Because it can expand the range of a protein’s interactions or modulate its activity, post-translational methylation of arginine residues in proteins must be duly coordinated and ‘decoded’ to ensure appropriate cellular interpretation of this biological cue. This can be achieved through modulation of the enzymatic activity/specificity of the protein arginine methyltransferases (PRMTs) and proper recognition of the methylation ‘mark’ by a subset of proteins containing ‘methyl-sensing’ protein modules known as ‘Tudor’ domains. In order to gain a better understanding of these regulatory mechanisms, we undertook a detailed biochemical characterization of the predominant member of the PRMT family, PRMT1, and of the novel Tudor domain-containing protein 3 (TDRD3). First, we found that PRMT1 function can be modulated by 1) the expression of up to seven PRMT1 isoforms (v1-7), each with a unique N-terminal region that confers distinct substrate specificity, and by 2) differential subcellular localization, as revealed by the presence of a nuclear export sequence unique to PRMT1v2. Second, our findings suggest that TDRD3 is recruited to cytoplasmic stress granules (SGs) in response to environmental stress potentially by engaging in methyl-dependent protein-protein interactions with proteins involved in the control of gene expression. We also found that arginine methylation may serve as a general regulator of overall SG dynamics. Finally, we uncovered that alteration of PRMT1, TDRD3, and global arginine methylation levels in breast cancer cells may be closely associated with disease progression and poor prognosis. Therefore, further studies into the pathophysiological consequences ensuing from misregulation of arginine methylation will likely lead to the development of novel strategies for the prevention and treatment of breast cancer.

Les protéines arginine méthyl transférases ("PRMTs") sont impliquées dans de nombreux processus cellulaires essentiels. La protéine CARMI ("Coactivator-associated arginine methyltransferase 1", appelée aussi "PRMT4") a été initialement identifiée par sa fonction co-activatrice de la transcription impliquantplusieurs récepteurs nucléaires des hormones. CARMI est une enzyme qui catalyse la réaction de méthylation sur les histones via un donneur de méthyl naturel, la S-adénosY-L -méthionine (SAM). De nombreux travaux ont montré que CARMI est surexprimée dans les cancers du sein et de la prostate. L' objectif de ce travail est la compréhension à l'échelle moléculaire du mode d'action de CARMI et l'étude du mécanisme de reconnaissances moléculaires et de transferts d' informations gouvernés par la protéine CARMI. La structure cristallographique obtenue de cette enzyme en présence de cofacteur, la S-AdénosyhHomocystéine ou la Sinefungine a eu un effet stabilisant. Ainsi, notre stratégie a été de créer des molécules hameçons basées sur le motif de la SAM capables d' ancrer un peptide mimant la séquence de l' histone H3, pour ensuite les tester en co-cristallisation avec CARMI. Ainsi, grâce à la diffraction aux rayons X, les interactions mises en jeu dans le complexe CARMlImolécules hameçons/peptide pourront être déterminées. Cette stratégie s'est effectuée en trois étapes : la première étape, décrite dans le chapitre 2, a consisté en la synthèse d'analogues de la SAM obtenus grâce à des modifications réalisées autour de l'atome de soufre. Ces composés nous ont permis d' explorer la « poche du sulfonium ». Puis la seconde étape, décrite dans le chapitre 3, a été la synthèse • d'analogues de bisubstrats nécessaires pour l'exploration de la « poche de l'arginine ». Dans une dernière étape, décrite dans les chapitres 4 et 5, nous avons abordé la synthèse d'adduits SAM-peptide pouf pouvoir étudier le « domaine de fixation du peptide ». Dans le quatrième chapitre, la méthode de choix est la création d'un lien covalent entre une molécule hameçon électrophile etun peptide par chimie de click in-situ : par réaction de cycloaddition de Huisgen; par réaction entre des molécules hameçons électrophiles capables de piéger un peptide cystéine ou un peptide arginine. Ces essais se sont révélés infructueux et une nouvelle stratégie a été employée en utilisant des molécules ancres. Dansle cinquième chapitre des molécules ancres ont donc été préformés pour ensuite être testés en cocristallisation dans CARMl. / Protein aginine methyltransferases (PRMTs) have been implicated in a variety of biological processes. Coactivator-associated arginine methyltransferase 1 (CARM1 , also known as PRMT4) was identified as an enhancer of the transcriptional activation by several nuclear hormone receptors CARM1 is an enzyme which methylates the arginines of histones via a natural methyl donor, the SAdenosyh-Methionine (SAM). Recent studies have shown that CARM-1 is over-expressed in breastumors and in hormone dependent prostate tumors. The goal of this work is to understand at the molecular level the mode of binding of substrate/product arginine-containing peptides, reflectingstates prior and subsequent to methylation and the detailed mechanism of action of this protein. Several crystal structures of the catalytic domain of CARM1 have shown that cofactor-binding, such as S-Adenosyl-L -Homocysteine or sinefungin, produces large conformational changes in the catalytic domain. These crystal structures clearly illustrate that SAM binding is a prerequisite for peptide binding and build up the productive peptide binding site. Our strategy was to design fishhook molecules derivatives of the SAM capable of anchoring a mimic peptide of the histone H3 in order to test the co-crystallization in CARM1. Consequently, thanks to X-Ray structure, interactions involvement in the complexe CARM1/fishhook molecule/peptide could be determined. This strategy was do ne in three steps: the first one, described in the chapter 2, consisted in synthesizing SAManalogues with several modifications around sulfur atom. These compounds permitted to explore the "sulfonium pocket". The second step, described in chapter 3, consisted in synthesizing analogues of bisubstrats to explore "arginine binding pocket". Finally, the last step, described in the chapter 4 and 5, consisted in synthesizing SAM-peptide adducts to study "peptide binding domain". ln the chapter 4, the chosen method is the creation of covalent link between this molecule and a peptide by click chemistry in-situ: by reaction of Huisgen's cycloaddition; by reaction between electrophilic fishhook molecules capable of capturing with a cystein or arginine peptides. Unfortunately, ail of these trials have been unsuccessful. Consequently SAM-peptide adducts were performed to be co-crystallized in CARM1. This part was described in the last chapter.

