L'obésité, le diabète de type II et le cancer sont considérés comme des fardeaux sociaux et économiques. Cette étude s'intéresse à la régulation hépatique du gène de la stéaroyl-CoA désaturase 1 (SCD1), enzyme-clé de la lipogenèse de nova (LDN) reconnue pour son implication dans ces pathologies. L'enzyme SCD1 transforme les acides gras saturés palmitate et stéarate en acides gras insaturés palmitoléate et oléate, respectivement. Principalement transcriptionnelle, sa régulation dépend de facteurs nutritionnels et hormonaux. Dans le cas présent, les voies de signalisation de l'insuline pouvant être impliquées dans la régulation du gène SCD1 sont étudiées. L'étude est réalisée chez des hépatocytes d'embryons de poulets et chez des hépatomes humains cultivés dans différentes conditions. Ces cellules sont transfectées avec des constructions de différentes parties promotrices du gène SCD1 clonées en amont du gène rapporteur luciférase. Incubées en présence d'insuline, qui active normalement les voies régulant la LDN, la transcription du gène SCD1 est augmentée d'environ 2,5 fois. Cette activation disparaît lorsque les cellules sont incubées avec les agents pharmacologiques LY249002 et Rapamycin qui inhibent respectivement la P13-K et la kinase mTOR. La transfection du dominant négatif p85 (sous-unité de P13-K) mène au même niveau d'inhibition que LY294002, alors que l'essai P13-K in vitro confirme l'inhibition de P13-K par ce dominant négatif. Lorsque les cellules sont incubées avec un agent inhibant la voie MAPK (PD98059), la transcription du gène SCD1 est doublée et ce, indépendamment de l'insuline. L'effet de l'inhibiteur a été confirmé par immunobuvardage dirigé contre les protéines Erk 1/2. En conclusion, l'utilisation de délétions du promoteur SCD1 a permis de cibler deux éléments de réponse à l'insuline (IREs) dépendants d'une voie P13-K/mTOR, ainsi qu'un site de régulation négative de la voie MAPK. Cette étude est une étape importante dans la caractérisation des mécanismes moléculaires liés au développement de plusieurs maladies majeures dans notre société: l'obésité, le diabète de type II et le cancer. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Lipogenèse de novo, Stéaroyl-CoA désaturase, Insuline, Inhibiteurs pharmacologiques, Transcription, Phosphatidylinositol 3-kinase (P13-K), Mammalian target of rapamycin (mTOR), Mitogen-activated protein kinase (MAPK).

The research project, The Enzymatic Synthesis of CoA Derivatives Using a New ATP Regeneration System, describes the multiple lab trials conducted to develop an ATP regeneration system using various concentrations of specific substrates and the two enzymes MatB and PrpE. Reactions were combined using different concentrations of ADP, the addition or removal of ADK (adenylate kinase), and the substitution of either MatB or PrpE. Further reactions were combined using trans-crotonyl, trifluoroacetic acid, butyric acid, acetic acid, and creatine phosphokinase. This report also includes the methods used and analysis of the different chromatographs of each sample tested. / text

Thesis (Ph.D.)--The University of Texas Southwestern Medical Center at Dallas, 2006. / Embargoed. Vita. Bibliography.

