[EN] Post-transcriptional modification of the wobble uridine (U34) of a tRNA set is an evolutionary conserved process, produced by homologous proteins from the MnmA/MTU1, MnmE/GTPBP3 and MnmG/MTO1 families. Mutations in the human genes MTU1 and GTPBP3 or MTO1 produce acute infantile liver failure, and hypertrophic cardiomyopathy and lactic acidosis, respectively, which usually cause lethality in the first months of life. It is assumed that the primary cause of these diseases is the lack of the modifications introduced by the MTU1 protein in position 2 (tiol) and GTPBP3 and MTO1 proteins (taurinomethylation) in position 5 at U34 in a subgroup of mt-tRNAs. Nevertheless, the molecular mechanisms underlying these diseases (and other diseases associated with such modifications) are not clear. The reason why the typical defects of oxidative phosphorylation (due to impaired mitochondrial translation) produce such wide range of phenotypes is still unknown. Our hypothesis sustains that the mitochondria-nucleus retrograde signaling pathways triggered by the hypomodification at position 2 and 5 of U34 are different, and that each nuclear response is modulated by the genetic and epigenetic programs of cells and organisms. In this work, we have used the nematode Caenorhabditis elegans as a model organism to study the effects of inactivating the homologue proteins to MTU1, GTPBP3 and MTO1, which we have named as MTTU-1, MTCU-1 and MTCU-2, respectively. We have proved that these nuclear encoded proteins are located in mitochondria and are involved in U34 modification of mt-tRNAs. The mtcu-1 and mtcu-2 mutants show a reduction in fertility, while the mttu-1 mutant shows a reduction in fertility and a lengthening of the reproductive cycle (both phenotypes are thermosensitive). The phenotypes exhibited by the mttu-1, mtcu-1 and mtcu-2 mutants support our hypothesis, in which the mttu-1 single mutation, on the one hand, and the mtcu-1 and mtcu-2 single mutations, on the other hand, trigger different retrograde signaling pathways which produce specific nucear expression. Thus, a nuclear dependent phenotypic trait (as transcription or mt-tRNAs stability) and the expression of nuclear genes as ucp-4, hsp-6, hsp-60 and other genes involved in mitochondrial metabolism show a differential pattern in both group of mutants. hsp-6 and hsp-60 genes (UPRmt markers) are downregulated in mttu-1 single mutant, which could be related to fertility and reproductive cycle thermosensitivity. The three single mutants exhibit reduced expression of glycolysis and ß-oxidation genes (usually more drastic in the mttu-1 mutant), an induction of a glutaminolysis marker, and an induction of the ucp-4 gene, which encodes a transporter of the succinate to the mitochondria. Due to all three single mutants display a mild OXPHOS dysfunction, we propose that the observed changes in the expression of genes involved in the mitochondrial metabolism reveal a TCA cycle reprogramming aimed to compensate the reduction of acetil-CoA (coming from glycolysis and fatty acid oxidation) though the activation of anaplerotic pathways characterized by the succinate import to mitochondria by UCP-4 and the incorporation of 2-oxoglurate from glutaminolysis. We also analyze the effects of the simultanous suppression of modifications at positions 2 and 5 of U34 in C. elegans. The double mutant mtcu-2;mttu-1 displayed a severe OXPHOS dysfunction and a 5-fold higher AMP/ATP ratio, which was associated with embryonic lethality, developmental arrest in primary larval stages, penetrant sterility in adults and extended lifespan. This lifespan extension is modulated by signaling pathways which depend on AMPK (specifically on AAK-1 catalitic subunit) and steroid hormones, through DAF-9 and DAF-12 proteins. This work shows the important gene reprogramming related to mitochondrial metabolism in response to U34 hypomodification of mt-tRNAs, and shows new connexions between signaling pathways that extend lifespan. / [ES] La modificación post-transcripcional de la uridina de tambaleo (U34) de ciertos tRNAs es un proceso conservado evolutivamente, realizado por proteínas homólogas de las familias MnmA/MTU1, MnmE/GTPBP3 y MnmG/MTO1, y biológicamente relevante. De hecho, mutaciones en los genes humanos MTU1 y GTPBP3 o MTO1 causan fallo hepático infantil agudo y cardiomiopatía hipertrófica infantil, respectivamente, que producen letalidad durante los primeros meses de