La dysfonction endothéliale vasculaire, processus majeur initiant l’athérosclérose, est étroitement liée à l’altération de la synthèse et/ou biodisponibilité du monoxyde d’azote (NO), dont l’arginine est le précurseur. Elle apparait aussi dès la phase postprandiale après un repas gras et sucré. Chez des sujets avec des facteurs de risque cardiométabolique, une supplémentation orale en arginine a un effet bénéfique sur des fonctions associées au NO. Aucune donnée ne permet cependant de lier la mise à disposition de l’arginine et la synthèse de NO, en situation normale ou de risque cardiométabolique. De plus, peu d’études ont étudié l’effet d’une supplémentation en arginine, dans un contexte nutritionnel (faible dose et libération lente) chez des sujets avec des facteurs de risque cardiométabolique. Ce travail vise donc à évaluer l’effet d’une supplémentation nutritionnelle en arginine, sur le métabolisme de l’arginine et la fonction endothéliale (FE), chez des sujets sains présentant des facteurs de risque cardiométabolique. Dans une première étude clinique, nous avons ainsi comparé la biodisponibilité de l’arginine ingérée et son utilisation pour la synthèse de NO, selon la présence de facteurs de risque cardiométabolique, et selon qu’elle était consommée sous une forme à libération immédiate (LI) ou sous une forme à libération prolongée (LP), mimant la mise à disposition naturelle de l’arginine alimentaire. Puis, dans une deuxième étude clinique, nous avons étudié l’effet de la supplémentation en arginine LP sur la FE à jeun et sur son altération postprandiale, chez des sujets sains présentant des facteurs de risque cardiométabolique; et si cet effet pourrait varier selon leur argininémie basale. Ce travail de thèse a ainsi mis en évidence une utilisation de l’arginine ingérée plus élevée pour la synthèse de NO chez les sujets avec des facteurs de risque cardiométabolique, et plus élevée avec la forme LP qu’avec la forme LI, en particulier chez ces sujets à risque. La deuxième étude, quant à elle, a révélé que les effets de la supplémentation en arginine-LP variaient selon l’argininémie basale des sujets présentant des facteurs de risque cardiométabolique. Chez les sujets avec une argininémie basale relativement plus faible, l’arginine-LP a atténué la diminution postprandiale de la FE et a conduit à une FE significativement meilleure à la fin de la période postprandiale. / Vascular endothelial dysfunction, the hallmark of early atherosclerosis, results from an impairment of the synthesis and/or bioavailability of nitric oxide (NO), the precursor of which is arginine. Endothelial dysfunction is also known to be induced transiently by a high-fat meal. In subject with cardiometabolic risk factors, oral arginine supplementation has a beneficial effect on NO-related physiological functions. However, no data relates the availability of arginine to the synthesis of NO in normal or cardiometabolic risk condition. In addition, few studies only have investigated the effect of arginine supplementation in a nutritional context (low dose and slow release) in subjects with cardiometabolic risk factors. This work aims to evaluate the effect of a nutritional arginine supplementation, on the arginine metabolism and endothelial function in healthy subjects with cardiometabolic risk factors. In a first clinical study, we have compared the bioavailability of oral arginine and its utilization for NO synthesis, as a function of the presence of cardiometabolic risk factors, and as a function of the form of release (immediate release, IR, as free arginine, or sustained release, SR, which mimics the slow release of dietary arginine). Then, in a second clinical study, we studied the effect of SR-arginine supplementation on fasting endothelial function and its postprandial alteration in healthy subjects with cardiometabolic factors. A further aim was to investigate whether this effect may vary according to the baseline arginine status of subjects. This thesis work has demonstrated a higher utilization of oral arginine for NO synthesis in subjects with cardiometabolic risk factors, and a higher utilization with the SR form, particularly in these subjects at risk. As to the second study, it showed that the SRarginine supplementation effects largely varied with baseline fasting arginine concentration of subjects with cardiometabolic risk factors. In subjects with a relatively lower baseline arginine concentration, SR-arginine attenuated the decrease in postprandial endothelial function and led to a significantly higher endothelial function at the end of the postprandial period.