A homeostase intracelular do colesterol é mantida, em células de mamíferos, por meio de uma série de mecanismos, porém pode haver uma ruptura deste balanço intracelular podendo levar ao desenvolvimento de doenças, como a lesão aterosclerótica que hoje em dia está acometendo grande parte da população mundial. A enzima HMG-CoA redutase, que possui cisteínas reativas em sua estrutura, pode ser alvo de estudos sobre o bloqueio do processo aterosclerótico, por meio de prostaglandinas da família das ciclopentenônicas (CP-PGs), particularmente a prostaglandina A2 (PGA2), que desviam completamente a síntese de novo de colesterol no sentido da síntese de fosfolípides em macrófagos/foam cells e outras preparações de macrófagos inflamatórios. Dessa forma o objetivo deste estudo foi investigar o efeito da PGA2 sobre a HMG-CoA redutase, em tipos celulares relacionados ao processo aterogênico, demonstrando seu mecanismo de ação por ligação covalente a cisteínas reativas desta enzima. Utilizaram-se macrófagos U937 que foram incubados ou não com LDL oxidada, tornando-se-os foam cells. As células foram tratadas com dose não tóxica (1 μM) de PGA2 ou com mistura controle por um período de 24 h. Outros grupos de macrófagos U937 e foam cells foram tratados com PGA2 biotinilada (com ou sem ditiotreitol, DTT 10 mM) na presença ou ausência de PGA2 (10 μM e 20 μM), por 2 h e 6 h. Foi feito também um estudo da atividade da HMG-CoA, através de kit específico e curvas de crescimentos celular em resposta PGA2. Nossos resultados mostraram aumento na imunodetecção de HMG-CoA redutase em macrófagos, quando tratados com LDL oxidada e tratados com PGA2 biotinilada por 2 h e 6 h, sendo que o tratamento com DTT produziu uma dramática redução na incorporação da mesma à enzima. A PGA2 foi capaz de reduzir a atividade da HMG-CoA em até 86,4% nas doses utilizadas. Macrófagos e foam cells tratados com PGA2 (1 μM) tiveram um aumento na proliferação celular quando comparados com aqueles que não receberam tratamento ou que foram tratadas com doses mais altas da PGA2. Nossos achados contribuem para esclarecer o mecanismo pelo qual ocorre a ação da PGA2 na reversão do processo aterosclerótico via inibição da enzima HMG-CoA redutase via ligação covalente nas cisteínas reativas desta enzima. / The intracellular cholesterol homeostasis is maintained in mammalian cells by a variety of mechanisms, although there may be intracellular breakdown of this balance that can lead to the development of diseases such as atherosclerotic lesions that are nowadays mostly affecting the population worldwide. The enzyme HMG-CoA reductase, which has critical reactive cysteines in its structure, may be target anti-atherosclerotic treatments , once cyclopentenonic prostaglandins (CP-PGs), particularly prostaglandin A2 (PGA2), which completely diverts de novo synthesis of cholesterol toward the direction of phospholipid synthesis in macrophages/foam cells in macrophage/foam cells and other inflammatory macrophage preparations. Thus the objective of this study was to investigate the effect of PGA2 on the HMG-CoA reductase in cell types related to the atherogenic process, demonstrating its mechanism of action by covalent binding to reactive cysteines of this enzyme. Macrophages U937 were used were incubated or not with LDL oxidized, making-up the foam cells. Cells were treated with non-toxic dose of PGA2 (1 μM) or with a mixture control for a period of 24 h. Other groups and U937 macrophage foam cells were treated with biotinylated PGA2 (with or without dithiothreitol, DTT, 10 mM) in the presence or absence of PGA2 (10 mM and 20 mM) for 2 h and 6 h. Was also made a study of the activity of HMG-CoA through kit specific and cell growth curves in response to PGA2. Our results showed the increase in immunodetection of HMG-CoA reductase into macrophages, when treated with LDL oxidized and treated with biotinylated PGA2 for 2 h and 6 h, and the DTT treatment produced a dramatic reduction the incorporation of the same enzyme. The PGA2 was able to reduce the activity of HMG-CoA to 86.4% at the doses used. Macrophages and foam cells treated with PGA2 (1 mM) had an increase in cell proliferation compared with those receiving no treatment or were treated with higher doses of PGA2. Our findings help to clarify the mechanism by which the action occurs in the PGA2 reversal of atherosclerosis by inhibiting the enzyme HMG-CoA reductase by way of covalent bond in the reactive cysteines of this enzyme.