vida. Se asume que la causa primaria de estas enfermedades es la ausencia de las modificaciones introducidas por la proteína MTU1 en la posición 2 (tiol) y las proteínas GTPBP3 y MTO1 (taurinometil) en la posición 5 de la U34 en un grupo de mt-tRNAs. Se desconocen los mecanismos subyacentes y las razones por las que el déficit de OXPHOS resultante en todos los casos (atribuido a alteraciones de la traducción mitocondrial de proteínas) produce fenotipos tan diversos. Nuestra hipótesis es que la señalización retrógrada mitocondria-núcleo promovida por la hipomodificación de los mt-tRNAs en 2 ó 5 de la U34 es diferente y la respuesta nuclear viene modulada por el programa genético y epigenético de células y organismos. Hemos utilizado el nematodo C. elegans como modelo para estudiar los efectos producidos por la inactivación de las proteínas homólogas de MTU1, GTPBP3 y MTO1 a las que hemos denominado MTTU-1, MTCU-1 y MTCU-2. Hemos comprobado que estas proteínas, codificadas por el núcleo, son de localización mitocondrial y están implicadas en la modificación de la U34 de los mt-tRNAs. Los mutantes mtcu-1 y mtcu-2 presentan una reducción en su fertilidad y, en el caso del mutante simple mttu-1, fenotipos asociados a termosensibilidad. Los fenotipos exhibidos por los mutantes mttu-1, mtcu-1 y mtcu-2 sustentan la hipótesis de que la mutación mttu-1, y las mutaciones mtcu-1 y mtcu-2 promueven señales retrógradas diferentes que producen patrones de expresión nuclear específicos. Así, un rasgo fenotípico dependiente de genes nucleares (como lo es la transcripción y/o estabilidad de los mt-tRNAs) y la expresión de genes nucleares como ucp-4, hsp-6, hsp-60 y otros implicados en el metabolismo mitocondrial muestran un patrón diferente en los dos grupos de mutantes. Los genes hsp-6 y hsp-60 (marcadores de la UPRmt) están regulados a la baja en el mutante mttu-1. Los tres mutantes simples exhiben una reducción en la expresión de genes de la glicólisis y de la ß-oxidación de los ácidos grasos, una inducción en un marcador de glutaminolisis y una inducción en el gen ucp-4 (mayor en mttu-1) implicado en el transporte de succinato a la mitocondria. Dado que los tres mutantes simples presentan una disfunción OXPHOS relativamente suave, proponemos que los cambios de expresión en genes que modulan el metabolismo mitocondrial revelan una reprogramación del ciclo del TCA que compensa la disminución en el aporte de acetil-CoA procedente de glicólisis y oxidación de ácidos grasos con la activación de rutas anapleróticas del ciclo del TCA (importe de succinato a la mitocondria por UCP-4 y aporte de ¿-cetoglutarato procedente de la glutaminolisis). También analizamos los efectos de la anulación simultánea de las modificaciones en las posiciones 2 y 5 de la U34. El doble mutante mttu-1;mtcu-2 presenta una disfunción OXPHOS severa, con una ratio AMP/ATP 5 veces superior al control, que resulta en letalidad embrionaria, detención del desarrollo en estadios larvarios tempranos y esterilidad completa en los adultos que presentan, por otra parte, una longevidad unas dos veces superior a la cepa control. Este incremento de la longevidad está modulado por rutas de señalización que dependen de la subunidad catalítica AAK-1 (AMPK), y de hormonas esteroideas (proteínas DAF-9 y DAF-12). El trabajo muestra la importante reprogramación de genes relacionados con el metabolismo mitocondrial en respuesta a la hipomodificación de la U34 de los mt-tRNAs y / [CAT] La modificació post-transcripcional de la uridina de balanceig (U34) de certs tRNAs és un procés conservat evolutivament realitzat per proteïnes homòlogues a les de les famílies MnmA/MTU1, MnmE/GTPBP3 i MnmG/MTO1 i biològicament relevant. De fet, mutacions en els gens humans MTU1 i GTPBP3 o MTO1 causen fallada hepàtica infantil aguda i cardiomiopatia hipertròfica infantil amb acidosis làctica, respectivament, que produïxen letalitat durant els primers mesos de vida. S'assumix que la causa primària d'aquestes malalties és l'absència de les modificacions introduïdes per la proteïna MTU1 a la posició 2 (tiol) i per les proteïnes GTPBP3 i MTO1 (taurinometil) a la posició 5 de la U34 en un grup de mt-tRNAs. Es desconeixen els mecanismes subjacents en estes malalties i les raons per les quals el dèficit de la OXPHOS resultant en tots els casos (atribuït a alteracions de la traducció mitocondrial de proteïnes) produïx fenotips tan diversos. La nostra hipòtesi és que la senyalització