Les cellules Natural Killer (NK), actrices majeures de la vigilance anti-tumorale, sont modulées par des facteurs nutritionnels et métaboliques. L'inhibition de leur activité favorise le développement tumoral. Un régime alimentaire hypercalorique induisant l'obésité est un facteur de risque de développer un cancer du sein. Au niveau du micro-environnement tumoral mammaire, la biodisponibilité en certaines molécules contrôle non seulement les cellules néoplasiques mais, également les cellules immunes infiltrées. Ainsi, la leptine, sécrétée à forte concentration par les adipocytes mammaires, pourrait favoriser la croissance tumorale et altérer les cellules NK. L'arginine fortement consommée par les cellules tumorales et les cellules suppresseurs dérivées des myéloïdes pourrait faire défaut aux cellules NK. L'objectif de cette thèse est de caractériser les activités fonctionnelles et métaboliques des cellules NK en situation de stress nutritionnel. Dans un premier temps, nous avons exploré, in vivo, l'impact d'un régime hypercalorique sur l'activité des cellules NK et sur le développement tumoral mammaire. Ensuite, nous avons cherché à identifier les potentielles altérations fonctionnelles des cellules NK en mimant, in vitro, les conditions retrouvées au niveau du micro-environnement tumoral telles que la présence de concentration élevée en leptine et la déplétion en arginine. Des souris Balb-c "nude" femelles ont été soumises à un régime hypercalorique (HC) versus une diète normo-calorique (NC) pendant 6 mois. Au bout de 5 mois, des cellules tumorales mammaires (MCF-7 ; groupes NCT et HCT) ou le véhicule (groupes NC et HC) ont été implantés au niveau de la quatrième paire de glandes mammaires. Sous régime HC, le développement tumoral s'accompagne d'une perte de masse grasse, de masse maigre et de poids corporel avec un volume et un poids de tumeur augmentés. Cette diète induit au niveau tumoral une sur-expression des ARNm d'enzymes impliquées dans la glycolyse et une sous-expression des acteurs du cycle de Krebs. Sous régime HC, l'expression de la caspase 3 clivée et des récepteurs des oestrogènes β et de la progestérone est réduite alors que celle du Ki67 est accrue. Les cellules NK des souris HC ont une cytotoxicité diminuée. Bien que la présence de tumeur stimule l'activité lytique des cellules NK, la cytotoxicité de ces cellules reste inférieure dans le groupe HCT comparativement à celle du groupe NCT. La leptine stimule, in vitro, de façon dose-dépendante l'activité métabolique des cellules NK. A fortes concentrations, elle active leur cytotoxicité vis-à-vis des cellules cibles MDA-MB-231. Cet effet passe par une stimulation de l'expression de TRAIL et de l'IFN-γ par les cellules NK. En revanche, vis-à-vis des cellules cibles MCF7, les cellules NK présentent une activité lytique réduite en présence de fortes concentrations de leptine, probablement en lien avec une réduction de l'expression de la perforine. En réponse à une déplétion en arginine dans le milieu de culture, la prolifération et la cytotoxicité des cellules NK sont abaissées. L'altération de la reconnaissance des cellules cibles par les récepteurs NKp46 et NKp30, la moindre transmission du signal activateur par la chaine ζ et la faible production d'IFN-γ peuvent expliquer l'inhibition de la cytotoxicité des cellules NK. Ainsi, un apport énergétique élevé favorise le développement tumoral mammaire notamment eninhibant la cytotoxicité des cellules NK. De plus, la leptine à fortes concentrations stimule ou réduit, in vitro, la cytotoxicité des cellules NK selon la nature des cellules cancéreuses mammaires cibles. Une déplétion en arginine, in vitro, quant à elle, inhibe la prolifération et la cytotoxicité des cellules NK. Ces travaux contribuent à mieux comprendre l'impact du micro-environnement sur la réponse antitumorale des cellules NK. / Natural killer (NK) cells are critical mediators of anti-tumor immunity. A high-calorie diet inducing obesity is associated with breast cancer development. NK cells are modulated by dietary and metabolic factors and a decrease in their lytic activity promotes mammary tumor development. In the breast microenvironment, high concentration of leptin can be secreted by mammary adipocytes and thereby could stimulate tumor growth and control immune cells. Arginine, strongly consumed by tumor and myeloid-derived suppressor cells, could be lacking to NK cells. The aim of this work is to characterize the functional and metabolic activities of NK cells in response to nutritional stress. Initially, we explored in vivo the impact of a high-calorie diet on NK cells activity and mammary tumor development. Then, we identified potential functional alterations in NK cells by mimicking the conditions found in the tumor microenvironment such as the presence of high leptin concentration and arginine depletion. Female Balb-c nude