A homeostase intracelular do colesterol é mantida, em células de mamíferos, por meio de uma série de mecanismos, porém pode haver uma ruptura deste balanço intracelular podendo levar ao desenvolvimento de doenças, como a lesão aterosclerótica que hoje em dia está acometendo grande parte da população mundial. A enzima HMG-CoA redutase, que possui cisteínas reativas em sua estrutura, pode ser alvo de estudos sobre o bloqueio do processo aterosclerótico, por meio de prostaglandinas da família das ciclopentenônicas (CP-PGs), particularmente a prostaglandina A2 (PGA2), que desviam completamente a síntese de novo de colesterol no sentido da síntese de fosfolípides em macrófagos/foam cells e outras preparações de macrófagos inflamatórios. Dessa forma o objetivo deste estudo foi investigar o efeito da PGA2 sobre a HMG-CoA redutase, em tipos celulares relacionados ao processo aterogênico, demonstrando seu mecanismo de ação por ligação covalente a cisteínas reativas desta enzima. Utilizaram-se macrófagos U937 que foram incubados ou não com LDL oxidada, tornando-se-os foam cells. As células foram tratadas com dose não tóxica (1 μM) de PGA2 ou com mistura controle por um período de 24 h. Outros grupos de macrófagos U937 e foam cells foram tratados com PGA2 biotinilada (com ou sem ditiotreitol, DTT 10 mM) na presença ou ausência de PGA2 (10 μM e 20 μM), por 2 h e 6 h. Foi feito também um estudo da atividade da HMG-CoA, através de kit específico e curvas de crescimentos celular em resposta PGA2. Nossos resultados mostraram aumento na imunodetecção de HMG-CoA redutase em macrófagos, quando tratados com LDL oxidada e tratados com PGA2 biotinilada por 2 h e 6 h, sendo que o tratamento com DTT produziu uma dramática redução na incorporação da mesma à enzima. A PGA2 foi capaz de reduzir a atividade da HMG-CoA em até 86,4% nas doses utilizadas. Macrófagos e foam cells tratados com PGA2 (1 μM) tiveram um aumento na proliferação celular quando comparados com aqueles que não receberam tratamento ou que foram tratadas com doses mais altas da PGA2. Nossos achados contribuem para esclarecer o mecanismo pelo qual ocorre a ação da PGA2 na reversão do processo aterosclerótico via inibição da enzima HMG-CoA redutase via ligação covalente nas cisteínas reativas desta enzima. / The intracellular cholesterol homeostasis is maintained in mammalian cells by a variety of mechanisms, although there may be intracellular breakdown of this balance that can lead to the development of diseases such as atherosclerotic lesions that are nowadays mostly affecting the population worldwide. The enzyme HMG-CoA reductase, which has critical reactive cysteines in its structure, may be target anti-atherosclerotic treatments , once cyclopentenonic prostaglandins (CP-PGs), particularly prostaglandin A2 (PGA2), which completely diverts de novo synthesis of cholesterol toward the direction of phospholipid synthesis in macrophages/foam cells in macrophage/foam cells and other inflammatory macrophage preparations. Thus the objective of this study was to investigate the effect of PGA2 on the HMG-CoA reductase in cell types related to the atherogenic process, demonstrating its mechanism of action by covalent binding to reactive cysteines of this enzyme. Macrophages U937 were used were incubated or not with LDL oxidized, making-up the foam cells. Cells were treated with non-toxic dose of PGA2 (1 μM) or with a mixture control for a period of 24 h. Other groups and U937 macrophage foam cells were treated with biotinylated PGA2 (with or without dithiothreitol, DTT, 10 mM) in the presence or absence of PGA2 (10 mM and 20 mM) for 2 h and 6 h. Was also made a study of the activity of HMG-CoA through kit specific and cell growth curves in response to PGA2. Our results showed the increase in immunodetection of HMG-CoA reductase into macrophages, when treated with LDL oxidized and treated with biotinylated PGA2 for 2 h and 6 h, and the DTT treatment produced a dramatic reduction the incorporation of the same enzyme. The PGA2 was able to reduce the activity of HMG-CoA to 86.4% at the doses used. Macrophages and foam cells treated with PGA2 (1 mM) had an increase in cell proliferation compared with those receiving no treatment or were treated with higher doses of PGA2. Our findings help to clarify the mechanism by which the action occurs in the PGA2 reversal of atherosclerosis by inhibiting the enzyme HMG-CoA reductase by way of covalent bond in the reactive cysteines of this enzyme.

