Escherichia coli ribosomal RNA (rRNA) is post-transcriptionally modified by site-specific enzymes. The role of most modifications is not known and little is known about how these enzymes recognize their target substrates. In this thesis, we have structurally and functionally characterized two S-adenosyl-methionine (SAM) dependent 23S rRNA methyltransferases (MTases) that act during the early stages of ribosome assembly in E. coli. RlmM methylates the 2'O-ribose of C2498 in 23S rRNA. We have solved crystal structures of apo RlmM at 1.9Å resolution and of an RlmM-SAM complex at 2.6Å resolution. The RlmM structure revealed an N-terminal THUMP domain and a C-terminal catalytic Rossmann-fold MTase domain. A continuous patch of conserved positive charge on the RlmM surface is likely used for RNA substrate recognition. The SAM-binding site is open and shallow, suggesting that the RNA substrate may be required for tight cofactor binding. Further, we have shown RlmM MTase activity on in vitro transcribed 23S rRNA and its domain V. RlmJ methylates the exocyclic N6 atom of A2030 in 23S rRNA. The 1.85Å crystal structure of RlmJ revealed a Rossmann-fold MTase domain with an inserted small subdomain unique to the RlmJ family. The 1.95Å structure of the RlmJ-SAH-AMP complex revealed that ligand binding induces structural rearrangements in the four loop regions surrounding the active site. The active site of RlmJ is similar to N6-adenine DNA MTases. We have shown RlmJ MTase activity on in vitro transcribed 23S rRNA and a minimal substrate corresponding to helix 72, specific for adenosine. Mutagenesis experiments show that residues Y4, H6, K18 and D164 are critical for catalytic activity. These findings have furthered our understanding of the structure, evolution, substrate recognition and mechanism of rRNA MTases.

Les sécrétines de l’hormone de croissance (GHRPs) sont de petits peptides synthétiques capables de stimuler la sécrétion de l’hormone de croissance à partir de l’hypophyse via leur liaison au récepteur de la ghréline GHS-R1a. Le GHRP hexaréline a été utilisé afin d’étudier la distribution tissulaire de GHS-R1a et son effet GH-indépendant. Ainsi, par cette approche, il a été déterminé que l’hexaréline était capable de se lier à un deuxième récepteur identifié comme étant le récepteur scavenger CD36. Ce récepteur possède une multitude de ligands dont les particules oxLDL et les acides gras à longue chaîne. CD36 est généralement reconnu pour son rôle dans l’athérogénèse et sa contribution à la formation de cellules spumeuses suite à l’internalisation des oxLDL dans les macrophages/monocytes. Auparavant, nous avions démontré que le traitement des macrophages avec l’hexaréline menait à l’activation de PPARƔ via sa liaison à GHS-R1a, mais aussi à CD36. De plus, une cascade d’activation impliquant LXRα et les transporteurs ABC provoquait également une augmentation de l’efflux du cholestérol. Une stimulation de la voie du transport inverse du cholestérol vers les particules HDL entraînait donc une diminution de l’engorgement des macrophages de lipides et la formation de cellules spumeuses. Puisque CD36 est exprimé dans de multiples tissus et qu’il est également responsable du captage des acides gras à longue chaîne, nous avons voulu étudier l’impact de l’hexaréline uniquement à travers sa liaison à CD36. Dans le but d’approfondir nos connaissances sur la régulation du métabolisme des lipides par CD36, nous avons choisi des types cellulaires jouant un rôle important dans l’homéostasie lipidique n’exprimant pas GHS-R1a, soient les adipocytes et les hépatocytes. L’ensemble de mes travaux démontre qu’en réponse à son interaction avec l’hexaréline, CD36 a le potentiel de réduire le contenu lipidique des adipocytes et des hépatocytes. Dans les cellules adipeuses, l'hexaréline augmente l’expression de plusieurs gènes impliqués dans la mobilisation et l’oxydation des acides gras, et induit également l’expression des marqueurs thermogéniques PGC-1α et UCP-1. De même, hexaréline augmente l’expression des gènes impliqués dans la biogenèse mitochondriale, un effet accompagné de changements morphologiques des mitochondries; des caractéristiques observées dans les types cellulaires ayant une grande capacité oxydative. Ces résultats démontrent que les adipocytes blancs traités avec hexaréline ont la capacité de se transformer en un phénotype similaire aux adipocytes bruns ayant l’habileté de brûler les acides gras plutôt que de les emmagasiner. Cet effet