mice were fed a high-caloric diet (HC) versus a standard caloric diet (SC) for 6 months. After five months, mammary tumor cells (MCF-7, SCT, HCT) or MatrigelTM (SC, HC) were implanted into the fourth mammary fat pads. The tumor development in HC diet-fed mice was associated with a decrease in body weight, body fat and lean mass and an increase in volume and weight of tumors. This diet induced tumor over-expression, at the transcriptional level, of enzymes involved in glycolysis and a down-expression of citrate cycle actors. Protein tumor levels of cleaved caspase 3, estrogen β and progesterone receptors were reduced while Ki67 was increased in the HC diet-fed mice. NK cell cytotoxicity of HC diet-fed mice was reduced. Although the presence of tumor stimulated NK cell lytic activity, this later was lower in the HCT group compared to the one of SCT mice. In vitro, leptin stimulated, in dose-dependent manner, the metabolic activity of NK cells. High leptin concentrations enhanced NK cell cytotoxicity against the MDA-MB-231 target cells. This phenomenon involved the increase of expression of TRAIL and IFN-γ in NK cells. However, against the MCF-7 target cells, NK cell lytic activity was reduced in the presence of high concentrations of leptin, probably in link to the decreased perforin expression. NK cell proliferation and cytotoxicity were impaired in response to arginine depletion. This inhibition of NK cell cytotoxicity could be linked to a low target cells recognition by NKp46 and NKp30, a reduced activating signal transmission by ζ chain and a low production of IFN-γ. Thus, high energy intake promotes mammary tumor development in particular by inhibiting NK cell cytotoxicity. In vitro, high leptin concentrations stimulate or reduce NK cell cytotoxicity according to the breast cancer cell targets. Furthermore, arginine depletion inhibits NK cell proliferation and cytotoxicity in vitro. These findings provide insight into the microenvironment impacts on NK cell antitumor response in tumor development.

Si l'intérêt nutritionnel des acides gras polyinsaturés (AGPI) n-3 dans la prise en charge et la prévention des dysfonctions associées au syndrome métabolique, est bien établi. Les mécanismes d'action spécifiques sous-jacents aux effets bénéfiques de cette famille d'acides gras sont encore en cours d'étude. L'objectif de ces travaux était d'explorer le rôle de l'acide alpha-linolénique ALA ou 18 :3 n-3, dans la modulation des voies affectant l'homéostasie de molécules bioactives dérivant du métabolisme secondaire des acides aminés (le monoxyde d'azote -NO- et le glutathion). L'hypothèse sous-jacente est que ces modulations pourraient expliquer, du moins en partie, le rôle des AGPI n-3 dans le maintien des fonctions biologiques contrôlées par ces métabolites (telles que la fonction endothéliale et le statut oxydant) et impliquées de près dans la physiopathologie du syndrome métabolique. Notre intérêt a porté particulièrement sur la voie de régulation génique via le PPARα et sur son implication dans le contrôle des gènes du métabolisme des acides aminés par l'ALA. Nous avons exploré chez la souris de type sauvage et invalidée pour le PPARα, l'effet de l'apport alimentaire d'ALA dans le cadre de régime normo- ou hyper-lipidiques sur les voies du métabolisme secondaire de l'arginine et de la cystéine. En parallèle nous nous sommes focalisés sur les effets de l'ALA au niveau vasculaire en utilisant un modèle de cellules endothéliales bovines en cultures. De ce travail de thèse s'est dégagé que l'ALA module effectivement le métabolisme secondaire de l'arginine et de la cystéine. L'apport d'ALA (à hauteur de 11% et 42% de l'apport énergétique) augmente la production de NO sans affecter l'expression hépatique des enzymes contrôlant l'utilisation de l'arginine (NOS et ARG). L'apport d'ALA (11%) augmente le pool hépatique du glutathion, alors que les plus forts apports d'ALA (42%) modulent l'expression des principales enzymes impliquées dans les voies d'utilisation de la cystéine (γGCL et CDO). Le PPARα ne semble pas être directement impliqué dans les effets observés de l'ALA, néanmoins, l'invalidation du PPARα rend le métabolisme secondaire des acides aminés plus sensible à la nature des acides gras alimentaire. Une meilleure biodisponibilité du NO et du glutathion suite à l'apport alimentaire d'ALA serait bénéfique pour la physiopathologie du syndrome métabolique. Il semble donc intéressant, à l'issus de ce travail, d'élaborer des études nutritionnelles validant ces effets de l'ALA chez l'homme dans une perspective de recommandations nutritionnelles. / *