retrògrada mitocondria-nucli promoguda per la hipomodificació dels mt-tRNAs en 2 o 5 de la U34 és diferent i la resposta nuclear en cada cas es dependent del programa genètic i epigenètic de cèl¿lules i organismes. Hem utilitzat el nematode C. elegans com a organisme model per a estudiar els efectes produïts per la inactivació de les proteïnes homòlogues de MTU1, GTPBP3 i MTO1 a les que hem denominat MTTU-1, MTCU-1 i MTCU-2. Hem comprovat que aquestes proteïnes, codificades pel nucli, són de localització mitocondrial i estan implicades en la modificació de la U34 dels mt-tRNAs. Els mutants mtcu-1 i mtcu-2 presenten una reducció en la seua fertilitat i, en el cas del mutant mttu-1, fenotipus associats a termosensibilitat. Els fenotipus exhibits pels mutants mttu-1, mtcu-1 i mtcu-2 sustenten la hipòtesi que la mutació mttu-1, i les mutacions mtcu-1 i mtcu-2 promouen senyals retrògrads diferents que produïxen patrons d'expressió nuclears específics. Així, un tret fenotípic dependent de gens nuclears (com ho és la transcripció i/o l'estabilitat dels mt-tRNAs) i l'expressió de gens nuclears com ucp-4, hsp-6, hsp-60 i altres implicats en el metabolisme mitocondrial mostren un patró diferent en els dos grups de mutants. Els gens hsp-6 i hsp-60 (marcadors de la UPRmt) estan regulats a la baixa en el mutant mttu-1. Els tres mutants simples exhibixen una reducció en l'expressió de gens de la glicòlisi i de la ß-oxidació dels àcids grassos, una inducció en un marcador de glutaminolisi i una inducció en el gen ucp-4 (major en el mutant mttu-1) implicat en el transport de succinat a la mitocondria. Atés que els tres mutants simples presenten una disfunció OXPHOS relativament suau, proposem que els canvis d'expressió en gens que modulen el metabolisme mitocondrial revelen una reprogramació del cicle del TCA que compensa la disminució en l'aportació d'acetil-CoA procedent de la glicòlisi i de l'oxidació d'àcids grassos amb l'activació de rutes anaplerótiques del cicle del TCA (importació de succinat a la mitocondria per UCP-4 i aportació de ¿-cetoglutarat de la glutaminolisi). També s'analitzen els efectes de l'anul¿lació simultània de les modificacions en 2 i 5 de la U34. El doble mutant mttu-1;mtcu-2 presenta una disfunció OXPHOS severa, amb una ràtio AMP/ATP 5 vegades superior al control, que resulta en letalitat embrionària, detenció del desenvolupament en estadis larvaris primerencs, esterilitat completa en els adults i una longevitat unes 2 vegades superior al control. Aquest increment de la longevitat està modulat per rutes de senyalització que depenen de la subunitat catalítica AAK-1 (AMPK), i d'hormones esteroidees (a través de les proteïnes DAF-9 i DAF-12). En resum, aquest treball mostra per primera vegada a nivell d'un animal model la important reprogramació de gens relacionats amb el metabolisme mitocondrial en resposta a la hipomodificació de la U34 dels mt-tRNAs i / Navarro González, MDC. (2016). Caenorhabditis elegans as a research tool to study mitochondrial diseases associated with defects in tRNA modification [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/61978 / TESIS

Introduction: Diagnosing inborn metabolic diseases, as a large subgroup of rare diseases, due to their rarity and wide variety of clinical manifestations, can be demanding and often prolonged. Objective: The aim of this work is, with the regard to clinical, biochemical and genetical aspects of selected rare diseases, to contribute to their rapid detection, widen the features of the natural course of the disease and contribute to their preventability. Material: This work includes cohort studies of patiens with cardiac manifestations in mitochondrial diseases, namely a group of 48 patients with TMEM70 protein deficiency, a group of 4 patients with Barth syndrome and individual cases of rare mitochondrial cardiomyopathies, thimidine kinase 2 deficiency and alanyl tRNA synthetase 2 deficiency. By determining the frequency, severity and type of heart disease, the phenotype was expanded, and the design of a therapeutic algorithm then made a positive impact on the prognosis of these patients. The work is also focused on the role of cardiac disease in the differential diagnosis of other genetically determined rare diseases, Marfan's syndrome and especially Pompe disease, where the emphasis is on early diagnosis, mainly due to the existence of an effective therapy. Focus on a broader differential diagnosis...