est également observé dans les tissus adipeux de souris et est dépendant de la présence de CD36. Dans les hépatocytes, nous avons démontré le potentiel de CD36 à moduler le métabolisme du cholestérol. En réponse au traitement des cellules avec hexaréline, une phosphorylation rapide de LKB1 et de l’AMPK est suivie d’une phosphorylation inhibitrice de l’HMG-CoA réductase (HMGR), l’enzyme clé dans la synthèse du cholestérol. De plus, la liaison d'hexaréline à CD36 provoque le recrutement d’insig-2 à HMGR, l’étape d’engagement dans sa dégradation. La dégradation de HMGR par hexaréline semble être dépendante de l’activité de PPARƔ et de l’AMPK. Dans le but d’élucider le mécanisme d’activation par hexaréline, nous avons démontré d’une part que sa liaison à CD36 provoque une déphosphorylation de Erk soulevant ainsi l’inhibition que celui-ci exerce sur PPARƔ et d’autre part, un recrutement de l’AMPK à PGC-1α expliquant ainsi une partie du mécanisme d’activation de PPARƔ par hexaréline. Les résultats générés dans cette thèse ont permis d’élucider de nouveaux mécanismes d’action de CD36 et d'approfondir nos connaissances de son influence dans la régulation du métabolisme des lipides. / Growth hormone releasing peptides (GHRPs) are small synthetic peptides aimed at stimulating GH release from the pituitary through their binding to ghrelin receptor known as growth hormone secretagogue receptor 1a (GHS-R1a). Using the GHRP, hexarelin to study tissue distribution of GHS-R1a and its GH-independent effect, it was observed that hexarelin was capable of binding to a second receptor identified as scavenger receptor CD36. While having multiple ligands, CD36 is mainly known for binding and internalizing oxLDL and long chain fatty acids. CD36 is thought to play a detrimental role in macrophage derived foam cell formation and development of atherosclerosis. Previously, we have shown that in macrophages, expressing both GHS-R1a and CD36, hexarelin promoted an activation of PPARƔ via GHS-R1a but also through its binding to CD36. This activation led to the induction of the LXRα-ABC transporters pathway and an increase in cholesterol efflux, reducing lipid-laden macrophage content. This positive effect on macrophages was reproduced in apolipoprotein E-null mice on a high fat diet treated with hexarelin. A significant reduction in the size of atherosclerotic lesions was observed while similar increases in the expression of PPARƔ, LXRα and ABC transporters occurred in isolated peritoneal macrophages. CD36 also plays a role in fatty acid uptake, and to further investigate the impact of the interaction of hexarelin with CD36, we aimed at evaluating the role of CD36 in regulating lipid metabolism in cells devoid of GHS-R1a such as adipocytes and hepatocytes. In the present thesis, we demonstrated through its interaction with hexarelin, the ability of CD36 to decrease intracellular lipid content in both adipocytes and hepatocytes. In adipocytes, hexarelin was able to increase the expression of several genes involved in fatty acid mobilization, fatty acid oxidation but also to induce the expression of the thermogenic markers, PGC-1α and UCP-1. In addition, hexarelin increased the expression of genes involved in mitochondrial biogenesis which was accompanied by mitochondrial morphological changes in agreement with what is usually seen in highly oxidative cells. In support of these findings, we also observed an increase in the activity of cytochrome c oxidase (a component of the respiratory chain) which could reflect an increase in oxidative phosphorylation. The results generated with cultured white adipocytes suggest the ability of hexarelin to promote changes toward a brown fat-like phenotype which also occurred in vivo and was dependent on the presence of CD36. In hepatocytes, CD36 was capable of regulating cholesterol metabolism by rapidly phosphorylating LKB1 and AMPK which subsequently resulted in the inactivating phosphorylation of HMG-CoA reductase, the rate-limiting enzyme in cholesterol synthesis. Hexarelin via CD36 also induced the recruitment of insig-2 to HMGR, the committed step in HMGR degradation while lifting the exerted inhibitory effect of Erk on nuclear receptor PPARƔ activity, and promoting the recruitment of AMPK to PPARƔ coactivator PGC-1α, suggesting an enhanced transcriptional potential of PPARƔ. The results generated during my graduate studies represent unique and novel mechanisms by which CD36 is capable of regulating lipid metabolism.

