Universiẗat, Diss., 2000--Dortmund. / Dateiformat: PDF.

Arsenic is the most pervasive environmental toxic substance. As a consequence of its ubiquity, nearly every organism has genes for resistance to inorganic arsenic. In one project I examined the role of glutaredoxin 2 (Grx2) in reduction of arsenate to arsenite. I demonstrated that Grx2 has both glutaredoxin thiol transfer activity and glutathione S-transferase (GST) activity. In a second project investigated arsenic resistance in a microbiome organism. I discovered that the human gut microflora B. vulgatus has eight continuous genes in its genome and these genes form an arsenical-inducible transcriptional unit. In two other projects I investigated the properties of two As(III) S-adenosylmethionine (SAM) methyltransferase (ArsM in microbes and AS3MT in animals). In this project we demonstrate that most fungal species have ArsM orthologs with only three conserved cysteine residues, and AfArsM from Aspergillus fumigatus methylates only MAs(III) and not As(III). For human, arsenic methylation process is thought to be protective from acute high-level arsenic exposure. However, with long term low-level exposure, hAS3MT is thought to produce intracellular methylarsenite (MAs(III)) and dimethylarsenite (DMAs(III)), which are considerably more toxic than inorganic As(III) and may contribute to arsenic-related diseases. Several single nucleotide polymorphisms (SNPs) in putative regulatory elements of the hAS3MT gene have been shown to be protective. In contrast, three previously identified exonic SNPs (R173W, M287T and T306I) may be deleterious. I identified five additional intragenic variants in hAS3MT (H51R, C61W, I136T, W203C and R251H). I purified the eight polymorphic hAS3MT proteins and characterized their enzymatic properties. Each enzyme had low methylation activity through decreased affinity for substrate, lower overall rates of catalysis and/or lower stability. I propose that amino acid substitutions in hAS3MT with decreased catalytic activity lead to detrimental responses to environmental arsenic and may increase the risk of arsenic-related diseases.

Protein arginine methylation is a posttranslational modification catalyzed by the family of proteins known as the protein arginine methyltransferases (PRMTs). Thousands of methylated arginines have been found in mammalian cells. Many targets of arginine regulation are involved in important cellular processes like transcription, RNA transport and processing, translation, cellular signaling, and DNA repair. Since PRMT dysregulation has been linked to a variety of disease states, understanding how the activity of the PRMTs is regulated is of paramount importance. PRMT1 is the predominant PRMT, responsible for about 85% of all arginine methylation in cells, but very little is known about how PRMT1 is regulated. Although a few methods to regulate PRMT1 activity have been reported, the details of interaction and regulatory mechanisms remain largely unknown. To better understand how PRMT1 is able to bind its substrates and how PRMT1 activity is regulated, we followed a mechanistic and structural biology approach to better understand how PRMT1 interacts with its substrates and protein regulators. In this study the regulation of Hmt1 methyltransferase activity by the Air1 and Air2 proteins was analyzed and only one was determined to affect Hmt1 activity. The posttranslational phosphorylation of Hmt1 had also been reported to affect Hmt1 activity in vivo and our preliminary studies suggest that additional factors may help influence the regulatory effect of phosphorylation. Lastly, we report a new method of PRMT regulation through the reversible oxidation of key PRMT1 cysteine residues. We are also able to show that this regulation occurs in cells and affects several PRMT isoforms.

No description available.

No description available.