Arginine decarboxylase is a key enzyme involved in the polyamine pathway of organisms. Pyruvoyl-dependent arginine decarboxylases are expressed in the form of proenzymes that self-cleave to form N-terminal [beta] and C-terminal [alpha] subunits generating an active pyruvoyl group at the [alpha] terminus. We have identified an archaeal homolog of a pyruvoyl-dependent arginine decarboxylase in Chlamydophila pneumoniae that could play a role in the persistence of the organism in the host. The recombinant enzyme showed highest activity at pH 3.4, which is the lowest optimum pH ever reported for a pyruvoyl dependent arginine decarboxylase. The proton-consuming decarboxylation raises intracellular pH, and thereby plays a role in acid-resistance. It could inhibit the pro-inflammatory nitric oxide synthase resulting in asymptomatic infection. A variant protein Thr⁵²Ser at the predicted cleavage site showed less pro-enzyme cleavage and activity compared to the wild-type. The homologs of arginine decarboxylase and flanking arginine-agmatine antiporter were also found in different biovariants of Chlamydia trachomatis. In the invasive L2 strain of C. trachomatis, the presence of a nonsense codon in the gene encoding arginine decarboxylase enzyme prevented the expression of an active enzyme. The variant protein with tryptophan replacing nonsense codon restored arginine decarboxylase activity. The non-invasive D strain of C. trachomatis had an intact arginine decarboxylase gene, but it was recombinantly expressed as a proenzyme that was uncleaved. The arginine-agmatine antiporters from both the strains were active and transported tritiated arginine into their cells. The polyamine pathway of the crenarchaeon Sulfolobus solfataricus uses arginine to make putrescine, but the organism lacks homologs of arginine decarboxylase. However, it has two paralogs of pyruvoyl dependent S-adenosylmethionine decarboxylase − SSO0536 and SSO0585. These enzymes were recombinantly expressed as pro-enzymes that self-cleaved into [beta] and [alpha] subunits. Even with a 47% amino acid sequence identity, the SSO0536 protein exhibited significant arginine decarboxylase activity whereas SSO0585 protein had significant S-adenosylmethionine decarboxylase activity. This is the first report of an S-adenosylmethionine decarboxylase enzyme showing alternative decarboxylase activity. The chimeric protein with the [alpha]-subunit of SSO0585 and [beta]-subunit of SSO0536 had arginine decarboxylase activity, suggesting that the residues responsible for substrate recognition are located in the amino terminus. / text

Reptiles are polyphyletic, and previous studies of renal anatomy and physiology in reptiles have covered a wide diversity of species of different phylogeny and habitat. To date, no study has examined the renal morphology and function of a group of closely related reptiles from different environments, yet this design has a number of advantages. Firstly, phylogenetic effects are reduced while adaptive specialisations in renal function or structure can be elucidated, and secondly, the variation in renal form and function between closely related species may be quantified in an effort to appreciate better the variation between more distantly related species. In this thesis, kidney morphology and renal function were studied in three Western Australian agamid lizards inhabiting environments differing in the availability of water. These key species were Pogona minor, Ctenophorus nuchalis and Ctenophorus salinarum. The renal anatomy of the three key lizards was characterised by determining glomerular diameter, volume density, surface area and number in each. Allometric relationships between kidney, colon and body mass were investigated in these and an additional 11 species of agamid lizard. Patterns of response to osmotic challenge were recorded by measuring renal variables such as urine flow rate, glomerular filtration rate and fractional reabsorption of filtrate among the three key species, and concurrent measurements of circulating arginine vasotocin in P. minor and C. nuchalis allowed the response of this hormone to homeostatic imbalance in these species to be gauged. The gross morphology and the glomerular characteristics of the kidneys was remarkably similar between species. Glomerular number and other characters varied as a function of body size rather than species, contrasting with reports in the literature suggesting that a given species has a particular number of glomeruli. ... Thus, kidney morphology is constrained among species and the response of each species to osmotic perturbation is similar. However, the mechanisms underlying antidiuresis and the hormonal control of this process differ subtly between species, and there is some evidence to suggest that P. minor is more adapted to a mesic environment than the other two lizards examined in this study. The hypothesis that renal form and function reflect the environment in which a lizard lives therefore receives partial support, although the reptilian bauplan is able to mitigate many of the forces that could potentially lead to renal specialisation.

