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1-acyl-glycerol-3-phosphate acyltransferase from rat liverBarden, Roland E. January 1969 (has links)
Thesis (Ph. D.)--University of Wisconsin--Madison, 1969. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.
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Enzymatic regulation of phosphatidylcholine synthesis via protein ubiquitinationButler, Phillip Louis 01 May 2010 (has links)
Pulmonary surfactant is a critical surface-active substance consisting of dipalmitoylphosphatidylcholine (DPPtdCho) and key apoproteins that are produced and secreted into the airspace from alveolar type II epithelial cells. Deficiency of the surfactant leads to severe lung atelectasis, ventilatory impairment, and gas-exchange abnormalities. The generation of DPPtdCho in cells occurs via two integral routes: the de novo and remodeling pathways. The interplay between these pathways has not been investigated. Overexpression of the remodeling enzyme, acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT1), in epithelia decreases de novo PtdCho synthesis without significantly altering cellular phospholipid mass; this occurs through increased degradation of cholinephosphotransferase (CPT1), the terminal enzyme of the de novo pathway. CPT1 is degraded by multi-ubuiquitination and trafficking via the lysosomal pathway. When expressed in lung epithelia, CPT1 mutants harboring arginine substitutions at multiple carboxyl-terminal lysine residues exhibited proteolytic resistance to effects of LPCAT1 overexpression. Cellular expression of these CPT1 mutants also restores de novo PtdCho synthesis to levels normally observed in lung epithelia. Further studies demonstrate that the SCF (Skip-Cullen-F-box) ubiquitin E3 ligase component, β-TrCP, was sufficient to degrade CPT1. Similar to CPT1, LPCAT1 levels are also regulated at the level of protein stability. However, LPCAT1 is a polyubiquitinated enzyme processed within the proteasome. Similar to CPT1, β-TrCP is the putative E3 ubiquitin ligase subunit responsible for LPCAT1 ubiquitination. β-TrCP appears to dock and ubiquitinate LPCAT1 within its amino-terminus. Collectively, these observations indicate the presence of cross-talk between the phospholipid remodeling and de novo pathways; this involves tight regulation by site-specific ubiquitination of indispensable regulatory enzymes catalyzed by SCF ubiquitin E3 ligase members that mechanistically provide homeostatic control of cellular phospholipid content.
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Diacylglycerol acyltransferase in relation to intramuscular fat deposition in the bovineMiddleton, Cameron K., University of Lethbridge. Faculty of Arts and Science January 1997 (has links)
Studies on the enzymology of fat foramtion in beef cattle may lead to the development of a marker for predicting the propensity of these animals to deposit intramuscular (i.m.) fat. Diacylglycerol acyltransferse (DFAT, EC 2.3.1.20) catalyzes the acylation of sn-1,2-diacylglycerol to form triacylglycerol (TAG). This thesis has focused on evaluating DGAT as a potential marker for predicting i.m. fat deposition in cattle and on characterization of the enzyme in three tissue types involved in TAG biosynthesis. Microsomes were isolated from subcutaneous (s.c.) adipose, i.m. adipose and muscle tissue of Wagyu crossbred cattle displaying a wide range of i.m. fat content (marbling). One aspect of this thesis focused on the relationship between total lipid present in meat samples from the pars costalis diaphragmatis (p.c.d.) and DGAT activity from s.c. adipose tissue, i.m. adipose tissue and muscle tissue of the p.c.d. Intramuscular adipose tissue DGAT activity exhibited a negative correlation with % TAG per gram wet weight p.c.d. (r=-.547;P<.01). There was, however, a positive correlation (r=.735;P<.001) between DGAT activity per unit wet weight i.m. adipose tissue and muscle DGAT activity per unit wet weight suggesting these two depots are coordinated in terms of TAG biosynthetic capacity. The second aspect of the thesis exmained the characteristics of microsomal DGAT from the three tissue types. Microsomes from several animals were combined and were used for enzymological studies of DGAT in an attempt to define possible differences in enzymatic properties among the three tissue types. Diacylglycerol acyltransferase from s.c. adipose tissue in the presence of 15 mM MgCl2 exhibited an enhanced preference for substrates containing oleoyl moieties relative to the other two tissue types. Diacylglycerol acyltransferase from s.c. adipose tissue alos differed slightly in sensitivity to N-ethylmaleimide and stability when compared to the other two tissues, muscle and i.m. adipose tissue. Collectively, these results suggested that either a different isoform of DGAT is expressed in s.c. adipose tissue as compared to i.m. adipose and muscle tissue, or that the endoplasmic reticulum micro-environments are sufficiently different to account for the depot specific properties of DGAT in bovine. The data presented in this thesis suggested that DGAT was not a suitable marker for the deposition of i.m. fat in mature cattle. Perhaps an examination of i.m. fat deposition in less mature animals will yield stronger, positive correlations between DGAT activity and the amount of i.m. fat present. / xvi, 109 leaves : ill. ; 28 cm.