La thèse décrit, dans le premier chapitre, les effets opposés sur la régulation de l'expression du gène du facteur tissulaire (TF) par la voie PI3K/Akt et la voie Erk1/2 in vitro. TF est une molécule clé pour initier la coagulation du sang. Son rôle est maintenant connu au développement embryonnaire, le maintien de l'intégrité vasculaire et la réparation tissulaire. De fait que divers cancers expriment des niveaux aberrantes du TF qui sont corrélés avec le pronostic, TF pourrait réellement favorise la croissance tumorale, l'angiogenèse et la métastase. En utilisant une lignée cellulaire épithéliale de cancer du sein MDA-MB-231, nous avons quantifié l'expression du gène du TF par le test luminescent, qPCR, western blot, et d'activité TF associée aux cellules in vitro. Nous avons constaté que 1) PI3K/Akt est la principale voie qui active l'expression des gènes TF. 2) L'activité Erk1 / 2 inhibe l'expression du gène TF; 3) le blocage de la voie Erk1/2par PD98059 induit une expression aberrante du gène du TF via sur-activation du récepteur du EGF; 4) cette sur-expression du TF peut être neutralisé par le blocage de l'EGFR et de la voie PI3K/Akt; 5) cette sur-expression induite par l'inhibition de Erk1/2est une caractéristique commune pour les lignées de cellules épithéliales cancéreuses testés, comme SKOV-3-3 et OVCAR ; 6) la forme soluble du TF suite à l'épissage alternatif représente une faible proportion de l'ARNm de TF totale et 7) Le niveau d’expression duTF des cellules MDA-MB-231 est corrélée à une activité procoagulante cellulaire et à l'invasivité des cellules in vitro. Cette étude a révélé une boucle de régulation négative de l’Erk1/2 vis-à-vis du l’activité du EGFR, ce qui suggère un effet indésirable des agents thérapeutiques ciblant l’Erk dans la clinique.La thèse décrit, dans le deuxième chapitre, les éléments de preuve de la fonction angiogénique de Hypb, une H3K36 méthyltransférase avec Hypb-/ - knockout souris.Ces souris ont montré une létalité embryonnaire à E10.5-E11.5 et de graves anomalies vasculaires dans le sac vitellin d'embryons et le placenta. Les expériences avec des cellules endothéliales HMEC-1 in vitro utilisant l’anti-Hypb siARN ont démontré défaut de migration et d’invasion cellulaire. En outre, les cellules traitées ont perdu la capacité de former des vaisseaux. Ces données sont bien cohérente avec l'analyse histologique de embryons Hypb-/- de souris dont le réseau complexe de ramification vaisseaux embryonnaires et la circulation sanguine étaient absentes. L'analyse génétique sur sac vitellin avec microarray ont suggéré une association entre le défaut de l'angiogenèse dans Hypb-/ - souris et la déréglementation de la sécrétion des protéines Angptl3 etCyr61, qui pourraient se lier à avß3. Il a également souligné le rôle de l'angiogénine,Angptl3 et Gja4 dans ce défaut, parce que lien de ces gènes à l'angiogenèse étaient démontrés. Mécanismes plausibles sont explorés. La régulation épigénétique de l'angiogenèse est une question importante parce qu'elle contrôle la régulation spatiale et temporelle de l'expression de milliers de gènes. Notre étude suggère clairement un rôle clé de Hypb et H3K36 méthylation et Hypb mécanismes liés aux processus de vascularisation chez les mammifères (vasculogenèse et l'angiogenèse). L'angiogenèse est importante pour la recherche fondamentale et application médicale dans les maladies cardio-vasculaires et de la thérapie anti-cancereuse. Nous espérons que de nouvelles stratégies thérapeutiques ciblant les voies épigénétiques permettra d'offrir de nouvelles voies thérapeutiques. / The thesis described, in the first chapter, an opposite regulatory effects of PI3K/Akt pathway and Erk1/2 pathway on tissue factor(TF) gene expression in vitro. TF is a keymolecule required to initiate blood coagulation, and is now accepted to be essential forembryo development, maintenance of vascular integrity and tissue repair. Since a variety of cancers show aberrant prognostics-correlated high levels of TF expression, it is believed that TF promotes tumor growth, angiogenesis and metastasis. Using an epithelial breast cancer cell line MDA-MB-231, we quantified TF gene expression by luminescent test, qPCR, western blot, and cell-associated TF activity in vitro. We found that 1) PI3K/Akt is the major pathway that activates TF gene expression. 2) Erk1/2activity inhibits TF gene expression; 3) blocking Erk1/2 by PD98059 aberrant lyupregulates TF gene expression via enhancing EGFR activity; 4) this enhanced TF expression can be neutralized by blocking EGFR and PI3K/Akt pathway activation; 5) TF upregulation induced by Erk inhibition is a common feature in the tested epithelial cancer cell lines SKOV-3 and OVCAR-3; 6) Soluble form of TF due to alternative splicing represents a small proportion of total TF mRNA and 7) The level of TF gene expression in MDA-MB-231 cells is correlated to cell procoagulant activity and cell invasiveness in vitro. This study revealed a negative feedback loop of Erk-mediated EGFR inhibitions,suggesting an undesirable effect of the agents targeting Erk in clinic.The thesis described, in the second chapter, the evidence of angiogenic function of Hypb,a H3K36 methyltransferase with Hypb-/- knockout mice. These mice demonstrated embryonic lethality at E10.5-E11.5 and severe vascular defects in the Hypb−/− embryo,yolk sac, and placenta. The experiments with endothelial cells HMEC-1 in vitro using anti-Hypb siRNA demonstrated defective cell migration and invasion. Furthermore, the treated cells lost the capacity of vessel formation. These data were well coherent with histological analysis of Hypb-/- mice embryos that lacked the intricate network of branching embryonic vessels and showed disrupted blood flow. The genetic microarray analysis on yolk sac suggested an association between the defect of angiogenesis inHypb-/- mice and deregulated Angptl3 and Cyr61 protein release, since both of which could bind to αvβ3. It also suggested the roles of angiogenin, Angptl3 and Gja4 in this defect because these angiogenesis-related genes were downregulated. Plausible mechanisms are discussed. The epigenetic regulation of angiogenesis is an important issue because it controls the spatial and temporal regulation of expression of thousands of genes. Our study clearly suggests a key role of Hypb and H3K36 methylation and Hypb-related mechanisms in the processes of mammalian vascularization (vasculogenesis and angiogenesis). Angiogenesis is important for both basic researchand medical application in cardiovascular diseases and cancer therapeutics. We believe that novel therapeutic strategies targeting epigenetic pathways will achieve real benefit in medical practices.