Most of the chemical reactions of living cells are catalyzed by protein enzymes. These enzymes are very efficient and display a high degree of specificity with respect to the reaction catalyzed. Cellular activities depend critically on the precise three-dimensional structure and function of thousands of enzymes. Many enzymes require binding of metal ions or small organic molecules for their function. The organic molecules that are indispensible components of catalysis by proteins are called coenzymes. Pyridoxal 5ʹ-phosphate (PLP) is a versatile coenzyme found in all living cells. PLP-dependent enzymes play a key role in the function of most of the enzymes catalyzing reactions in the metabolic pathways of amino acid synthesis and degradation. The enzyme pyridoxal kinase serves to make available the co-enzyme PLP to apo-PLP dependent enzymes. Because of their key role in cellular function and their medical importance, the structure and function of PLP-dependent enzymes have been extensively investigated. In the past decade, detailed investigations on the structure and function of several PLP-dependent enzymes have been carried out in our laboratory. The enzymes studied are B. subtilis serinehydroxymethyl transferase (SHMT), S. typhimurium acetylornithine aminotransferase (AcOAT), S. typhimurium and E. coli diaminopropionate ammonia lyase (DAPAL), S. typhimurium D-serine dehydratase (DSD), S. typhimurium D-cysteine desulfhydrase (DCyD) and S. typhimurium arginine decarboxylase (ArgD). The extensive studies conducted on PLP-dependent enzymes in our laboratory during the past decade has not only resulted in deeper understanding of their structure and function but also raised several new questions regarding substrate recognition, reaction specificity, role of active site residues in the catalytic reaction, mechanism of catalysis and potential applications of these enzymes. This thesis is an attempt to answer some of these questions. The thesis also presents the structure and function of a new protein, Salmonella typhimurium pyridoxal kinase, the enzyme that provides PLP for PLP-dependent enzymes. Single crystal X-ray diffraction technique is the most powerful tool currently available for the elucidation of the three-dimensional structures of proteins and other biological macromolecules and for revealing the relationship between their structure and function. X-ray diffraction studies have provided in depth understanding of the topology of secondary structural elements in the three-dimensional structures of proteins, the hierarchical organization of protein domains, structural basis for the substrate specificity of enzymes, intricate details of mechanisms of enzyme catalyzed reactions, allosteric regulation of enzyme activity, mechanisms of feed-back inhibition, structural basis of protein stability, symmetry of oligomeric proteins and their possible biological implications and a myriad of other biochemical and biophysical properties of proteins. The work reported in this thesis is primarily based on X-ray diffraction studies. X-ray crystal structure investigations are complemented by spectral and biochemical studies on the catalyzed reactions. The thesis begins with an introduction to PLP-dependent enzymes and presentation of a brief summary of the earlier work carried out in our laboratory on PLP-dependent enzymes (Chapter 1). A brief description of earlier functional classification of PLP-dependent enzymes and the more recent classification of these enzymes into the four groups based on their three-dimensional structure is provided. Although enzymes belonging to these four structural classes have evolved from independent evolutionary lineages, they share some common features near their active sites and in the mode of PLP binding. Earlier work carried out elsewhere on pyridoxal kinase and its key role in maintaining PLP at a low concentration in the cytosol is presented. Different mechanisms that have been proposed for the transfer of PLP from pyridoxal kinase to other apo PLP-dependent enzymes are briefly described. The experimental procedures and computational methods used during the course of these investigations to obtain the results reported in chapters 3-6 are presented in Chapter 2. Most of these methods are applicable to the isolation of plasmids, cloning, over expression, protein purification, mutant construction, crystallization, X-ray diffraction data collection and processing, structure elucidation and refinement, validation and structural analysis presented in the next three chapters. Various programs and protocols used for data processing, structure determination, refinement, model building, structure validation and analysis are also briefly described. In chapter 3, the role of a number of active site residues in the reaction catalyzed by EcDAPAL, a fold type II PLP-dependent enzyme, the structure of which was determined earlier in the laboratory is explored by mutational, biochemical and structural analyses. Earlier studies had established the probable role of Asp120 and Lys77 in the reaction leading to the breakdown of D-DAP and L-DAP, respectively (Bisht et al., 2012). To further validate the earlier observations, a number of active site mutants were generated for Asp 120 (D120N, D120C, D120S and D120T), Asp 189 (D189N, D189C, D189S and D189T), Lys77 (K77T, K77H, K77R and K77A), His 123 (H123L) and Tyr 168 (Y168F). The structure of D120N mutant crystal obtained after soaking in crystallization cocktail containing D-DAP revealed the presence of an intact external aldimine complex at the active site supporting the earlier proposal that Asp120 is the base abstracting the Cα proton from the D-isomer of DAP. Biochemical and structural observations suggested that none of the Asp189 mutants may bind PLP and were catalytically inactive suggesting an essential role for Asp189 in catalysis. In contrast to type I PLP-dependent enzymes, none of the Lys 77 mutants of EcDAPAL could bind PLP either covalently or non-covalently and were inactive with both the isomers of DAP. Thus, Lys77 appears to be important for both PLP binding and catalysis. H123L mutant formed an external aldimine with D-DAP and a gem-diamine complex with L-DAP indicating that this residue is also crucial for catalysis. These studies have provided additional support to the catalytic mechanism of EcDAPAL proposed earlier. The next Chapter 4 explores the structure, function and catalytic mechanism of Salmonella typhimurium DAPAL (StDAPAL). The protein was purified