Loss-of-function genetic screens are a powerful approach to identify the genes involved in biological processes. For nearly a century, forward genetic screens in model organisms have provided enormous insight into many cellular processes. However, the difficulty in generating and recovering bi-allelic mutations in diploid cells severely hindered the performance of forward genetic screens in mammalian cells. The development of a retroviral gene-trap vector to mutagenise the human near-haploid KBM7 cell line transformed forward genetic screens in human cells. The re-purposing of the microbial CRISPR/Cas9 system now offers an effective method to generate gene knockouts in diploid cells. Here, I performed a head-to-head comparison of retroviral gene-trap mutagenesis screens and genome-wide CRISPR knockout screens in KBM7 cells. The two screening approaches were equally effective at identifying genes required for the endoplasmic reticulum (ER)-associated degradation of MHC class I molecules. The ER-resident enzyme HMG-CoA reductase (HMGCR) catalyses the rate-limiting step in the cholesterol biosynthesis pathway and is targeted therapeutically by statins. To maintain cholesterol homeostasis, the expression of HMGCR is tightly regulated by sterols transcriptionally and post-translationally. Sterols induce the association of HMGCR with Insig proteins, which recruit E3 ubiquitin ligase complexes to mediate degradation of HMGCR by the ubiquitin proteasome system. However, the identity of the E3 ligase(s) responsible for HMGCR ubiquitination is controversial. Here, I use a series of genome-wide CRISPR knockout screens using a fluorescently-tagged HMGCR exogenous reporter and an endogenous HMGCR knock-in as an unbiased approach to identify the E3 ligases and any additional components required for HMGCR degradation. The CRISPR screens identified a role for the poorly characterised ERAD E3 ligase RNF145. I found RNF145 to be functionally redundant with gp78, an E3 ligase previously implicated in HMGCR degradation, and the loss of both E3 ligases was required to significantly inhibit the sterol-induced degradation and ubiquitination of HMGCR. A focused E3 ligase CRISPR screen revealed that the combined loss of gp78, RNF145 and Hrd1 was required to completely block the sterol-induced degradation of HMGCR. I present a model to account for this apparent complexity.

A homeostase intracelular do colesterol é mantida, em células de mamíferos, por meio de uma série de mecanismos, porém pode haver uma ruptura deste balanço intracelular podendo levar ao desenvolvimento de doenças, como a lesão aterosclerótica que hoje em dia está acometendo grande parte da população mundial. A enzima HMG-CoA redutase, que possui cisteínas reativas em sua estrutura, pode ser alvo de estudos sobre o bloqueio do processo aterosclerótico, por meio de prostaglandinas da família das ciclopentenônicas (CP-PGs), particularmente a prostaglandina A2 (PGA2), que desviam completamente a síntese de novo de colesterol no sentido da síntese de fosfolípides em macrófagos/foam cells e outras preparações de macrófagos inflamatórios. Dessa forma o objetivo deste estudo foi investigar o efeito da PGA2 sobre a HMG-CoA redutase, em tipos celulares relacionados ao processo aterogênico, demonstrando seu mecanismo de ação por ligação covalente a cisteínas reativas desta enzima. Utilizaram-se macrófagos U937 que foram incubados ou não com LDL oxidada, tornando-se-os foam cells. As células foram tratadas com dose não tóxica (1 μM) de PGA2 ou com mistura controle por um período de 24 h. Outros grupos de macrófagos U937 e foam cells foram tratados com PGA2 biotinilada (com ou sem ditiotreitol, DTT 10 mM) na presença ou ausência de PGA2 (10 μM e 20 μM), por 2 h e 6 h. Foi feito também um estudo da atividade da HMG-CoA, através de kit específico e curvas de crescimentos celular em resposta PGA2. Nossos resultados mostraram aumento na imunodetecção de HMG-CoA redutase em macrófagos, quando tratados com LDL oxidada e tratados com PGA2 biotinilada por 2 h e 6 h, sendo que o tratamento com DTT produziu uma dramática redução na incorporação da mesma à enzima. A PGA2 foi capaz de reduzir a atividade da HMG-CoA em até 86,4% nas doses utilizadas. Macrófagos e foam cells tratados com PGA2 (1 μM) tiveram um aumento na proliferação celular quando comparados com aqueles que não receberam tratamento ou que foram tratadas com doses mais altas da PGA2. Nossos achados contribuem para esclarecer o mecanismo pelo qual ocorre a ação da PGA2 na reversão do processo aterosclerótico via inibição da enzima HMG-CoA redutase via ligação covalente nas cisteínas reativas desta enzima. / The intracellular cholesterol homeostasis is maintained in mammalian cells by a variety of mechanisms, although there may be intracellular breakdown of this balance that can lead to the development of diseases such as atherosclerotic lesions that are nowadays mostly affecting the population worldwide. The enzyme HMG-CoA reductase, which has critical reactive cysteines in its structure, may be target anti-atherosclerotic treatments , once cyclopentenonic prostaglandins (CP-PGs), particularly prostaglandin A2 (PGA2), which completely diverts de novo synthesis of cholesterol toward the direction of phospholipid synthesis in macrophages/foam cells in macrophage/foam cells and other inflammatory macrophage preparations. Thus the objective of this study was to investigate the effect of PGA2 on the HMG-CoA reductase in cell types related to the atherogenic process, demonstrating its mechanism of action by covalent binding to reactive cysteines of this enzyme. Macrophages U937 were used were incubated or not with LDL oxidized, making-up the foam cells. Cells were treated with non-toxic dose of PGA2 (1 μM) or with a mixture control for a period of 24 h. Other groups and U937 macrophage foam cells were treated with biotinylated PGA2 (with or without dithiothreitol, DTT, 10 mM) in the presence or absence of PGA2 (10 mM and 20 mM) for 2 h and 6 h. Was also made a study of the activity of HMG-CoA through kit specific and cell growth curves in response to PGA2. Our results showed the increase in immunodetection of HMG-CoA reductase into macrophages, when treated with LDL oxidized and treated with biotinylated PGA2 for 2 h and 6 h, and the DTT treatment produced a dramatic reduction the incorporation of the same enzyme. The PGA2 was able to reduce the activity of HMG-CoA to 86.4% at the doses used. Macrophages and foam cells treated with PGA2 (1 mM) had an increase in cell proliferation compared with those receiving no treatment or were treated with higher doses of PGA2. Our findings help to clarify the mechanism by which the action occurs in the PGA2 reversal of atherosclerosis by inhibiting the enzyme HMG-CoA reductase by way of covalent bond in the reactive cysteines of this enzyme.