The thesis involves computer simulation and theoretical studies of phase transition in soft-condensed matter systems and theoretical understanding of enzyme kinetics along with kinetic proofreading of tRNA-aminoacylation in biological systems. Based on the system and phenomena of interest, the work has be classified into the following four major parts: I. Surface phenomena and surface energy of vapor-liquid interface. II. Condensation of vapor in two and three dimensions. III. Liquid-solid phase transition in polydisperse systems. IV. Enzyme catalysis and kinetic proofreading in biosynthesis. Above mentioned four parts have further been divided into thirteen chapters. In the following we provide a brief chapter-wise outline of the thesis. Part I deals with surface tension and interfacial properties of vapor-liquid interface for Lennard-Jones (LJ) fluid in both two and three dimensions. In Chapter 1, we provide a brief overview of vapor-liquid interface and existing theoretical and computer simulation studies of surface/line tension. In this chapter we also discuss about the existing experimental studies. In Chapter 2, we present computer simulation studies of surface tension in two dimensional Lennard-Jones system. The sensitivity of line tension on range (potential cut-off) of interparticle interaction is discussed in this chapter. We present Density Functional Theory (DFT) of line tension of vapor-liquid interface based on Weeks-Chandler-Anderson (WCA) and Barker-Hendersen (BH) perturbation techniques. We compare the DFT prediction with the computer simulation results. In general, WCA approach has been found to be successful for 3D system in predicting the surface tension. In 2D, however, it does not give good agreement either for phase diagram or for the line tension. In fact, BH also does not give accurate values of the coexistence parameters, however, it predicts better line tension compared to WCA. In Chapter 3 we present both theoretical and computer simulation studies of gas-liquid surface tension for three dimensional Lennard-Jones fluid. We perform non-equilibrium computer simulation study following Transition Matrix Monte Carlo (TMMC) method to obtain surface tension for various ranges of potential and introduce a new scaling relation of surface tension in order to capture both the temperature and interparticle interaction range dependence. The scaling shows excellent agreement with the simulation result and it can also predict the critical temperature with sufficient accuracy. The width of the gas-liquid interface is found to be insensitive to the range of the potential, whereas the density separation of the bulk vapor and liquid phases increases with increasing range of potential. Thus, the major contribution comes from the increasing density separation of the bulk vapor and liquid phases. Part II consists of four chapters, where we focus on the age old problem of nucleation, from the perspective of thermodynamics and kinetics. We account for the rich history of the problem in the introductory Chapter 4. In this chapter we describe various types and examples of the nucleation phenomena, and a brief account of the major theoretical approaches used so far. We begin with the most successful Classical Nucleation Theory (CNT), and then move on to more recent applications of Density Functional Theory (DFT) and other mean-field types of models. We present various experimental techniques used in the literature to obtain rate of nucleation. We conclude with a comparison between the experiments, theories and computational studies. In the next chapter (Chapter 5) we attempt to understand the mechanism of the gas-liquid nucleation in three dimension at large metastability from microscopic point of view. Here we study the nature of sequential growth of all liquid-like clusters (not just the largest cluster) at different degrees of metastability. Therefore, we have ordered the clusters according to their decreasing sizes and identified them in terms of kth largest cluster where, k = 1 denotes the largest cluster in the system, k = 2 represents the second largest and k = 3 is the third largest and so on. We have studied both the free energies and the trajectories of the liquid-like clusters in this extended set of order parameters. We further define Fkl(n) as the free energy of the kth largest cluster with size n. Classical nucleation theory provides an expression of unconditional free energy of a single cluster, F (n) (the free energy of formation of a cluster of size n), which is an intensive property of the system. The study of our conditional free energy surfaces, Fkl(n), reveals a more detailed, microscopic picture of the system’s cluster size distribution that is necessary to understand the kinetics of nucleation and growth at large metastability. The rate of nucleation shows a cross over at kinetic spinodal (the limit of metastability, ∆F1 l = 0). Below kinetic spinodal only one (largest) cluster crosses the critical size through activation whereas above this point more than one cluster grow simultaneously through barrierless diffusion. We present a theoretical analysis of the free energy of kth largest cluster based on order statistics. The theoretical predictions are in excellent agreement with computer simulation results for the range of supersaturation we studied. While the previous chapter focuses on relatively well-studied nucleation mechanism in 3dimensional (3D) LJ system at large metastability, in Chapter 6 we present our studies on the characteristics of the nucleation phenomena in two dimensional Lennard-Jones fluid for different ranges of interparticle interaction. Using various Monte Carlo (MC) methods, we calculate the free energy barrier of nucleation and bulk densities of equilibrium liquid and vapor phases, and also investigate the size and shape of the critical nuclei. We find an interesting interplay between the range of interaction potential and the extent of metastability. The free energy barrier of nucleation strongly depends on the range of