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The ontogeny of acyl coenzyme A: cholesterol acyltransferase in rat liver, intestine, adipose tissue, and aortaLittle, Marie-Térèse E. January 1990 (has links)
Epidemiological studies have shown that cholesterol is a major risk factor for the development of atherosclerosis. Since the atherosclerotic plaque develops over a long period interventions early in life may be of some benefit. In addition, it has been shown that the enzymes involved in cholesterol metabolism can be manipulated in early life. Therefore, studies of the developmental patterns of the key enzymes in cholesterol metabolism are of great importance. Acyl coenzyme A: cholesterol acyltransferase (ACAT) is the primary enzyme which catalyzes the conversion of free cholesterol to cholesterol esters in cells. A better understanding of the role and control of ACAT during development is needed in order to trace the possible causes in early life that lead to atherosclerosis in the adult.
This research focused on the developmental pattern of ACAT in the rat liver, intestine, brown and white adipose tissue (BAT and WAT, respectively) and aorta. Age specific changes were observed in the rat liver, intestine and BAT. The rat liver and intestine possess significant amounts of ACAT activity throughout development and there appears to be marked variations in activity during this time. The rat BAT and WAT appear to be devoid of ACAT activity throughout development with the exception of adult BAT. Due to the small amount of the aortic tissue samples and/or the insensitivity of the assay, no definite conclusions could be made from this aortic study.
In searching for factors that might control the ACAT enzyme the immediate effects of short-term manipulation of diet on the activity of ACAT were studied. The rats were all weaned early on day 18 to one of the following diets: Purina Rat Chow, high carbohydrate (HG) , high fat (HF) , or 2% cholesterol. The HF was the only diet that consistently increased hepatic ACAT activity in all the age groups. The cholesterol diets significantly increased the activity of ACAT in the 22 and 25 day old rats. The HG diet increased the activity of ACAT in the 22, 25, and 30 day old rats. No significant differences were observed between the adult control and HG diet groups. Feeding rats a HF or HG diet precipitated a dramatic drop in intestinal ACAT activity in the 22 day old animals. These effects were not observed in the older animals. The high cholesterol diet had no significant effect on the intestinal enzyme's activity in 22 day old rats. There was no significant change in the BAT and WAT ACAT activity with the experimental diets with the exception that all the experimental diets decreased ACAT activity in the adult BAT. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate
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IMPROVED SYNTHESIS OF ACETYL-COA AND MALONYL-COA ANALOGS AND THEIR USE TO STUDY STRUCTURE-FUNCTION RELATIONSHIPS OF ACYLTRANSFERASESAaron B Benjamin (9175175) 29 July 2020 (has links)
Thioesters are highly reactive centers for acyl-CoAs
which allows them to be utilized in a variety of differing enzyme chemistries.