Η HDL είναι ένα μείγμα λιποπρωτεϊνικών σωματιδίων υψηλής πυκνότητας, που ανάλογα με τη σύσταση τους σε λιπίδια μπορούν να είναι δισκοειδή ή σφαιρικά. Η κύρια αθηροπροστατευτική δράση της HDL, οφείλεται στο γεγονός ότι η συγκεκριμένη λιποπρωτεΐνη συλλέγει την περίσσεια χοληστερόλης από τους περιφερικούς ιστούς και τη μεταφέρει στο ήπαρ όπου καταβολίζεται. Επιπλέον, έχει αντιφλεγμονώδη και αντιοξειδωτική δράση. Η κύρια πρωτεΐνη της HDL είναι η απολιποπρωτεΐνη Α-Ι (apoA-I). Ωστόσο, πρόσφατα αποδείχθηκε ότι σε πειραματόζωα με έλλειψη στην apoA-I και κατά συνέπεια στην κλασσική HDL, η απολιποπρωτεΐνη Ε (apoE) αλληλεπιδρά με τον μεταφορέα λιπιδίων ABCA1 προάγοντας την de novo σύνθεση HDL σωματιδίων. Στην παρούσα μελέτη, στόχος ήταν η εύρεση της περιοχής της apoE που είναι υπεύθυνη για την λειτουργική αλληλεπίδραση με τον ABCA1 για το σχηματισμό HDL. Για το σκοπό αυτό, ανασυνδυασμένοι αδενοϊοί που εξέφραζαν καρβοξυ-τελικές συντετμημένες μορφές της apoE4 (AdGFP-E4[1-259], AdGFP-E4[1-229], AdGFP-E4[1-202], AdGFP-E4[1-185]), χορηγήθηκαν σε ποντίκια με έλλειψη στην ApoA-I σε δόση 8x108 pfu και πέντε μέρες μετά τη μόλυνση δείγματα πλάσματος αναλύθηκαν για το σχηματισμό HDL. Κλασματοποίηση των λιποπρωτεϊνών του πλάσματος με υπερφυγοκέντρηση σε διαβάθμιση πυκνότητας καθώς και FPLC χρωματογραφία αποκάλυψε ότι όλες οι συντετμημένες μορφές της apoE4 προάγουν το σχηματισμό HDL. Ανάλυση ηλεκτρονικής μικροσκοπίας με αρνητική χρώση των HDL κλασμάτων, επιβεβαίωσε ότι όλες οι συντετμημένες μορφές της apoE4 είναι ικανές να προάγουν το σχηματισμό σωματιδίων με διάμετρο στην περιοχή της HDL. Τα δεδομένα αυτά οδηγούν στο συμπέρασμα ότι η αμινοτελική περιοχή της apoE που εκτείνεται από τα αμινοξέα 1 έως 185 αρκεί για το σχηματισμό HDL σωματιδίων in vivo. Αυτά τα ευρήματα, ανοίγουν το δρόμο στην έρευνα για το σχεδιασμό βιολογικών φαρμάκων με βάση την apoE για τη θεραπεία της δυσλιπιδαιμίας, της αθηροσκλήρωσης και της στεφανιαίας νόσου. / HDL is a mixture of high density lipoprotein particles that depending on the lipid composition may be discoidal or spherical. The main atheroprotective property of HDL is reverse cholesterol transport, a process that unloads excess cholesterol from peripheral tissues and transports it to the liver for catabolism. HDL has also anti-inflammatory and antioxidant properties. The main protein of HDL is apolipoprotein A-I (apoA-I). However, recently it was shown that in the absence of apoA-I and consequently classical HDL, apolipoprotein E (apoE) interacts functionally with the lipid transporter ABCA1, promoting the de novo synthesis of HDL-like particles. The present study focused on the identification of the domain of apoE that is responsible for the functional interaction with ABCA1 and the formation of apoE-containing HDL. Recombinant attenuated adenoviruses expressing carboxy-terminal truncated forms of apoE4 (apoE4[1-259], apoE4[1-229], apoE4[1-202], and apoE4[1-185]) were administered to apoA-I-deficient mice at a low dose of 8x108 pfu and five days post-infection plasma samples were isolated and analyzed for HDL formation. Fractionation of plasma lipoproteins of the infected mice by density gradient ultracentrifugation and FPLC revealed that all forms were capable of promoting HDL formation. Negative staining electron microscopy analysis of the HDL density fractions confirmed that all C-terminal truncated forms of apoE4 promoted the formation of particles with diameters in the HDL region. Taken together, these data establish that the aminoterminal 1 to 185 region of apoE suffices for the formation of HDL particles in vivo. These findings may have important ramifications in the design of apoE-based biological drugs for the treatment of dyslipidemia, atherosclerosis and coronary heart disease.