from a construct carrying a hexa-histidine tag at the C-terminus by Ni-NTA chromatography. The purified protein was demonstrated to be homogeneous by SDS-PAGE and MALDI-TOF. Crystals of StDAPAL belonging to the C-centred monoclinic space group (C121) with four molecules in the asymmetric unit were obtained by the micro batch method and used for collecting X-ray diffracting data. The crystal structure was determined by molecular replacement using the homologous enzyme from E. coli (PDB code 4D9M, Bisht et al., 2012), which shares a sequence identity of 50% with the S. typhimurium enzyme as the phasing model in the program Phaser (McCoy et al., 2007) of the CCP4 suite. The model was refined with Refmac5 of CCP4 suite to R and Rfree values of 25.5% and 30.9%, respectively. A superposition of the structure so obtained over EcDAPAL revealed that the two structures are very similar. A sulfate molecule bound to the active site of StDAPAL could be located. The position of the sulfate corresponds to that of the carboxyl group of aminoacrylate intermediate of EcDAPAL (4D9M). The PLP was bound to Lys78 as an internal aldimine. Since the active sites of the two protomers in fold type II PLP-dependent enzymes are independent, it might be possible to obtain functional monomers of EcDAPAL. With this view, mutation of a conserved Trp (Trp399) present in the dimeric interface resulted in the destabilization of the dimeric interface and partial conversion of the dimeric protein to a monomeric protein. However, the monomeric species of EcDAPALW399R was unable to bind PLP and hence did not possess any catalytic activity. This highlights the importance of dimeric organization for efficient binding of PLP as well as for the activity of the enzyme. A remarkable difference between EcDAPAL and StDAPAL is the absence of a disulfide bond between residues Cys271 and Cys299 in StDAPAL equivalent to the bond formed between Cys265 and Cys291 in EcDAPAL. Mutation of Cys265 and Cys291 of EcDAPAL to Ser did not affect the activity of the enzyme towards either of the isomers of the substrate indicating that the disulfide bond is not crucial for enzyme activity. The stability of the loop corresponding residues 261-295 of EcDAPAL was believed to be promoted by the disulfide bond. However, the equivalent loop was found to be ordered in StDAPAL even though the disulfide bond is absent. In contrast to StDAPAL, EcDAPAL did not show any metal dependent activity. The previous two chapters dealt with fold type II PLP-dependent enzymes. In contrast, Chapter 5 deals with revisiting the structure and function of a fold type I PLP-dependent enzyme, Salmonella typhimurium arginine decarboxylase (StADC). ADC is a very large polypeptide in comparison with other fold type I enzymes. It is induced when the bacterium is subjected to low pH and plays a major role in protecting the cells from acid stress. The structure of StADC was determined but not satisfactorily refined by Dr. S. R. Bharat earlier. The X-ray diffraction data collected by Bharat needed to be improved and the structure needed to be further refined and compared with the homologous E. coli enzyme. Therefore, the entire process of data processing, structure solution and refinement was repeated. The refined structure of StADC was found to correspond to the apo form of the enzyme with only a phosphate molecule occupying the position equivalent to that of 5’ phosphate of PLP observed in EcADC holo enzyme structure. This allowed examination of structural changes that accompany PLP binding and formation of an internal aldimine. The apo to holo transition in StADC involves the movement and ordering of two loops consisting of residues 151-164 and 191-196 which are in the linker and PLP binding domains of the protein, respectively. Phosphate binding by itself appears to be insufficient for these structural changes. These two loops are close to the PLP binding site of the other protomer of the dimer. Hence, these movements are probably important for the catalytic function of the enzyme. Holo ADC has been found as a decamer in other studies. The decameric form of the apo-StADC suggests that PLP binding may not be essential for the oligomeric state of the protein. ADC appears to reduce proton concentration inside the cell in two ways; (i) by surface charge neutralization and (ii) by arginine decarboxylation by extracting a proton from the cytoplasm. The resulting product agmatine is exchanged for extra cellular arginine by arginine-agmatine antiporter. The low sequence identity and lack of structural similarity of the inducible and constitutive forms of ADC from S. typhimurium shows that these are unlikely to be products of divergent evolution. The final chapter 6 of the thesis presents the work carried out on S. typhimurium pyridoxal kinase (PLK). In the salvage pathway of pyridoxal 5’phosphate (PLP), PLP is produced as the product of the reaction catalyzed by PLK using PL, PN and PM as substrates. Thus, PLK plays the critical role of ensuring availability of PLP to the large number of PLP-dependent enzymes. S. typhimurium PLK was purified to homogeneity, crystallized in its native as well as ligand bound forms. It was necessary to circumvent an unusual problem caused by spots arising from a contaminant crystal to obtain the structure of the native crystals of PLK that belonged to the P212121 space group with two protomers in the crystal asymmetric unit. It was then straight forward to determine the ligand bound structures of StPLK (space group P43212) obtained by co-crystallization with ATP, PL and Mg2+ by molecular replacement using the wild type structure as the phasing model. The structures obtained by co-crystallization revealed the presence of ADP, Mg2+ and a PL bound to the active site Lys233 via a Schiff base (internal aldimine). This is the first structure in which the presence of an internal aldimine in the active site of PLK has been observed. Formation of the internal aldimine might be one way to prevent the release of excess PLP and protecting the cell from PLP induced toxicity. The enzyme was shown to be inhibited by the product which will also help in maintaining PLP concentration at low levels. It was also demonstrated that PLK interacts with apo-PLP-dependent enzymes. This observation supports possible direct transfer of PLP from PLK to PLP-dependent enzymes. The thesis ends with an appendix where the work carried out during the course of the thesis work but not as part of the thesis is briefly described.