As a result of this reactivity, structure-function studies of enzymes using acyl-CoA
substrates is difficult. When acyl-CoAs are used in structure-function studies,
they often result in a hydrolyzed CoA substrate fragment bound in the active
site or require only one of multiple substrates in order to be bound. This results
in a lack of information regarding enzyme interactions with the key thioester
and acyl chain. To overcome this challenging problem, I have synthesized
acetyl- and malonyl-CoA analogs where the thioester has been replaced by an ester
(oxygen), amide (nitrogen), or carbonyl (carbon) in a way that is easier,
cheaper, and more efficient than performed previously. In addition, we used our
synthetic analogs to study a enzymes which span different acyltransferase
mechanisms in a combination of kinetics and structure. With this work, it was
determined that the amide analogs were stable in all enzymes it was utilized
for, while the ester analogs were mostly stable, except the acetyl analog in
KasIII, where it acted as a pseudo substrate. As such, these synthetic analogs may
have future potential in either type of enzyme for structure-function studies,
albeit limited for the acetyl ester analog.
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Transient expression of avocado DGAT1 and PDAT1 in N. benthamiana leavesRahman, Md Mahbubur, Shockey, Jay, Kilaru, Aruna 04 April 2018 (has links)
The avocado mesocarp contains up to 60-70% oil by dry weight where triacylglycerol (TAG) is the major constituent. This neutral lipid, TAG is utilized by plants for the carbon and energy source when stores in seed tissue. There is significant human nutritional demand for vegetable oil, but its use in the production of renewable biomaterials and fuels has intensified the need to increase oil production. In plants, the final and committed step in TAG biosynthesis is catalyzed by diacylglycerol acyltransferases (DGAT) and/or a phospholipid: diacylglycerol acyltransferases (PDAT). However, the regulation of TAG biosynthesis is not well-studied in nonseed tissues such as mesocarp of avocado. Based on the transcriptome data of Persea americana it is hypothesized that both DGAT and PDAT are likely to catalyze the conversion of diacylglycerol to TAG. In this study, putative DGAT1 and PDAT1 were identified and comprehensive in silico analyses were conducted to determine the respective start codons, full-length coding sequences, transmembrane domains, predicted protein structures and phylogenetic relationships with other known DGAT1s and PDAT1s. These data reveal that the putative DGAT1 and PDAT1 of a basal angiosperm species retain features that are conserved not only among angiosperms but also other eukaryotes. For transient expression, DGAT1 and PDAT1 were transformed into N. benthamiana leaves by agrobacterium-mediated transformation. Lipid droplet was visualized by Nile Red staining and lipid content and compositions were analyzed by TLC and GC-MS. It was found that avocado DGAT1 and PDAT1 increase lipid content significantly when expressed in tobacco leaves. These results suggest that avocado DGAT1 and PDAT1 are functional and synthesize TAG when expressed in planta.
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Structural Basis for Protein Recognition, Acyl-substrate Delivery, and Product Release by ACP in the Biosynthesis of Lipid AMasoudi, S. Ali January 2014 (has links)
<p>Acyl-carrier-protein (ACP) is the principal transporter of fatty acids, coordinating acyl-transfer among a vast network of diverse enzymes and biochemical processes. ACP association with protein partners is thought to be exceedingly transient. This paradigm has posed challenges for understanding the molecular basis for acyl-delivery and dissociation. During biosynthesis of the lipid A component (endotoxin) of lipopolysaccharides, ACP shuttles acyl-intermediates thioester-linked to its 4'-phosphopantetheine arm among four acyltransferases: LpxA, LpxD, LpxL, and LpxM. LpxA and LpxD are essential cytoplasmic enzymes, which not only provide an excellent model system to study ACP-based interaction, but also offer an important therapeutic target for development of novel antibiotics. The current dissertation reports the crystal structures of three forms of <italic>Escherichia coli</italic> ACP engaging LpxD, which represent stalled substrate and breakage products along the reaction coordinate. The structures reveal the intricate interactions at the interface that