No description available.

La protéine BamB est une lipoprotéine de membrane externe appartenant au complexe BAM (β-Barrel Assembly Machinery) et impliquée dans l’assemblage des protéines de membrane externe (PME), la sensibilité aux antibiotiques, le contrôle de l’expression des trois systèmes de sécrétion de type III (T3SS) et la virulence de Salmonella. Chez E. coli, au sein du complexe BAM, elle interagit directement avec la protéine BamA. De plus, chez cette bactérie, BamB présente une activité sérine-thréonine kinase. Afin de mieux caractériser le rôle de BamB, nos objectifs ont été d’étudier (1) l’impact de l’altération de l’interaction de BamB avec le complexe BAM ou de sa séquestration dans le cytoplasme sur l’ensemble des rôles décrits de BamB et (2) l’activité kinase putative de BamB chez Salmonella. Nos résultats montrent que certains rôles de BamB sont dissociables entre eux et que l’interaction BamA/BamB n’est pas requise pour le rôle de BamB dans le contrôle de l’expression des T3SS, la virulence de Salmonella et l’assemblage des PME à la membrane externe. Aucune activité kinase ni aucune activité cytoplasmique de la protéine n’a pu être formellement démontrée. / BamB is an outer-membrane lipoprotein belonging to the BAM complex (β-Barrel Assembly Machinery). In Salmonella, it is involved in the assembly of outer membrane proteins (OMP), in antibiotic susceptibility, in the transcriptional control of the three Type-Three-Secretion-Systems (T3SS) related genes and also in virulence. In E. coli, BamB interacts directly with the BamA protein. Moreover, BamB has been shown to have a serine-threonin kinase activity in this bacterium. In order to better characterize the roles of the BamB protein, our purposes were to study (1) the impact of the alteration of the interaction of BamB with the BAM complex or of its cytoplasmic sequestration and (2) its putative kinase activity in Salmonella. Our results show that some of the BamB roles are dissociable and that the BamA/BamB interaction is not required for T3SS expression, Salmonella virulence or OMP assembly in the outer membrane. Currently, neither a kinase activity nor a cytoplasmic activity has been clearly demonstrated for this protein.

The studies presented in this thesis addressed mainly two aspects of Piwi-interacting RNA (piRNA) biology in the Drosophilagermline. We investigated the role of the piRNA pathway in embryonic axis specification. piRNAs mediate silencing of retrotransposons and the Stellate locus. Mutations in the Drosophila piRNA pathway genes armitage and aubergine disrupt embryonic axis specification, triggering defects in microtubule polarization and asymmetric localization of mRNA and protein determinants in the developing oocyte. Mutations in the ATR/Chk2 DNA damage signal transduction pathway dramatically suppress these axis specification defects, but do not restore retrotransposon or Stellatesilencing. Furthermore, piRNA pathway mutations lead to germline-specific accumulation of γ-H2Av foci characteristic of DNA damage. We conclude that piRNA based gene silencing is not required for axis specification, and that the critical developmental function for this pathway is to suppress DNA damage signaling in the germline. We have also identified a new member of the piRNA pathway. We show that mutations in rhino, which encodes a rapidly evolving Heterochromatin Protein 1 (HP1) chromo box protein, lead to germline specific DNA break accumulation, trigger Chk2 kinase dependent defects in axis specification, and disrupt germline localization of Piwi proteins. Mutations in rhino and the piRNA pathway gene armitage disrupt silencing of all major transposon families, but do not alter expression of euchromatic or heterochromatic protein coding genes. Deep sequencing studies show that rhino mutations significantly reduce or eliminate anti-sense piRNAs derived from the majority of transposable elements in the Drosophila genome, and lead to a dramatic reduction in piRNAs derived from major piRNA production clusters on chromosomes 2R and 4. Rhino protein localizes to distinct nuclear foci, and associates with the chromosome 2R and 4 clusters by chromatin immunoprecipitation. The Rhino HP1 homologue is therefore required for piRNA biogenesis, transposon silencing, and maintenance of germline genome integrity.