Les "protéine arginine méthyltransférases" (PRMT) sont impliquées dans de nombreux processus cellulaires : transcription, maturation et transport des ARN, traduction, transduction du signal, réplication et réparation de l'ADN, et apoptose. Différents travaux ont montré que des dérégulations de ces mécanismes impliquant les PRMT peuvent induire certains cancers, faisant de ces enzymes de nouvelles cibles potentielles en chimiothérapie. Il s’avère donc crucial de comprendre le mode d’action des PRMT à l’échelle atomique, à la fois au niveau fondamental et pour le développement de nouveaux médicaments. Les travaux décrits ici s’intéressent à la protéine PRMT4/CARM1 et s’appuient sur des études structurales par bio-cristallographie, pour comprendre les mécanismes de la réaction de méthylation catalysée par CARM1 et découvrir des inhibiteurs spécifiques, mais aussi sur des études en solution, pour caractériser l’interaction entre CARM1 et ses substrats. / Protein arginine methyltransferases (PRMTs) are involved in several cellular mechanisms: transcription, RNA maturation and transport, translation, signal transduction, DNA replication and repair, and apoptosis. Different studies showed that deregulation of those mechanisms involving PRMTs can induce some cancers, making these enzymes new potential targets for chemotherapy. It is therefore crucial to understand the mode of action of PRMTs at the atomic scale, both at the fundamental level and for the development of new drugs. The studies described here focus on PRMT4/CARM1 and rely on structural studies by bio-crystallography, in order to understand the methylation mechanisms catalyzed by CARM1 and to discover specific inhibitors, but also on in vitro studies, to characterize the interaction between CARM1 and its substrates.

Le travail de la thèse présente une série de (bio)capteurs conductimétriques, à base de la clinoptilolite, pour la détermination de l’ammonium, de l’urée et de la L-arginine. La clinoptilolite, le matériau nanométrique, possédant des propriétés de la sorption intrinsèque et une capacité d’échange cationique vis-à-vis des espèces ammonium, a été d’abord utilisée pour la réalisation d’un microcapteur conductimétrique sélectif à NH4+. Ci-après, une application de ce nanomatériau dans les biocapteurs est favorable pour le fonctionnement dans les solutions tampons multicomposants. Parmi plusieurs variantes de biocapteurs à l’urée à base de la zéolite, la plus intéressante est le biocapteur, dans lequel la couche de la clinoptilolite, déposée sur le transducteur, a été recouverte par le dépôt de la couche de l’uréase et de la zéolite. Pour l’élaboration d’un biocapteur conductimétrique hautement sensible pour la détermination de la L-arginine, l’arginase et l’uréase ont été co-réticulées sur le transducteur. Une détermination quantitative de la L-arginine dans une solution buvable « Arginine Veyron » a montré un fort accord avec les données fournies par le producteur. Une procédure détaillée de l’optimisation du biocapteur conductimétrique pour la détection de la L-arginine dans le sérum bovin a été proposée. La clinoptilolite a été également appliquée comme un modificateur dans la co-immobilisation de l’arginase et l’uréase pour améliorer les caractéristiques analytiques de biocapteur conductimétriques pour la détermination de la L-arginine / Currentwork presents a serie of conductometric (bio)sensors based on clinoptilolite, for ammonium, urea and L-arginine determination. Clinoptilolite, a nanoscale material possessing exceptional sorption and cation-exchange properties toward ammonium species, was initially used for the development of NH4+-selective conductometric microsensor. The clinoptilolite-based microsensor was selective toward ammonium in the presence of interferences that are commonly found along with ammonium in natural waters. Hereafter, an application of this nanomaterial in biosensors is favorable for operation in multicomponent buffer solutions. Among the several variants of the urea biosensors based on zeolite, considerably better characteristics were obtained for the biosensor comprising a clinoptilolite adlayer and an upper layer of immobilized urease and zeolite. In the work, for first time was developed a highly sensitive conductometric biosensor for L-arginine determination based on arginase and urease co-immobilized in a single membrane. The results of a quantitative determination of L-arginine in a drinkable solution “Arginine Veyron”, obtained by the biosensor, were in high correlation with the data provided by the producer. The L-arginine conductometric biosensor was optimized for the serum analysis. Clinoptilolite was also applied as a modifier in co-immobilization of arginase and urease for the improvement of analytical characteristics of the conductometric biosensor for L-arginine determination