optimally position ACP for acyl-delivery and directly involve the pantetheinyl group. Conformational differences among the stalled ACPs provide the molecular basis for the association-dissociation process. An unanticipated conformational shift of 4'-phosphopantetheine groups within the LpxD catalytic chamber reveals an unprecedented role of ACP in product release. Moreover, the crystal structure of <italic>E. coli</italic> LpxA in complex with one form of ACP (holo-ACP) is presented. The structure reveals three molecules of holo-ACP localize to the C-terminal domain of the LpxA homotrimer, and shows the functional role of this domain is two-fold: ACP recognition and nucleotide binding of UDP-GlcNAc. A comparison with the LpxD:ACP complexes uncovers that ACP utilizes different surface residues for recognition even amongst closely related acyltransferases, yet still relies on "electrostatic steering" for docking to its enzyme partner. Insights gleaned from the presented structures have provided not only a better understanding of ACP interaction with acyltransferases, but also has identified the "drugable molecular landscape" for the development of novel antibiotics against infective bacteria.</p> / Dissertation
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Etude de la synthèse des précurseurs majeurs à la synthèse des lipides membraniares : l'acide lysophosphatidique et l'acide phosphatidique chez Toxoplasma gondii / L'auteur n'a pas fourni de titre anglaisAmiar, Souad 01 December 2016 (has links)
Les Apicomplexa sont des parasites intracellulaires obligatoires. Ils peuvent être responsables d’importantes maladies infectieuses. Toxoplasma gondii par exemple, se développe au sein de la cellule hôte, dans une niche protectrice « la vacuole parasitophore » jusqu’à épuisement des ressources de la cellule hôte ou il provoque sa sortie pour ré envahir à nouveau, c’est la phase aigüe de la toxoplasmose. Afin de répondre à leur besoins nutritifs nécessaires à cette expansion rapide, le parasite combine de manière intéressante et très complexe les voies de synthèse de novo et d’import des nutriments depuis la cellule hôte. Dans le cas des lipides, le parasite en a besoin en une importante quantité pour assurer la ségrégation des organites, la formation des nouvelles membranes filles et l’expansion de la membrane de la vacuole parasitophore pendant la division. La synthèse de novo des lipides a été reportée essentielles pour le parasite tout comme la synthèse de novo des acides gras via la voie procaryote de synthèse des acides gras FASII dans l’apicoplaste.Dans cette étude nous apportant des éléments intéressants qui relient la voie de synthèse FASII et la synthèse des lipides. Nous avons pu démontrer que l’apicoplaste possède une voie de synthèse des précurseurs important voire essentielles à la synthèse de tous les lipides membranaires, qui est principalement le LPA dans le cas de T. gondii. Les enzymes acyltransférase impliquées dans la synthèse de ces précurseurs sont TgATS1 et TgATS2 pour former le LPA et le PA respectivement. Elles sont ortologues aux enzymes précédemment caractérisées chez les bactéries et le chloroplaste des plantes et algues. Les modifications de ces enzymes et les analyses de lipidique et de spectrométrie de masse, ont révélé le rôle l’implication de ces enzymes dans la synthèse des phospholipides membranaires à partir des acides gras néo synthétisés de novo (le C14:0). Cette étude présente aussi des résultats préliminaires sur une voie de synthèse du PA dans le réticulum endoplasmique. La être de TgATS2 n’est pas létale et elle est compensée par augmentation de l’abondance des acides gras C16 :0 et C18 :0 dans la fraction des phospholipides extraits. Ces informations suggèrent une importante collaboration entre l’apicoplaste et le réticulum endoplasmique pour la synthèse des lipides nécessaires pour le développement intracellulaire du parasite. / Apicomplexa phylum includes a large number of obligate intracellular parasites responsible for important human and animal diseases, especially malaria and toxoplasmosis. There is current no efficient vaccine against these parasites. Severe toxoplasmosis caused by Toxoplasma gondii, occurs in immunocompromised individuals and during congenital infection. T. gondii is dependent on large amounts of lipids for its intracellular development within the host cell. These lipids are acquired by a combination of host lipid scavenging and de novo biosynthetic pathways. T. gondii is able to de novo synthesis of fatty acid via a prokaryotic FASII pathway in the apicoplast, a relict non-photosynthetic plastid. Genome mining suggests that the apicoplast can generate phosphatidic acid, central phospholipid precursor. Our recent work confirmed that the apicoplast harbors the first step of PA synthesis via a glycerol-3-phosphate acyltransferase enzyme called ATS1 by homology to chloroplast enzyme, which generates lysophosphatidic acid (LPA). This essential LPA can be exported from the apicoplast for the de novo bulk synthesis of phospholipids sustaining parasite membrane biogenesis (Amiar et al. Plos Path. 2016). T. gondii genome encodes for two other acyltransferases named sn-acylglycerol 3-Phosphate acyltransferases (AGPAT). AGPATs ensure the second step of PA synthesis using LPA. In this work we showed that these enzymes are localized in the Endoplasmic Reticulum and the apicoplast (named AGPAT and ATS2, respectively). A genetic disruption of ATS2 using CRISPR-Cas9 strategy affects parasite growth and normal cytokinesis. Lipidomic analysis using mass spectrometry combined to stable isotope labelling of ATS2-KO reveals an important reduction of lipids containing apicoplast-generated fatty acid C14:0. However, an increase of lipids containing C16 and C18 fatty acids was observed, suggesting a compensation of ATS2 loss by AGPAT activity in ER. These data indicated an important collaboration between apicoplast and ER for lipid synthesis that involves massive lipid trafficking between the two organelles.
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Identification of Acyltransferases Associated with Triacylglycerol Biosynthesis in AvocadoSung, Ha-Jung 01 December 2013 (has links) (PDF)
A variety of plants synthesize and store oil in the form of triacylglycerols (TAG) in their seed and nonseed tissues that are commonly used as vegetable oils. In seed tissues, an acyl CoA-dependent diacylglycerol (DAG) acyltransferase (DGAT) and/or -independent phospholipid:DGAT (PDAT) catalyze the conversion of DAG to TAG. In avocado fruit, which stores up to 70% oil by dry weight in mesocarp, it is hypothesized that both DGAT and PDAT are likely involved in TAG synthesis. To investigate, TAG content and composition and transcript levels for the acyltransferases in avocado fruit were quantified by gas chromatography and real-time polymerase chain reaction, respectively. Temporal, tissue-specific and phenotypic comparisons revealed that while DGAT1 gene expression was specifically associated with TAG accumulation, PDAT also correlated with higher levels of polyunsaturated fatty acid; DGAT2 was barely detectable. These studies suggest that TAG biosynthesis in nonseed tissues of avocado involves acyl CoA-dependent and -independent reactions.
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Isolation, Physical and Chemical Characterization of Lecithin:Cholesterol Acyltransferase from Human PlasmaChong, Kui Song 12 1900 (has links)
The physiological role of LCAT has been the subject of a number of recent articles (Glomset, 1979; Nilsson-Ehle et al., 1980). According to most current theories, the enzyme functions in combination with high-density lipoproteins in the reverse cholesterol transport pathway which presumably returns peripheral cholesterol to the liver where cholesterol catabolism takes place. Despite the exciting potential for studies on the catalytic function and the nature of the enzyme-substrate complex, the mechanism of action of LCAT remains largely unexplored. The relatively slow progress in the elucidation of the LCAT reaction mechanism is likely to be due to the difficulties in the isolation of the enzyme in sufficient quantities. Consequently, considerably less is known about the physical and chemical properties of the enzyme. Therefore, the first objective of this investigation was to isolate and purify sufficient amount of enzyme for subsequent characterization studies. The second objective of this investigation was to characterize the physical properties of the enzyme by techniques including analytical ultracentrifugation, ultraviolet spectroscopy, and circular dichroism and fluorescence spectroscopy. The third objective of this investigation was to characterize the chemical properties of the enzyme which deals with the amino acid and carbohydrate composition and with some basic structural features that are related to the chemical composition of LCAT.
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