Les ribosomes sont des macrocomplexes ribonucléoprotéiques sophistiqués, essentiels pour décoder l’information génétique et la traduire en protéines fonctionnelles. Chez les organismes eucaryotes, le ribosome est constitué de deux sous-unités, la petite (40S) et la grande (60S). Leur biogenèse est un processus fondamental, très complexe, qui mène à la synthèse et l’assemblage de 4 ARNr et 80 protéines ribosomiques (79 chez la levure). La biogenèse du ribosome a longtemps été étudiée chez Saccharomyces cerevisiae. Près de 20 ans de recherches ont été nécessaires à la communauté scientifique pour identifier les quelques 200 facteurs de synthèse du ribosome levurien. Alors que le schéma global de cette voie de biosynthèse semble conservé chez les organismes eucaryotes, de nombreux éléments suggèrent qu’elle serait plus élaborée chez l’homme et nécessiterait un plus grand nombre de facteurs que chez la levure. De plus, la caractérisation de nombreuses ribosomopathies, ou maladies du ribosome prédisposant aux cancers, a suscité un intérêt accru pour l’étude de la voie de biosynthèse du ribosome dans le paradigme expérimental le plus approprié, la cellule humaine.<p><p>Au cours de ma thèse de doctorat, j’ai contribué à un projet systématique d’identification de facteurs d’assemblage (FA) du ribosome chez l’homme. Pratiquement, nous avons identifié 286 FA humains, dont beaucoup sont homologues aux facteurs levuriens connus, et 74 sont sans équivalent chez la levure. Par ailleurs, j’ai caractérisé en détail certains facteurs. En particulier, Trm112 pour lequel j’ai montré qu’il agit comme un stabilisateur de la méthyltransférase (MTase) Bud23, spécifique à l’ARNr 18S de la sous-unité levurienne 40S. J’ai également participé à la caractérisation de mutations à l’interface du complexe Bud23-Trm112. Enfin, j’ai contribué à l’étude de trois FA que nous avons identifiés chez l’homme, DIMT1L et WBSCR22-TRMT112. J’ai montré que ces protéines sont les orthologues des MTases levuriennes Dim1 et Bud23-Trm112, qu’elles sont requises pour la synthèse et la modification de l’ARNr mature de la petite sous-unité ribosomique, et qu’elles seraient impliquées dans un mécanisme conservé contrôlant la qualité de la voie de biosynthèse du ribosome.<p><p>La totalité des FA que nous avons identifiés en cellule humaine sont à la disposition de la communauté scientifique dans une base de données en ligne accessible sur la page www.RibosomeSynthesis.com. Nous espérons que cette ressource contribuera à une meilleure compréhension des mécanismes moléculaires sous-jacents au développement des ribosomopathies et à l’élaboration d’agents thérapeutiques efficaces.<p> / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished

A cardiomiopatia Chagásica Crônica (CCC) é a principal complicação decorrente da infecção pelo protozoário hemoflagelado Trypanosoma cruzi (T. cruzi). Trata-se de uma cardiomiopatia dilatada, caracterizada por um intenso infiltrado inflamatório, fibrose, dilatação das câmaras cardíacas, hipertrofia de cardiomiócitos e anormalidades de condução. Sua fisiopatologia é complexa e ainda não se consegue explicar porque apenas 30% dos pacientes infectados desenvolvem essa complicação. Nesse contexto, nosso laboratório descreveu pela primeira vez uma redução na expressão de microRNAs (miRNAs) enriquecidos em músculo (myomiRs) no miocárdio de pacientes com CCC. Sabendo-se que disfunções na biogênese de miRNAs em modelos animais levam ao desenvolvimento de cardiomiopatia do tipo dilatada com redução da expressão de myomiRs, hipotetizou-se que a CCC em humanos estaria associada a um prejuízo na biogênese de miRNAs no miocárdio. Dessa forma, amostras de ventrículo esquerdo de miocárdio de pacientes com CCC (n=16) e controles não-cardiomiopatas (n=6) foram utilizadas para avaliar: 1) a expressão gênica e proteica da maquinaria da biogênese de miRNAs (Drosha, Exportina-5, RAN, Dicer1, TRBP, PACT e Argonauta2), por qPCR e western blotting, respectivamente; 2) a expressão do transcrito primário (pri-miRNA), precursor (pré-miRNA) e miRNA maduro de myomiRs (miR-1, -133a, -133b, -208a, -208b, e -499); 3) o perfil de miRNAs diferencialmente expressos em CCC utilizando qPCR array; e 4) a interação dos miRNAs diferencialmente expressos com disfunções características da CCC (fibrose, miocardite, arritmia e hipertrofia) por meio de análises de bioinformática. Nossos resultados apontam para uma não-alteração nas etapas nucleares da biogênese de miRNAs (transcrição, edição e transporte), já que não foram encontradas alterações na expressão de pri- e pré-miRNAs de myomiRs, bem como dos componentes protéicos da biogênese, Drosha, Exportina-5 e RAN. Entretanto, observou-se uma disfunção na segunda etapa de edição da biogênese, citoplasmática, caracterizada por uma redução de 2/3 nos níveis protéicos de Dicer1, a qual não foi acompanhada por uma redução na expressão de seu RNA mensageiro. Evidenciou-se ainda, uma redução na expressão de 97,5% dos miRNAs maduros diferencialmente expressos no miocárdio de