Many higher plants can synthesize methyl chloride gas via a common metabolic route, also known as the biological chloride methylation. The reaction is catalyzed by an S-adenosyl-L- methionine (AdoMet) dependent halide/thiol methyltransferase (H/TMT). It is speculated that plants use chloride methylation to remove excess chloride via volatilization and hence maintain homeostatic levels of cytoplasmic chloride ion, suggesting a role of H/TMT in salt tolerance. In this project, the effect of engineering a Brassica oleracea thiol methyltransferase (BoTMT) into tomato was studied to determine the physiological relevance of this enzyme in conferring salt tolerance. Transgenic tomato plants acquired the ability to release methyl chloride in response to NaCl treatment, but exhibited no greater tolerance to NaCl, based on several morphological and physiological measurements, as compared to the wild-type plants. The results indicate that AdoMet dependent chloride methylation is unlikely to contribute to an increase in salt tolerance in higher plants.

Many higher plants can synthesize methyl chloride gas via a common metabolic route, also known as the biological chloride methylation. The reaction is catalyzed by an S-adenosyl-L- methionine (AdoMet) dependent halide/thiol methyltransferase (H/TMT). It is speculated that plants use chloride methylation to remove excess chloride via volatilization and hence maintain homeostatic levels of cytoplasmic chloride ion, suggesting a role of H/TMT in salt tolerance. In this project, the effect of engineering a Brassica oleracea thiol methyltransferase (BoTMT) into tomato was studied to determine the physiological relevance of this enzyme in conferring salt tolerance. Transgenic tomato plants acquired the ability to release methyl chloride in response to NaCl treatment, but exhibited no greater tolerance to NaCl, based on several morphological and physiological measurements, as compared to the wild-type plants. The results indicate that AdoMet dependent chloride methylation is unlikely to contribute to an increase in salt tolerance in higher plants.