interaction potential. The study is carried out at an intermediate level of supersaturation (away from the kinetic spinodal limit). A surprisingly large cutoff (rc � 7.0�, where � is the diameter of LJ particles) in the truncation of the LJ potential is required to obtain converged results. A lower cutoff leads to a substantial deviation in the values of the nucleation barrier, and characteristics of the critical cluster (with respect to full range of interaction). We observe that in 2D system CNT fails to provide a reliable estimate of the free energy barrier. While it is known to slightly overestimate the nucleation barrier in 3D, it underestimates the barrier by � 50% at the saturation ratio S =1.1 (defined as S = P/Pc, where Pc is the coexistence pressure) and at the reduced temperature T � =0.427 (defined as T � = kBT/�, where � is the depth of the potential well). The reason for the marked inadequacy of the CNT in 2D can be attributed to the non-circular nature of the critical clusters. Although the shape becomes increasingly circular and the clusters become more compact with increase in cutoff radius, an appreciable non-circular nature remains even for full potential (without truncation) to make the predictions of CNT inaccurate. In Chapter 7 we report the computer simulation study of nucleation in three dimensional LJ system. At a fixed supersaturation the free energy barrier of nucleation increases with increasing range of interparticle interaction. On increasing range of intermolecular interaction, the kinetic spinodal where the mechanism of nucleation changes from activated barrier crossing to barrierless diffusion, shifts towards the deep metastable region. Both the critical cluster size and pre-critical minimum in the free energy surface of kth largest cluster shift towards the smaller size at their respective kinetic spinodal as we increase the range of potential. We find only a weak non-trivial (other than supersaturation and surface tension) contribution to the free energy barrier of nucleation. Part III consists of two chapters and focuses on the liquid-solid phase transition of polydisperse fluid. In Chapter 8 we introduce polydisperse systems and their classification based on different identities. We describe the importance and abundance of polydisperse system in nature. The theoretical modeling of different polydisperse systems and their extent of applicability have also been presented. We have discussed about the various factors which control the phase diagram and various phenomena related to the structure and phase transition. In Chapter 9 we present computer simulation study on freezing/melting of Lennard-Jones (LJ) fluid at different polydispersities. The freezing/melting of polydisperse LJ fluids presents an interesting case study, because, as the polydispersity increases the energy-entropy balance becomes increasingly unfavorable for the solid to exist as a stable phase. The energy of the solid increases due to build up of strain energy because of increasing mismatch in size of the neighbors, while the entropy of the liquid increases. These two factors lead to the existence of a terminal polydispersity. We find beyond the terminal ploydispersity, δ. 0.11system remains in the disorder state even at very high pressure and low temperature. The terminal polydispersity obtained in the present study is close to the experimental value (δt. ≈ 12%). Interestingly, contrary to hard sphere polydisperse fluid, LJ fluid does not exhibit reentrant melting. The last part (Part IV) of the thesis consists of three chapters that deal with the enzyme catalysis and kinetic proofreading of tRNA-aminoacyl synthetases. In Chapter 10 we describe protein synthesis process in biological system and corresponding two processes: aminoacylation of tRNA and translation of amino acid in ribosome. Our interest is to understand the enzyme catalysis involved in aminoacylation of tRNA in the process of protein synthesis. We present the classification of 20 aminoacyl-tRNA synthetases into two classes based on their structure and mode of binding to ATP and tRNA. We discuss all the steps involved in whole tRNA-aminoacylation process. Then we introduce kinetic proofreading during aminoacylation reaction. In Chapter 11 we theoretically analyze the single turn over and steady state reaction mechanism of two classes of aminoacyl-tRNA synthetases. Class I enzymes not only differ in their structure but they also differ with respect to the pre-steady kinetics compared to class II enzymes. We find that the strong binding of product to class I enzymes causes the product release step to be rate limiting step leading to the burst of product formation in pre-steady reaction. On the other hand class II enzymes do not show any burst kinetics. The present study based on time dependent probability statistics is successful in explaining all the experimental results quantitatively. In Chapter 12 we present an augmented kinetic scheme and then employ methods of time dependent probability statistics to understand the mechanism of kinetic proofreading of isoleucyl-tRNA synthetase (IRS) which belongs to class I. We investigate that the enhanced hydrolysis of wrong substrate (Val) enables IRS to discriminate the correct substrate (Ile) and wrong substrate (Val) efficiently. It has been observed that an extra CP1 editing domain serves as an activating domain towards enhanced hydrolysis of Val. The present study is able to explain most of the existing experimental observations. In the concluding note, Chapter 13 lists a few relevant problems that may prove worthwhile to be addressed in future. In the Appendices, we present two of the techniques used in our present computer simulation and theoretical studies. Appendix A describes Grand Canonical Transition Matrix Monte Carlo (GC-TMMC) method which is employed in computer simulation studies of nucleation and surface tension. In Appendix B we present the probabilistic method of waiting time distribution computation used in enzyme catalysis and kinetic proofreading.

Malgré l’intérêt et la diversité des propriétés pharmacologiques des 2,5-dicétopipérazines (DKP), les voies de biosynthèse de ces molécules d’origine microbienne sont très peu connues. L’objectif de mes travaux de thèse a été i) de documenter de nouvelles voies de biosynthèse de DKP qui se caractérisent par la présence d’une synthase de cyclodipeptides (CDPS) travaillant souvent de concert avec une ou plusieurs enzymes de modification des cyclodipeptides et ii) d’explorer la diversité chimique codée par ces voies. Dans un premier temps, je me suis intéressée aux CDPS. Après la sélection par bioinformatique de candidats dans les bases de données génomiques, j’ai pu identifier 51 nouvelles CDPS actives et montrer que ces enzymes peuvent incorporer 17 des 20 acides aminés naturels. Par ailleurs, ce travail a permis de mieux caractériser la famille des CDPS, de définir l’existence de plusieurs sous-familles aux signatures fonctionnelles spécifiques et d’établir les premiers éléments d’un code de spécificité pour la synthèse de cyclodipeptides. Dans un second temps, je me suis attachée à caractériser les enzymes de modification associées aux nouvelles CDPS et, en particulier, les dioxygénases dépendant du Fe(II) et du 2-oxoglutarate (OG) qui sont très représentées dans ces voies. J’ai ainsi pu détecter une activité in vivo pour 11 OG et poursuivre la caractérisation in vitro pour l’une de ces OG, ce qui a permis de caractériser les DKP qu’elle synthétise et d’ainsi montrer la complexité des modifications chimiques introduites. L’ensemble de ces travaux a donc permis d’identifier et de caractériser de nouvelles voies de biosynthèse qui donnent accès à une diversité accrue de DKP. / Despite the interest and diversity of the pharmacological properties of 2,5-diketopiperazines (DKPs), the biosynthetic pathways of these microbial molecules are poorly documented. The aim of my doctoral work was i) to identify new DKP biosynthetic pathways that are characterized by the presence of a cyclodipeptide synthase (CDPS) often associated with one or more cyclodipeptide-tailoring enzymes and ii) to explore the chemical diversity encoded by these pathways. First of all, my study focused on CDPSs. After the bioinformatics-based selection of candidates, 51 novel CDPS were characterized, revealing the incorporation of 17 of the 20 proteinogenic amino acids. Moreover, this work has allowed a better characterization of the CDPS family, by showing the existence of several subfamilies with specific functional signatures and laying the foundations of a specificity conferring code for the synthesis of cyclodipeptides. Second, I characterized the tailoring enzymes associated with the newly identified CDPSs and, in particular, the Fe(II) and oxoglutarate dependent dioxygenases (OGs) that are highly represented in these pathways. I detected the in vivo activity for 11 OGs and characterized the in vitro activity for one of them, showing the complexity of the chemical modifications introduced into the cyclodipeptide. This work has led to identify and characterize novel biosynthetic pathways that provide access to a greater diversity of DKPs.

Cyclodipeptides and their derivatives, the diketopiperazines (DKPs), constitute a large class of secondary metabolites with noteworthy biological activities that are mainly synthesized by microorganisms. The biosynthetic pathways of some DKPs contain cyclodipeptide synthases (CDPSs), a newly defined family of enzymes. CDPSs hijack aminoacyl-tRNAs from their essential role in ribosomal protein synthesis to catalyze the formation of the two peptide bonds of various cyclodipeptides. The aim of the work presented in this thesis manuscript is to characterize the CDPS family. At first, the structural and mechanistic characterization of the first identified CDPS, AlbC of Streptomyces noursei, is presented. Then, the results obtained with three other CDPSs, each of which having suitable properties to increase our understanding of the CDPS family, are described. The CDPS Ndas_1148 of Nocardiopsis dassonvillei extends our knowledge of the molecular bases of the CDPS specificity. The CDPS AlbC-IMI of S. sp. IMI 351155 is a good model to analyze the interaction of each of the two substrates required for the formation of a cyclodipeptide. Finally, the characterization of the CDPS Nvec-CDPS2 from Nematostella vectensis provides the first example of enzymes of animal origin involved in nonribosomal peptide synthesis.

Les aminoacyl-ARNt synthetases (aaRS) sont impliquées dans le mécanismes de la traduction. Dans les cellules humaines, il existe deux jeux de gènes nucléaires codant pour les aaRS : un pour les aaRS cytosolique (cyt), le second pour les aaRS mitochondriales (mt). Les aaRS mt sont traduites dans le cytosole, adressées et importées dans la mitochondrie.Mutations dans 9 gènes d’aaRS mt ont été démontrées comme responsables de pathologies mitochondriales. Certaines des mutations n’affectent pas la propriété originelle d’aminoacylation. Il a été proposé que certaines de ces mutations puissent affecter des propriétés alternatives.Alors l’organisation des aaRS cyt est bien étudiée et que des implications dans des fonctions alternatives établies pour certaines d’entre elles, les connaissances quant aux aaRS mt restent parcimonieuses. L’objectif principal de ce manuscrit de thèse est: (i) révéler d’organisation sous-mt de l’AspRS mt; (ii) étendre l’analyse de l’organisation sous-mt à l’ensemble des aaRS mt; et (iii) contribuer à la compréhension de mécanismes moléculaires sous-jacents à certaines pathologies. Ces travaux ouvrent la porte vers d’autres investigations de l’organisation des aaRS à l’intérieur de la mitochondrie. Ces contributions seront utiles à la meilleure compréhension de mécanismes moléculaires sous-jacents à pathologies mitochondriales. / Aminoacyl-tRNA synthetases (aaRSs) are housekeeping enzymes involved in translation. In human cells, 2 different sets of nuclear genes code for aaRSs. One codes for cytosolic (cyt) aaRSs, and the second one codes for aaRSs of mitochondrial (mt) location. Mt-aaRSs are translated in the cytosol, targeted and imported into mitochondria.Mutations in 9 mt-aaRSs have been described. Some of the mutations do not display significant influence on the housekeeping aminoacylation activity. It has been proposed that those mutations affect alternative functions.Alternate functions have been described for cyt-aaRSs. While the organization of cyt-aaRSs is explored and their involvement into alternate functions established, the properties of the human mt-aaRSs remain unknown. On one site, this thesis integrate mt-AspRS into new functional networks (sub-mitochondrial localization and partnership). On the other site, it expand the view of the sub-mitochondrial organization to the full set of mt-aaRSs and should ultimately shed light into the molecular mechanisms underlying some of the pathologies. These results open the door for additional investigations to gain a complete view about the sub-mitochondrial organization of aaRSs. Those contributions will be of help for the understanding of molecular mechanisms underlying some mitochondrial disorders.

Les travaux présentés dans cette thèse ont eu pour objectif de contribuer à la compréhension du lien entre l’aspartyl-ARNt synthétase mitochondriale (AspRSmt) humaine et le syndrome LBSL, en étudiant les propriétés de cette enzyme au niveau cellulaire. Les objectifs étaient : 1) d’explorer l’organisation de l’AspRSmt dans la mitochondrie (Chapitre 1), 2) d’identifier la forme mature de l’AspRSmt après son import, ainsi que la localisation sous-mitochondriale de cette enzyme (Chapitre 2), 3) d’évaluer l’impact de quelques mutations, impliquées dans le syndrome LBSL, sur les propriétés de l’AspRSmt (Chapitre 3). Nous avons démontré que l’AspRSmt existe sous différentes formes de produits de maturation, et qu’elle est retrouvée, au moins, dans deux complexes, suggérant potentiellement différents partenaires et/ou fonctions pour cette enzyme. Nous avons établi la localisation sous-mitochondriale de l’AspRSmt, et démontré que cette dernière est doublement localisée avec une fraction soluble et une fraction périphérique interagissant avec la membrane. Nous avons également découvert que, sous certaines conditions de stress, l’AspRSmt est relarguée de la mitochondrie et pourrait avoir un lien avec le processus d’apoptose. En outre, nous avons évalué l’impact de quelques mutations, impliquées dans le syndrome LBSL, et trouvé qu’elles n’ont pas d’effet significatif sur les propriétés de l’AspRSmt. L’ensemble des résultats souligne, d’une part, les lacunes restant à combler concernant les propriétés de l’AspRSmt dans la compréhension du lien mutations/pathologie (LBSL), et d’autre part, suggère fortement l’existence d’une éventuelle fonction non canonique (alternative) de l’AspRSmt. / The aim of the PhD project was to contribute to the understanding of the link between mutations in the human mitochondrial aspartyl-tRNA synthetase (mt-AspRS) and LBSL disease, by studying the properties of this enzyme at the cellular level. Our objectives were: 1) to explore the organization of mt-AspRS in mitochondria (Chapter 1), 2) to identify the mature form of mt-AspRS after its import, and to characterize its submitochondrial localization (Chapter 2), 3) to assess, in cellulo, the impact of some LBSL-causing mutations on some properties of mt-AspRS (Chapter 3). We showed that mt-AspRS is processed into different mature forms, and that mt-AspRS belongs to two complexes likely suggesting different partners and/or functions. We demonstrated that mt-AspRS is dually localized with soluble and peripherally membrane-associated fractions. We also demonstrated that, under stress conditions, mt-AspRS is released outside mitochondria with a possible link to the apoptosis. Furthermore, we assessed the impact of some LBSL-causing mutation on some cellular properties of mt-AspRS, and showed that most mutations do not have a significant impact. This underscores the need for more studies about mt-AspRS properties, and strongly suggests a potential non-canonical (alternative) function of the enzyme.

Les protéines sont responsables de pratiquement toutes les fonctions performées au sein du corps cellulaire et de ses alentours. Le contrôle de l’expression génique détermine l’abondance, la localisation et le moment de la production de protéines dans la cellule. Il s’agit de l’un des processus centraux à la régulation de la physiologie et du fonctionnement cellulaire. La moindre perte de balance dans ce complexe système engendre des conséquences majeures sur l’intégrité cellulaire, menant au développement de plusieurs maladies parfois incurables. La traduction de l’ARN messager en produit protéique constitue la dernière étape de l’expression génique. Elle est régulée de plusieurs façons, intrinsèques et extrinsèques à la séquence. Il s’agit également du processus cellulaire le plus coûteux en termes d’énergie. Le profilage des ribosomes (Ribo-Seq) figure parmi les récentes et prometteuses technologies ayant permis une meilleure étude des mécanismes de régulation de la traduction. Ces résultats contiennent toutefois la présence de variabilité et de bruits de nature infondée. Ce travail présente la mise en place d’une stratégie permettant la dissociation de signaux d’origine biologique de ceux ayant une origine technique. Ceci est effectué au travers de la mise en place de profiles consensus de densité ribosomale extrait d’une analyse comparative de plusieurs expériences de Ribo-Seq chez la levure (Saccharomyces cerevisiae). Les signaux biologiques dérivés par les profils consensus correspondent avec les signatures de pauses ribosomales connues, telles que les scores de repliements de l’ARNm et la charge des acides aminés. Épatamment, notre stratégie a également permis l’identification de séquences différentiellement transcrites (DT). Ces dernières jouent un rôle sur la cinétique de la phase d’élongation de la traduction, elles comportent notamment une surreprésentation de codons associés aux modifications des ARNs de transfert (tRNAs). Elles se retrouvent d’ailleurs impliquées dans le maintien de l’homéostase cellulaire, ayant une présence marquée chez des gènes prenants part aux mécanismes de biosynthèse de la macromolécule ribosomale ainsi que chez les ARNms aux sublocalisations cellulaires précises, notamment chez les mitochondries et le réticulum endoplasmique (ER). En plus de démontrer les possibilités de découvertes offertes par la technique du Ribo-Seq, cette étude présente une évidence de la nature dynamique et hétérogène du processus de traduction chez la cellule eucaryote. Elle démontre également le rôle de l’information directement encodée dans la séquence dans l’optimisation générale de l’homéostasie cellulaire. / Proteins are responsible for virtually all functions performed within and in the surroundings of a cell. The control of gene expression, which determines the amount, localisation and timing of protein production in the cell, is the central processes in the regulation of cellular physiology and function. Any disturbance in this complex system can generate important consequences on cellular integrity, sometimes leading to incurable diseases. The translation of messenger RNA into a protein product is the last step of the gene expression mechanism. It can be regulated in manifold ways, both intrinsically and extrinsically to the transcript sequence. It is also the costliest cellular process in terms of energy. Ribosome profiling (Ribo-Seq) is one of the recent and promising technologies making it possible to better study the mechanisms of translation regulation. Its results have however been shown to display variability in reproducibility and to contain noise of uncharted sources. This work presents the implementation of a strategy for dissociating signals of biological origin from those of technical origin. This is performed by the computation of a consensus profile of ribosomal density derived from a comparative analysis of several Ribo-Seq experiments in yeast (Saccharomyces cerevisiae). The biological signals derived by the consensus profiles correspond with signatures of known ribosomal pauses, such as mRNA folding strength and amino acid charges. Amazingly, our strategy also enabled the identification of differentially transcribed (DT) sequences. The latter have shown an over-representation of codons associated with modifications of transfer RNAs (tRNAs). They are also involved in the control of cellular homeostasis, exhibiting a marked presence in genes involved in ribosome biosynthesis as well as in mRNAs with precise translation sub-localization, particularly in mitochondria and the endoplasmic reticulum (ER). In addition to demonstrating the possibilities of discovery offered by the Ribo-Seq technique, this study also presents evidence of the dynamic and heterogeneous nature of the translation process in the eukaryotic cell. It also showcases its diverse regulatory mechanisms and the role of information directly encoded in the sequence in the general optimization of cellular homeostasis.

Systematic studies of basal nonsense suppression, orthogonality of tRNAPyl variants, and cross recognition between codons and tRNA anticodons are reported. E. coli displays detectable basal amber and opal suppression but shows a negligible ochre suppression. Although detectable, basal amber suppression is fully inhibited when a pyrrolysyl-tRNA synthetase (PylRS)-tRNAPyl_CUA pair is genetically encoded. trnaPyl_CUA is aminoacylated by an E. coli aminoacyl-tRNA synthetase at a low level, however, this misaminoacylation is fully inhibited when both PylRS and its substrate are present. Besides that it is fully orthogonal in E. coli and can be coupled with PylRS to genetically incorporate a NAA at an ochre codon, tRNAPyl_UUA is not able to recognize an UAG codon to induce amber suppression. This observation is in direct conflict with the wobble base pair hypothesis and enables using an evolved M. jannaschii tyrosyl-tRNA synthetase-tRNAPyl_UUA pair and the wild type or evolved PylRS-tRNAPyl_UUA pair to genetically incorporate two different NAAs at amber and ochre codons. tRNAPyl_UCA is charged by E. coli tryptophanyl-tRNA synthetase, thus not orthogonal in E. coli. Mutagenic studies of trnaPyl_UCA led to the discovery of its G73U form which shows a higher orthogonality. Mutating trnaPyl_CUA to trnaPyl_UCCU not only leads to the loss of the relative orthogonality of tRNAPyl in E. coli but also abolishes its aminoacylation by PylRS.

No description available.