and by characterizing a unique fusion protein consisting of PPT1 and a dolichol metabolizing enzyme (DOLPP1) in Schizosaccharomyces pombe, which we named pdf1 (for ppt1-dolpp1 fusion1). To do this, first, I characterized S. pombe pdf1 by ablating the pdf1 gene and studying the function of each of the proteins (PPT1 and DOLPP1) independently. These results revealed that PPT1 and DOLPP1 may be co-regulated in lower organisms but the functional relationship in higher eukaryotes remains unclear. To further explore the relationship between PPT1 and DOLPP1 in mammalian cells, I cloned the mammalian ortholog of DOLPP1 and subsequently characterized the mouse Dolpp1p. Finally, I characterized and analyzed LLOs in various mouse models of NCL by FACE (fluorophore assisted carbohydrate electrophoresis). It was shown that LLOs accumulated in PPT1-deficient mouse brain and the level of LLO accumulation was 14.5-fold higher as compared to wild type brain. Despite the striking accumulation of LLOs in PPT1-deficient brain compared to age-matched controls, I also found that the LLOs encompassed only 0.3% of the autofluorescent storage material by mass. Therefore, the abnormal dolichol catabolism is most likely a secondary phenotype to PPT1 deficiency during the pathogenesis of infantile Batten disease.Item Genetic Reduction of Cholesterol Synthesis in the Mouse Brain Does Not Affect Amyloid Formation in an Alzheimer’s Disease Model, but Does Extend Lifespan(2011-08-26T17:35:30Z) Warren, Rebekkah Lynn; Russell, David W.In vitro alterations in cellular cholesterol content or synthesis affect the cleavage of amyloid precursor protein (APP) to amyloidogenic peptides characteristic of Alzheimer’s disease (AD). To determine whether a decrease in cholesterol synthesis would affect APP processing in vivo, we crossed cholesterol 24-hydroxylase knockout (KO) mice, which exhibit a 50 percent reduction in sterol synthesis, with transgenic mice (B6.Cg-Tg(APPswe, PSEN1E9)85Dbo/J) that develop AD and followed progression of the disease and lipid metabolism in the offspring. APP expression and amyloid plaque deposition in the cortex and hippocampus of 3- to 15-month-old male and female AD mice were similar in the presence and absence of cholesterol 24-hydroxylase. At 15 months of age, a modest but statistically significant decline in insoluble A-beta 40 and A-beta 42 peptide levels was detected in the hippocampus but not cortex of KO/AD mice versus WT/AD mice. Amyloid plaque accumulation did not affect brain sterol or fatty acid synthesis rates in 24-hydroxylase WT or KO mice. Unexpectedly, loss of one or two 24-hydroxylase alleles increased longevity in AD mice. These studies suggest that reducing de novo cholesterol synthesis in the brain will not substantially alter the course of AD, but may confer a survival advantage.