pacientes com CCC, incluindo myomiRs. As análises in silico revelaram haver participação dos miRNAs diferencialmente expressos em disfunções associadas a CCC, com destaque para a fibrose miocárdica, nodo central da rede. Experimentos adicionais preliminares sugeriram o acúmulo de adutos de 4-hidroxi-2-nonenal, decorrente do estresse oxidativo e de uma menor atividade da enzima aldeído desidrogenase 2, como uma possível causa para as alterações encontradas. Este é o primeiro estudo a caracterizar a biogênese de microRNAs em uma cardiomiopatia. Além disso, demonstrou-se que uma redução global do perfil dos miRNAs maduros diferencialmente expressos, decorrente uma disfunção na enzima Dicer1, está associada a eventos patológicos característicos da CCC. Estes mecanismos apresentam relevância biológica e terapêutica, podendo ser possivelmente compartilhados com cardiomiopatias de outras etiologias / Chronic Chagas disease cardiomyopathy (CCC) is the most severe complication of the infection by the haemoflagellate protozoan Trypanosoma cruzi. This dilated cardiomyopathy is characterized by an intense inflammatory infiltrate, fibrosis, dilation of cardiac chambers, cardiomyocyte hypertrophy and conduction abnormalities. Its pathophysiology is complex and why only 30% of patients experience this complication remains an open question. In this regard, our laboratory described for the first time a reduction in the expression of muscle-enriched microRNAs (myomiRs) in human CCC myocardium. Knowing that biogenesis dysfunction and myomiR reduced expression have been associated to the development of dilated cardiomyopathy in animal models, we hypothesized that an impairment of myocardial microRNA biogenesis would be associated to CCC. Hence, left ventricle tissue samples from CCC patients (16) and non-cardiomyopathy donors (6) were used to analyze: 1) mRNA and protein expression, by qPCR and western blotting, of canonical microRNA biogenesis machinery (Drosha, Exportin-5, RAN, Dicer1, TRBP, PACT, AGO2); 2) primary transcript (pri-miR), precursor (pre-miR) and mature microRNA expression of myomiRs (miR-1, -133a, -133b, -208a, -208b, e -499); 3) mature microRNA profile using qPCR array; and 4) the interaction between differentially expressed mature microRNAs and hallmark CCC dysfunctions (fibrosis, myocarditis, hypertrophy and arrhythmia) using bioinformatics tools. Our results point to a non-dysfunction of biogenesis nuclear steps (transcription, editing and transport), since expression of pri-, pre-microRNAs, Drosha, Exportin-5 and Ran are similar between CCC patients and controls. However, we observed an alteration in the cytoplasmic editing step, characterized by a 2/3 reduction in Dicer1 protein levels. In addition, a major downregulation of differentially expressed mature microRNAs (97,5%) was noticed. In silico analysis revealed an association between differentially expressed microRNAs and CCC hallmarks, particularly fibrosis, a central node in the network. Additional preliminary data suggest 4-hydroxi-2-nonenal myocardial accumulation, resulting from oxidative stress and aldehyde dehydrogenase 2 lower activity, as a possible cause for the alterations here described. This is the first study to conduct a comprehensive analysis of microRNA biogenesis machinery in a cardiomyopathy. Moreover, we have shown a major reduction in the expression of mature microRNAs, due to lower Dicer1 protein levels, to be associated to CCC hallmark dysfunctions. These mechanisms are biologically and therapeutically relevant, and may be shared with cardiomyopathies from different etiologies

Mitochondria are essential eukaryotic organelles, acting as the sites for numerous crucial metabolic and signalling pathways. The biogenesis of mitochondria requires efficient targeting of several hundreds of proteins from the cytosol, to their varied functional locations within the organelle. The translocation of localized proteins across the inner membrane, and their subsequent folding is achieved by the ATP-dependent function of mitochondrial Hsp70 (mtHsp70). It is a bonafide member of the Hsp70 chaperone family, which are involved in a multitude of functions, together aimed at protein quality control and maintenance of cellular homeostasis. These varied functions of Hsp70 proteins require binding to exposed hydrophobic patches in substrate polypeptides thus preventing non-productive associations. The interaction with substrates occurs through the substrate-binding domain (SBD) and is regulated by the ATPase activity of the nucleotide-binding domain (NBD), through a series of conformational changes. Conversely, substrate binding to the SBD also stimulates ATP hydrolysis, and thereby the core activities of the two domains are regulated by mutual allosteric signalling. This mechanism of bidirectional inter-domain communication is indispensable for Hsp70 function, which is characterized by cycles of substrate binding and release, coupled to cycles of ATP binding and hydrolysis. The process of allosteric