Thesis (Ph. D.)--University of Canterbury, 2006. / Typescript (photocopy). "20-July-2006." Includes bibliographical references. Also available via the World Wide Web.

La traduction des ARNm est un processus très complexe qui en plus des nombreux facteurs impliqués, nécessite également des étapes de maturation des protéines et ARN pour la production fidèle des protéines. Parmi ces évènements, des modifications post-transcriptionnelles et post-traductionnelles, dont la méthylation est la plus fréquente, sont trouvées dans tous les composants et principalement chez les eucaryotes. Le rôle des méthylations dans la traduction est parfaitement illustré par la protéine Trm112, qui est un activateur essentiel pour la fonction de 4 méthyltransférases (MTase) (Trm9, Trm11, Bud23 et Mtq2) qui modifient des facteurs impliqués dans la synthèse des protéines. Chez la levure, les complexes Trm9-Trm112 et Trm11-Trm112 catalysent la formation de mcm5U34 et m2G10, respectivement sur certains ARNts. Le complexe Bud23-Trm112 modifie l’ARNr 18S pour former la m7G1575 tandis que le complexe Mtq2-Trm112 modifie le facteur de terminaison de classe I eRF1sur la chaine latérale de la glutamine du motif GGQ. Jusqu’à présent, des études structurales et fonctionnelles du réseau d’interaction de la protéine Trm112 se sont uniquement focalisées chez les eucaryotes alors que cette protéine est trouvée dans les 3 domaines du vivant. Dans cette étude, des expériences de co-immunoprécipitations couplées à de la LC-MS/MS ont permis d’étudier le réseau d’interaction de la protéine Trm112 chez l’archée H. volcanii. Celui-ci s’avère être composé de plus de MTase que chez les eucaryotes. Pour la première fois, la structure cristallographique d’un complexe Trm112-MTase d’archée a été déterminée, révélant un mode d’interaction conservé par rapport aux complexes eucaryotes malgré une très faible identité de séquence. De façon très intéressante, un des partenaires de Trm112 chez H. volcanii est orthologue d’une protéine humaine dont nous avons pu démontré qu’elle est une nouveau partenaire de la protéine TRMT112 humaine / Methylation is a widely distributed modification found in a variety of substrates involved in different steps of eukaryotic protein translation. Methylation reactions are catalyzed by enzymes called methyltransferases (MTases) generally using S-adenosyl-L- methionine (SAM or AdoMet) as the methyl donor. The effects of methylation on translation are perfectly illustrated by the Trm112 protein, which is an activating platform, essential for the function of four SAM-dependent MTases (Trm9, Trm11, Bud23 and Mtq2) modifying factors participated in protein synthesis. The Trm9-Trm112 and Trm11-Trm112 complexes methylate some tRNAs to form mcm5U34 and m2G10 respectively. The Bud23-Trm112 complex modifies 18S rRNA to form m7G1715 while the Mtq2-Trm112 complex methylates class I translation termination factor eRF1 at glutamine side chain of GGQ motif. Until now, the study of Trm112 network in eukaryotes has been quite clear structurally and functionally, however, little is known for corresponding proteins in Archaea.My PhD project aims to characterize the Trm112 network in archaea using Haloferax volcanii as a model organism and to decipher the mechanisms of substrate modification by Trm112-MTase complexes. This will help understanding the roles of these enzymes in protein synthesis from an evolutionary point of view.Towards this goal, I have generated several H. volcanii strains (Δtrm112, Δtrm112 Trm112-Flag, …). Co-immunoprecipitation of Trm112-Flag coupled to mass spectrometry allowed me identifying a significant number of methyltransferases (MTases), including putative orthologues of eukaryotic Trm112 partners, as potential interactors. I have next validated these new partners by biochemical approaches (co-purification, enzymatic assays, …) and determined the crystal structure for one Trm112-MTase complex. I have then convincing evidences that H. volcanii Trm12 has more MTase partners than the eukaryotic one. My work opens new routes towards the characterization of the role of Trm112 in archaea but has also led to the identification of a new MTase partner of the eukaryotic Trm112.