Class of 2012 Abstract / Objectives: To evaluate the literature with respect to drug-drug interactions of the hydroxymethylglutaryl CoA reductase inhibitors atorvastatin, fluvastatin, lovastatin, pitavastitin, pravastatin, simvastatin, and rosuvastatin with warfarin. Methods: This descriptive retrospective study identified articles reporting on each drug-drug interaction from the online databases PubMed (1970 – February 2012) and the drug compendia Micromedex and Facts & Comparisons. The studies included in this investigation were primary literature reports, written in English with human subjects. All studies included were evaluated using the van Roon 5-point quality of evidence scale developed in the Netherlands to assess drug-drug interactions. This scale rates the study type from lowest to highest quality, from zero to four. Case-reports were evaluated using the Drug Interaction Probability Scale (DIPS). The DIPS tool uses 10 questions to evaluate the probability that an adverse event is caused by a drug-drug interaction. Results: Twenty studies met the inclusion criteria. One study involved atorvastatin, four for fluvastatin, three for lovastatin, 2 for pitavastatin, 1 for pravastatin, 5 for rosuvastatin, and 6 for simvastatin. The mean van Roon quality of evidence score was 2.1+/- 0.74, the mean score for atorvastatin, pitavastatin, and pravastatin was 3, with the mean score of fluvastatin, lovastatin, rosuvastatin, and simvastatin was 2. 70% of the literature reviewed were case-reports or letters. Conclusions: The studies and reports supporting HMG-CoA reductase inhibitors and warfarin drug-drug interactions are most commonly case-reports and are of low quality and quantity.

The immunologic relatedness of various cofactor-binding sites of enzymes requiring different nucleotide cofactors was examined. Chicken antibodies specific for NADPH- or CoA-binding domains were raised using an NADPH- or CoA-requiring enzyme as an immunogen. Antibodies specific for either NADPH- or CoA-binding domains were isolated by immunoaffinity chromatography of the respective antisera using unrelated NADPH- or CoA-requiring enzymes as affinity ligands. The reactivities of the NADPH- and CoA-binding-site-specific antibodies with a variety of enzymes that required different cofactors was shown on Western blots of SDS-PAGE of the enzymes. Variable cross-reactivities were observed among all nucleotide-cofactor requiring enzymes with each specific cofactor-domain-antibody population. Numerous proteins not physiologically associated with nucleotide cofactors, including acyl carrier protein, were completely unreactive. Proteins that bound phosphoryl compounds either as substrates or cofactors showed varying degrees of reactivity with each population of specific antibodies. These included aldolase, ribulose-1,5-bisphosphate carboxylase/oxygenase, ribonuclease A, carbonic anhydrase and triosephosphate isomerase. The immunologic cross-reactivity suggested that these proteins share a common structural feature, probably a primary structure epitope, since the proteins had been subjected to denaturing polyacrylamide gel electrophoresis. A candidate for this common structural feature is a glycine-rich sequence comprising a phosphate binding loop.

No description available.