regulation in Hsp70 proteins has been comprehensively investigated, especially in the bacterial homolog, DnaK. However, the in vivo functional significance of inter-domain communication in the eukaryotic mtHsp70 system and the mechanism of its regulation remain unexplored. Furthermore, the complex physiological implications of impairment in allosteric communication and their correlation with diverse disease conditions, including Myelodysplastic syndrome (MDS), and Parkinson’s disease (PD), are yet to be elucidated. Based on this brief introduction, the primary research objectives set out in the present thesis were to: 1. uncover the regulation of ligand-modulated allosteric communication between the two domains of mtHsp70; and its in vivo significance in the context of protein import into the organelle. (Chapter 2) 2. understand the role of mtHsp70 in progression of Parkinson’s disease; and to study the modulation of α-synuclein toxicity by the protein quality control function of the mtHsp70 chaperone network. (Chapters 3 and 4) We have employed a battery of genetic and biochemical approaches to investigate the above questions using the Saccharomyces cerevisiae mtHsp70 protein, Ssc1; an essential protein that is involved in a plethora of critical functions in this eukaryotic model system. Objective 1: Structural studies, primarily in bacterial DnaK, have yielded mechanistic insights into its interactions with ligands and cochaperones, as well as conformational transitions in different ligand-bound states. In recent years, the availability of crystal structures of full-length DnaK and detailed information from NMR studies and single-molecule resolution spectroscopic analyses (both DnaK and eukaryotic Hsp70s), have significantly contributed to our understanding of the inter-domain interface, critical residues and contacts, and the energetics of the entire process of ligand-modulated conformational changes. Although eukaryotic mtHsp70s have a high degree of conservation with DnaK, they possess significant differences in their conformational and biochemical properties. They are essential for a vast repertoire of physiological functions, which are distinctly different from their bacterial counterpart. Using a combined in vivo and in vitro approach, we have uncovered specific structural elements within mtHsp70s, which are required for allosteric modulation of the chaperone cycle and maintenance of in vivo functions of the protein. Foremost, we demonstrate that a conserved SBD loop, L4,5 plays a critical role in inter-domain communication, and multiple mutations in this loop result in significant growth and protein translocation defects. The mutants are associated with a specific set of altered biochemical properties, which are indicative of impaired inter-domain communication. Using the loop L4,5 mutant, E467A as a template for genetic screening, we report a series of intragenic suppressor mutations, which are capable of correcting a distinct subset of the altered properties, and thereby leading to restoration of in vivo functions, including growth, preprotein import and mitochondria biogenesis. The suppressors modify the altered conformational landscape associated with E467A, and also provide us with information regarding unique aspects governing the regulation of allosteric communication, especially in physiological contexts. Strikingly, they reveal that restoration of communication in the NBD to SBD direction is sufficient for function, when the protein is primed in a high ATPase activity state. In this unique scenario, the requirement for ATPase stimulation upon substrate binding is rendered unnecessary, thereby making conformational changes in the SBD to NBD direction, dispensable for function. Further, we provide evidence to show that loop L4,5 functions synergistically with the linker region, working in tandem for organization of the inter-domain interface and propagation of communication. Together, our analyses provide the first insights into regulation of allosteric inter-domain communication in vivo and their implications in mitochondrial protein translocation and organelle biogenesis. Objective 2: Point mutations in the loop L4,5 have been associated with Myelodysplastic syndrome. Additionally, a mutation isolated in clinical cases of Parkinson’s disease was found to be impaired in allosteric communication. These observations further highlight the importance of efficient inter-domain communication in mtHsp70 in the complex physiological scenario of eukaryotic cells. Independent clinical screens of PD patients have revealed unique point mutations in the mtHsp70 and a strong association of the gene locus with the disease progression. This is also correlated with decreased mtHsp70 levels in affected neurons and the interactions of this protein with established PD-candidate proteins like α-synuclein and Dj-1. Further, mitochondrial dysfunction is a common phenomenon associated with neurodegenerative disorders. To understand the specific role of mtHsp70 in PD, we