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The effect of [alpha]-aminoadipate [delta]-semialdehyde synthase knockdown on the lysine requirement and urate oxidase knockdown on oxidate stress in a murine hepatic cell lineCleveland, Beth Marie. January 2007 (has links)
Thesis (Ph. D.)--West Virginia University, 2007. / Title from document title page. Document formatted into pages; contains vii, 112 p. : ill. (some col.). Includes abstract. Includes bibliographical references.
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Chemical probes for histone lysine demethylasesGerken, Philip January 2016 (has links)
The primary objective of this DPhil research project was to develop selective and cell-active inhibitors of the histone lysine demethylase KDM2A, which could potentially lead to the discovery of a novel chemical probe. Chapter one of this thesis introduces the role of histone lysine demethylases (KDMs) in the epigenetic regulation of gene expression and discusses the value of chemical probes as tools to study these enzymes. Chapter two describes the synthesis of a library of indoline-based KDM2A inhibitors using a modular synthetic approach to explore key structure-activity relationships and a chiral counterion-mediated strategy to synthesize lead candidates enantioselectively. Chapter three discusses investigations into the cellular activity of lead compounds and explores strategies to address limitations associated with cytotoxicity and promiscuity. Chapter four describes the application of a variety of experimental techniques to identify the mode of target inhibition. Finally, chapter five focuses on the development of an enantioselective C-acylation reaction to access spirocyclic fragments asymmetrically.
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Studies of Nε-Lysine Acetylation Modification on Escherichia coli Topoisomerase IZhou, Qingxuan 28 June 2017 (has links)
Escherichia coli topoisomerase I (TopA), a regulator of global and local DNA supercoiling, is modified by Nε-Lysine acetylation. The sirtuin protein deacetylase CobB can reverse both enzymatic and non-enzymatic lysine acetylation modifications. Here, we explored the effect of lysine acetylation on E. coli topoisomerase I through analysis of TopA relaxation activity and protein expression in cell extract of wild-type and a ΔcobB mutant strains. We showed that the absence of deacetylase CobB in a ΔcobB mutant reduced intracellular TopA relaxation activity while elevating TopA expression and topA gene transcripts levels. Acetyl phosphate mediated lysine acetylation decreased the activity of purified TopA in vitro, and the interaction with purified CobB protected TopA from such inactivation. We explored the physiological significance of TopA acetylation on DNA supercoiling by two-dimensional gel analysis and on cell growth rate by growth curve analysis. We found that the absence of CobB increased negative DNA supercoiling. The slow growth phenotype of the ∆cobB mutant can be partially compensated by overexpression of recombinant TopA. In addition, the specific activity of TopA expressed from His-tagged fusion construct in the chromosome was inversely proportional to the degree of in vivo lysine acetylation during growth transition and growth arrest. Investigation of TopA relaxation mechanism using nuclease footprinting and TopA oxidative crosslinking suggested the potential association of TopA acetylation in catalysis. Mass spectrometry analysis of in vitro acetyl phosphate acetylated TopA identified abundant lysine acetylation sites. Substitution of lysine residues by site-directed mutagenesis was used to model the effect of acetylation on individual lysine residues. Our results showed that substitution of Lys-484 with alanine reduced the relaxation activity, suggesting the reduction of TopA relaxation activity by acetylation was probably in part due to acetylation on Lys-484. These findings demonstrate that E. coli topoisomerase I is modulated by lysine acetylation and the prevention of TopA inactivation from excess lysine acetylation and consequent increase in negative DNA supercoiling is an important physiological function of the sirtuin deacetylase CobB.
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Enrichment and Identification of Methylation at the Proteome LevelStar, Alexandra 22 January 2016 (has links)
Methylation is a post-translational modification which occurs on lysine and arginine residues. Methylation is difficult to detect due to its low abundance and lack of charge. Our laboratory previously developed a novel enrichment approach, ProMENADe, for lysine and arginine methylation in the human embryonic kidney (HEK) 293T cell line which is coupled with mass spectrometry.
Simplifying a lysate with subcellular fractionation prior to enrichment increased the identification of methylation sites by 39.5% while using multiple proteases for digestion increased identification by 27%. Combining these methods yielded a 47.2% increase. Analysis at the 1% methylation level FDR filtered for C-terminal methylation identified 169 sites and further analysis revealed 74 of these sites overlap with the PhosphoSite database. This ProMENADe enrichment strategy yielded 95 novel methylation sites to the field and can be a key tool in the field of methylation allowing for the enrichment and identification of methylated proteins.
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Molecular and functional characterization of set domain proteins in the epigenetic regulation of Arabidopsis thaliana development / Caractérisation moléculaire et fonctionnelle des protéines à domaine SET dans la régulation épigénétique du développement d' Arabidopsis thalianaShafiq, Sarfraz 12 April 2012 (has links)
Alors que les méthylations sur différents résidus lysine des histones (par exemple H3K4, H3K27 et H3K36) sont bien connues pour exercer diverses fonctions biologiques, leurs interactions et/ou leur mode d’actions demeurent encore peu caractérisés. Par la génétique et des outils de biologie moléculaire, nous visons à étudier les rôles et interconnections des méthylations des H3K4, H3K27 et H3K36 dans la transcription, la croissance de la plante et la régulation du développement chez Arabidopsis thaliana.La première partie de ma thèse est centrée sur les rôles et interconnections des méthylations de H3K4 and K36.ATX1 et ATX2 sont des méthyltransférases de H3K4 alors que SDG8 est une méthyltransférase de H3K36.L’analyse de doubles mutants a révélé que sdg8 est dominant sur atx1 et atx2 pour le temps de floraison et larégulation de la prolifération cellulaire. La triméthylation de H3K36 (H3K36me3) est partiellement dépendante de H3K4me3 mais non-réciproquement. SDG25 a une double activité H3K4me3 et H3K36me3 et les déméthylases de H3K4, LDL1 et LDL2, sont des antagonistes de l’activité de SDG25. Les triples mutants sdg25ldl1ldl2 fleurissent plus tôt que la lignée sauvage, mais plus tard que sdg25 et montrent une augmentation de taille de cellule similaire à celle des mutants ldl1ldl2.La deuxième partie de ma thèse se concentre sur les rôles et interconnections entre les méthylations H3K4/K36 et H3K27. CLF catalyse les H3K27me3 au sein du complexe PRC2. Les doubles mutants sdg8clf etsdg25clf fleurissent plus tôt que les mutants simples et montrent un nombre réduit de cellules par feuille. Unniveau plus élevé de H3K4me3 et dans une moindre mesure de H3K36me3 a été observé dans le cas de déposition de H3K27me3 réduite, et de la même façon, une déposition de H3K4me3/H3K36me3 réduite augmente aussi le niveau de H3K27me3. Distinct du rôle antagoniste rapporté auparavant entre CLF et ATX1,CLF n’a pas montré d’antagonisme avec SDG25 ou SDG8.La dernière partie de ma thèse est centrée sur le mécanisme de SDG26 dans la régulation du temps de floraison. Mes résultats ont montré que SDG26 est une méthyltransférase H3K4 et/ou H3K36 spécifique de la chromatine de SOC1, un intégrateur de la floraison actif. De manière similaire à SDG25 et SDG8, SDG26 ne travaillait pas de façon antagoniste avec CLF. L’analyse de doubles mutants a révélé que sdg26 domine atx2mais sdg25, atx1 and clf est dominant sur sdg26 pours le temps de floraison et la régulation de la prolifération cellulaire. Les triples mutants sdg26ldl1ldl2 fleurissaient encore plus tard que les mutants sdg26 et ldl1ldl2 et a révélé que sdg26 est dominant sur ldl1ldl2 lors de la régulation de la prolifération cellulaire. Les analysesd’interaction avec les autres composants de PRC2, VEL1 et VRN5, ont révélé que sdg26vel1 et sdg26vrn5 fleurissaient encore plus tard que les mutants simples dans des conditions de jours courts et de vernalisation. Ensemble, mes résultats révèlent des couches additionnelles de complexité de redondance et de diversification de fonctions entre et au sein des méthyltransférases et déméthylases, pour la transcription, le temps de floraison et la régulation de la prolifération cellulaire chez Arabidopsis. / While methylations at different lysine residues of histones (e.g. H3K4, H3K27 and H3K36) are well known to exert diverse biological functions, their interactions and/or ensemble-actions remain poorly characterized so far.Using genetic and molecular biology tools, we aim to investigate roles and ‘crosstalks’ of H3K4, H3K27 andH3K36 methylations in transcription and plant growth and development regulation in Arabidopsis thaliana.The first part of my thesis focuses on the roles and crosstalks between H3K4 and K36 methylations.ATX1 and ATX2 are H3K4 methyltransferases while SDG8 is a H3K36 methyltransferase. Double mutant analysis revealed that sdg8 dominates over atx1 and atx2 in flowering time and cell proliferation regulation.H3K36 trimethylation (H3K36me3) is partially dependent on H3K4me3 but not vice versa. SDG25 has a dualH3K4me3 and H3K36me3 activity and the H3K4-demethylases LDL1 and LDL2 antagonize SDG25 activity.The sdg25ldl1ldl2 triple mutants flowered earlier than wild type but later than sdg25 and showed an increased cell size similarly to ldl1ldl2 mutantsThe second part of my thesis focuses on the roles and crosstalks between H3K4/K36 and H3K27methylations. CLF within PRC2 complex catalyzes H3K27me3. The sdg8clf and sdg25clf double mutants flowered earlier than the single mutants and showed a reduced number of cells per leaf. An increased level ofH3K4me3 and to a less extent H3K36me3 was observed upon impaired H3K27me3 deposition, and similarly impaired H3K4me3/H3K36me3 deposition also enhanced H3K27me3 level. Distinct from previously reported antagonistic role between CLF and ATX1, CLF did not show antagonism with SDG25 or SDG8.The last part of my thesis focuses on mechanism of SDG26 in flowering time regulation. My result showed that SDG26 is a H3K4 and/or H3K36 methyltransferase specific at chromatin of SOC1, an activeflowering integrator. Similarly to SDG25 and SDG8, SDG26 did not work antagonistically with CLF. Double mutant analysis revealed that sdg26 dominates over atx2 while sdg25, atx1 and clf dominate over sdg26 inflowering time and cell proliferation regulation. The sdg26ldl1ldl2 triple mutants flowered even later than thesdg26 and ldl1ldl2 mutants and showed that sdg26 dominates over ldl1ldl2 in cell proliferation regulation.Interaction analysis with the other PRC2 components VEL1 and VRN5 revealed that sdg26vel1 and sdg26vrn5flowered even later than the single mutants under short day and vernalization conditions.Together, my study revealed additional layers of complexity of overlap and non-overlap functions between and within methyltransferases and demethylases in transcription, flowering time and cell proliferation regulation in Arabidopsis.
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The dissociation of ammonium salts and their effect on the physiology and biochemistry of L-lysine synthesis by Corynebacterium glutamicum FP6Kenyon, Colin Peter January 1994 (has links)
The availability and assimilation of NH₄⁺ plays an integral role in the growth of microorganisms and the production of amino acids by these organisms. This study investigated the dissociation of NH₄⁺in aqueous solution, its availability and effect on the enzymes of NH₄⁺ assimilation and its influence on lysine production by Corynebacterium glutamicum.In aqueous solution the extent of dissociation of NH₄C1, {NH₄)₂S0₄ and (NH₄)₂HP0₄ increases with decreasing concentration. A model is proposed for the dissociation of these molecules. It is believed that at very low concentrations, dissociation to NH₃ plus the respective counter-ions occurs. At these low concentrations the NH₃ acts as the substrate for glutamine synthetase. At the higher concentrations dissociation is to NH₄⁺ which is the substrate for glutamate dehydrogenase. At these higher concentrations the enzyme activities obtained for glutamate dehydrogenase, at equivalent concentrations of the above ammonium salts, were different when based on the total concentration of NH₄⁺, and similar when based on the concentration of free NH₄⁺. L-Iysine occurs in the +1 ionic form, at pH 7,2. The lysine which is produced during fermentation associates with the anionic counter-ion of the ammonium salt used. The concentration of the free NH₄⁺ in the media appears to affect both the rate of lysine synthesis as well as the yield. The lysine fermentation occurs in two stages; a growth (or replicative) phase, during which very little lysine is produced, and a lysine synthesis (or maturation) phase. During the lysine synthesis phase there is no cell replication, however an increase in the mass of the biomass produced is apparent. Evidence is provided for the possible concomitant synthesis of the the cell wall polymer, glycerol teichoic acid, and lysine. On the basis of this evidence, a nucleotide balance is proposed for lysine and teichoic acid synthesis. The replicative phase and the maturation phase have to be effectively separated to obtain optimal lysine yields and titres. It is believed that teichoic acid synthesis during the replicative phase must be kept to a minimum for optimal yields and titres to be obtained, and on completion of the cell wall and therefore teichoic acid synthesis, lysine synthesis ceases. As the production of lysine appears to be affected by the NH₄⁺ concentration in the culture media, it is proposed that a futile cycle may exist around the transport and assimilation of the NH₄⁺. If the fermentations are run at low free NH₄⁺ concentrations, it was shown that lysine yields of 0,66, on the glucose utilised, are attainable during the fermentation.
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A Systems Level Characterization of the Saccharomyces Cerevisiae NuA4 Lysine AcetyltransferaseMitchell, Leslie January 2011 (has links)
Lysine acetylation is a post-translational modification (PTM) studied extensively in the context of histone proteins as a regulator of chromatin dynamics. Recent proteomic studies have revealed that as much as 10% of prokaryotic and mammalian proteins undergo lysine acetylation, and as such, the study of its biological consequences is rapidly expanding to include virtually all cellular processes. Unravelling the complex regulatory network governed by lysine acetylation will require an in depth knowledge of the lysine acetyltransferase enzymes that mediate catalysis, and moreover the development of methods that can identify enzyme-substrate relationships in vivo. This is complex task and will be aided significantly through the use of model organisms and systems biology approaches. The work presented in this thesis explores the function of the highly conserved NuA4 lysine acetyltransferase enzyme complex in the model organism Saccharomyces cerevisiae using systems biology approaches. By exploiting genetic screening tools available to the budding yeast model, I have systematically assessed the cellular roles of NuA4, thereby identifying novel cellular processes impacted by the function of the complex, such as vesicle-mediated transport and the stress response, and moreover identified specific pathways and proteins that are impacted by NuA4 KAT activity, including cytokinesis through the regulation of septin protein dynamics. Moreover, I have developed a mass spectrometry-based technique to identify NuA4-dependent acetylation sites amongst proteins that physically interact with NuA4 in vivo. Together this work demonstrates the diversity of processes impacted by NuA4 function in vivo and moreover highlights the utility of global screening techniques to characterize KAT function.
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Structural and Biochemical Dissection of the KMT2 Core ComplexZhang, Pamela Peng January 2015 (has links)
Histone H3 lysine 4 (H3K4) methylation is an evolutionarily conserved mark commonly associated with transcription activation in eukaryotes. In mammals, this post-translational modification is deposited by the KMT2 family of H3K4 methyltransferases. Biochemical studies have shown that the enzymatic activity of the KMT2 enzymes is regulated by a core complex of four evolutionarily conserved proteins: WDR5, RbBP5, ASH2L and DPY30, collectively known as WRAD, which are all important for global H3K4 methylation. However, how these proteins interact and regulate the activity of the KMT2 enzymes is not well investigated. During my PhD, I have used structural and biochemical approaches to determine the interactions underlying formation of the core complex and regulation of KMT2 enzymatic activity. My research have shown that 1) WDR5 uses two peptide-binding clefts on opposite sides of its β-propeller domain to bridge the KMT2 enzymes to the regulatory subunit RbBP5, 2) the WDR5 peptidyl-arginine-binding cleft exhibits plasticity to accommodate the binding of all KMT2 enzymes and 3) RbBP5 S350 phosphorylation stimulates formation of the RbBP5-ASH2L complex and H3K4 methylation by the mammalian KMT2 enzymes. Collectively, these studies have provided the structural basis for understanding the important interactions governing KMT2 complex assembly and activity.
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The effect of liquid rumen-protected lysine supplementation on lactation performance of Holstein cowsVenter, Richardt 13 August 2009 (has links)
Thirty high-producing multiparous Holstein cows were used in a completely randomized block design to compare a lysine deficient total mixed ration, which was sufficient in methionine, to the same diet supplemented with a rumen protected lysine product. The CPM-Dairy prediction model was used to estimate the nutrient requirements and adequacy or deficiency of amino acids. During the 21-day prepartum transition period, cows were fed 4 kg (dry basis) of the lysine deficient diet plus Eragrostis curvula hay ad lib. After calving, cows were fed the lysine deficient diet for the first three weeks and were then blocked according to the average production from day 19-21. Fifteen cows were allocated to each treatment and blocked into 15 groups of two each. Data on production parameters were analyzed for all cows and also separately for cows in the 10 highest production blocks. The experimental period was from day 22 to 120 postpartum. Lysine supplementation resulted in an optimal dietary lysine : methionine ratio in metabolisable protein of 7.2 : 2.4. Lysine supplementation did not affect dry matter intake, milk production, milk fat percentage, milk protein percentage, milk urea nitrogen, body weight or body condition score; but decreased the non-casein nitrogen and whey content of milk. Furthermore, milk casein, which is the milk nitrogen fraction most sensitive towards increased duodenal supply of lysine and methionine, was not affected. The rumen protected lysine product evaluated did not improve cow productivity, probably because the product was either unprotected from rumen degradation, or overprotected to the extent that the lysine was not available for absorption in the small intestine; or absorbed but could not be metabolised. Copyright / Dissertation (MSc(Agric))--University of Pretoria, 2009. / Animal and Wildlife Sciences / unrestricted
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Effect of Dietary Lysine on Plasma Amino Acid Profile and Selected Metabolites of Late-Stage Finishing PigsRegmi, Naresh 14 August 2015 (has links)
The objective of this study was to evaluate the effects of dietary lysine on plasma concentrations of amino acid (AAs) and other metabolites of finishing pigs. Eighteen crossbred late-stage finishing pigs (9 barrows and 9 gilts) were assigned to three dietary treatments, Diets I (lysine-deficient), II (lysineequate), and III (lysine-excess), according to a randomized complete block design with gender as block. After 4 weeks on trial, jugular vein blood were collected and centrifuged for plasma samples, which were analyzed for concentrations of 24 AAs using HPLC method and 6 selected metabolites related to major nutrient metabolism using ACE Alera Clinical Chemistry System. Dietary lysine had a significant effect on plasma concentrations of 13 AAs and 3 metabolites (albumin, cholesterol, and urea nitrogen). The patterns of AA and metabolite concentrations alteration indicated complex metabolic interactions between lysine and other AAs, which subsequently had effects on other metabolites and animal growth performance.
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