have developed a yeast model for studying the disease variants in isolation from other players of the multifactorial disease, and in complete absence of the wild type protein. We generated two analogous PD-mutations in Ssc1, R103W and P486S; which recapitulated the symptoms of mitochondrial dysfunction in affected neurons, including cell death, inner membrane depolarization, increased generation of ROS, and respiratory incompetence. At the molecular level, we observed an increased aggregation propensity of R103W, while P486S exhibited futile enhanced interaction with J-protein cochaperone partners thereby resulting in loss of chaperoning activity and impaired mitochondrial protein quality control. Remarkably, these altered biochemical properties mimicked similar defects in the human mtHsp70 variants, therefore, affirming the involvement of mtHsp70 in PD progression. To further investigate the relevance of impaired mitochondrial protein quality control in PD, we have explored whether mtHsp70 can act as a genetic modifier of α-synuclein toxicity. It is known that α-synuclein can act as an unfolded substrate for the Hsp70 chaperone system and also deposits as intracellular aggregates in PD-affected brains. Intriguingly, it is known to translocate into mitochondria under conditions of neuronal stress in spite of lacking a canonical mitochondrial signal sequence. Utilizing our yeast-PD model, we find that targeting of α-synuclein A30P disease variant into mitochondria leads to a severe mitochondrial dysfunction phenotype in the wild type Ssc1 background, but not the P486S mutant background. This results in multiple cellular manifestations, which are reversed upon overexpression of the Ssc1 chaperone. Significantly, increasing the J-protein cochaperone availability also leads to reversal of the mutant-associated defects. However, the simultaneous overexpression of both together does not additively improve the protective effects; highlighting the importance of the relative availability of chaperone and cochaperone proteins in preventing aggregation. Our analyses further reveal that while both the wild type and P486S Ssc1 proteins are equally capable of delaying aggregation of α-synuclein, only the wild-type chaperone is better able to prevent aggregation in the presence of its J-protein cochaperone, leading to accumulation of soluble oligomeric species. These observations raised the intriguing possibility, that the reduced chaperoning ability of the proline to serine PD-mutant is, in fact, a compensatory adaptation, favoring the aggregation of α-synuclein over its more toxic soluble oligomeric form. We verify this hypothesis with the aggregation kinetics of A30P α-synuclein, whose intrinsically lower aggregation tendency results in a pronounced delay in aggregation with the wild-type chaperone, thereby strongly favoring the toxic oligomeric species and correlating with the observed lethality in yeast cells. In conclusion, our study provides a model of α-synuclein aggregation-related toxicity and its modulation by the extent of protein quality control within the mitochondrial matrix, through the action of the mtHsp70 chaperone network.

La biogenèse du ribosome est un processus complexe et dynamique qui nécessite de nombreuses étapes de maturation et de modification des ARNr ainsi que l’assemblage et le transport des RNPs précurseurs. Un ribosome mature contient une centaine de pièces, ARN et protéines confondus, mais son assemblage requiert l’intervention de plus de 400 facteurs de synthèse. De part le coût énergétique important de ce processus, plusieurs voies de régulation interviennent pour contrôler la biogenèse des ribosomes en fonction des conditions nutritives. L’une des voies les plus connue est la voie TOR (Target of rapamycin). Cette voie de régulation agît principalement au niveau de la transcription des différents intervenants de la biogenèse :les ARNr, les protéines ribosomiques mais aussi les facteurs de synthèse. Ces facteurs, ayant une action transitoire dans la maturation des ribosomes, sont, par économie, recyclés pour la synthèse de nouveaux ribosomes. Nous nous sommes donc intéressés au devenir de ces facteurs, plus particulièrement de ceux intervenants dans la biogenèse de la petite sous unité, lorsque les conditions environnementales sont inadaptées à la croissance cellulaire. Ainsi, nous avons pu montré, pour quatre facteurs particuliers :Dim2, Rrp12, Hrr25 et Fap7, que leur localisation est dépendante de la synthèse ribosomique. Ainsi, lors de carence en sources nutritives, l’inhibition de la synthèse et de l’activité ribosomique entraîne un confinement de ces facteurs ribosomiques dans le nucléole ou dans des corps cytoplasmiques. En outre, la localisation particulière des facteurs ribosomiques Hrr25 et Fap7 dans les P-bodies en phase de croissance saturée laisse penser que ces corps cytoplasmiques sont le lieu de dégradation des pré-ribosomes lorsque les carences nutritives perdurent. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished