Orientador: Jorg Kobarg / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-07T08:03:42Z (GMT). No. of bitstreams: 1 Passos_DarioOliveirados_D.pdf: 4831709 bytes, checksum: 0aa3e031d4e82dc447636416b68401e8 (MD5) Previous issue date: 2006 / Resumo: A proteína Ki-1/57 que é encontrada tanto no núcleo quanto no citoplasma está associada com atividade de proteína quinase serina/treonina e é fosforilada nestes resíduos após ativação celular. Neste trabalho verificamos que Ki-1/57 interage com a proteína Chromatin-Helicase-DNA-binding domain 3 (CHD3) e com a proteína adaptadora/sinalizadora RACK1 no núcleo. Pelo sistema do duplo híbrido de levedura (SDHL) a proteína arginina metiltransferase 1 (PRMT1) foi selecionada como outra proteína de interação. A PRMT1 integra uma família representada por nove enzimas humanas que catalisam reações de metilação em resíduos de arginina. Em seguida, usando agora a PRMT1 como isca - no SDHL - identificamos as proteínas Ki-1/57 e hnRNPQ, juntamente com outras 13. A maioria delas contêm motivos ¿RGG-box¿ em suas seqüências de aminoácidos, que são conhecidos alvos para metilação. Posteriormente verificamos que Ki-1/57 e seu provável parálogo CGI-55 conservam dois motivos ¿RGG/RXR-box¿ e que são substratos in vitro para a metilação de argininas pela PRMT1. Estudos de mapeamento mostraram que todos os fragmentos contendo o motivo ¿RGG/RXR-box¿ interagem com a PRMT1 e são alvos à metilação in vitro. Ki-1/57 endógena, imunoprecipitada de células L540, mostrou ser metilada in vivo, além de ser um alvo a metilação pela PRMT1 in vitro, somente quando as células são previamente tratadas com o inibidor da metilação Adox. Tratamento das células Hela com o inibidor da metilação (Adox) causa desaparecimento da imuno-marcação citoplasmática de Ki-1/57 e relativa redistribuição do parálogo CGI-55 para o citosol. Assim, pode ser especulado que a metilação destas proteínas deve ser um evento importante para suas localizações subcelulares e conseqüentemente para suas funções. Em resumo, nossos dados sugerem que o SDHL é um método efetivo na identificação de novos substratos celulares para a PRMT1 e poderia ser estendido para a identificação e caracterização de novos substratos para os outros integrantes da família das PRMTs humanas / Abstract: The protein Ki-1/57 that is found both in the cytoplasm and nucleus is associated with serine/threonine protein kinase activity and gets phosphorylated on serine and threonine residues upon cellular activation. We demonstrated that Ki-1/57 interacts with the Chromatin-Helicase-DNA-binding domain protein 3 (CHD3) and with the adaptor/signaling protein RACK1 in the nucleus. By utilizing the yeast two-hybrid system (YTHS), we were further able to find the protein arginine-methylatranseferase-1 (PRMT1) as another interacting protein. PRMT1 is a member of the family constituted by 9 human enzymes that catalyze methylation reactions on arginine residues. Afterwards, by using PRMT1 as bait in the YTHS we identified both Ki-1/57 and NSAP1 as interacting proteins, along with 13 other proteins. The majority of them present RGG-box clusters in their amino acid sequences, which are known to be targets for arginine methylation. We further found that Ki-1/57 and its putative paralogue CGI-55 have two RGG/RXR-box clusters conserved between them and that they are substrates for arginine-methylation by PRMT1 in vitro. In mapping studies, we observed that all Ki-1/57 protein fragments containing the RGG/RXRbox clusters interact with PRMT1 and are targets for methylation in vitro. Endogenous cellular Ki-1/57 seems to be methylated in vivo and is a target for methylation by PRMT1 in vitro, only when cells have been previously treated with the methylation inhibitor Adox. Treatment of Hela cells with the inhibitor of methylation (Adox) causes the disappearance of the immuno-staining of Ki-1/57 in the cytoplasm and a relative redistribution of the paralogue CGI-55 to the cytosol. It can therefore be speculated that the methylation of these proteins is important for their sub-cellular localization and in consequence for their function. In summary our data suggest that the YTHS is an effective method for the identification of novel cellular PRMT substrates and could be extended for the identification and characterization of novel substrates to the other components of the human PRMT1 family / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

O estudo de venenos de artrópodes é de grande interesse para melhorar os tratamentos contra envenenamentos e oferece uma ótima ferramenta para melhor compreensão dos sistemas nervoso e imunológico, coagulação sanguínea e respostas inflamatórias. As abelhas são um dos animais venenosos mais estudados e a elucidação do seu proteoma é de interesse na elucidação de reações tóxicas e alérgicas a ferroadas. O número de acidentes envolvendo estes insetos é crescente, tendo ultrapassado 20.000 notificações entre 2001 e 2006 em todo o país e, apesar disso, não há um tratamento específico para estas vítimas, nem mesmo uma identificação completa dos antígenos presentes nesse veneno. O perfil protéico descrito até então apresenta cerca de 40 proteínas. O objetivo deste trabalho foi identificar o perfil protéico do veneno de abelhas utilizando a união da abordagem proteômica e da cromatografia de afinidade. Identificar também as proteínas alergênicas deste veneno e algumas modificações pós-traducionais como fosforilação e glicosilação. Além disso, um soro antiveneno específico foi produzido e sua ação neutralizadora testada. O veneno de abelhas foi separado por cromatografia de afinidade utilizando o soro antiveneno imobilizado em coluna de Sepharose 4B. Para identificação das proteínas foram utilizadas técnicas de 2D-SDS-PAGE, MALDI TOF/TOF e nanoESI-LC/MS-MS. Ensaios de Western Blotting foram realizados para identificar as proteínas alergênicas e fosforiladas. A utilização da cromatografia de afinidade permitiu a identificação 2 de proteínas pouco abundantes. Foram identificadas 54 proteínas, dentre as quais 9 nunca haviam sido descritas neste veneno, como MRJP-2, alfaglicosidase, transferinas, proteases, quinases e um inibidor de protease. Após a identificação destas proteínas foi possível propor um provável mecanismo de ação deste veneno. Dentre as proteínas identificadas como alergênicas, a MRJP-8 foi identificada pela primeira vez, juntamente com fatores relacionados ao PDGF e VEGF. Os resultados dos ensaios de neutralização de atividades citotóxicas, hemolíticas e miotóxicas mostraram a eficiência do soro antiveneno produzido. Chegou-se a um volume de 5,7 mL de soro antiveneno necessários para neutralizar a ação tóxica provocada por 100 ferroadas de abelhas. Este valor está na mesma faixa de eficiência dos melhores antivenenos (ofídicos, aracnídicos e escorpionídicos) produzidos no Brasil e no mundo. O lote de soro antiveneno produzido mostrou resultados satisfatórios para ser utilizado nos testes clínicos / The aim of this work was to identify the protein profile of honeybee venom, and detect allergenic proteins and post-translational modifications. Furthermore specific antivenom was produced and potency tests were performed in order to check its power of neutralization of toxic activities of venom. They were identified 54 proteins, 9 that have never been reported before in this venom. After identification of these proteins it was possible to outline a feasible mechanism of action of venom. For the first time MRJP-8, transferrin, PDGF and VEGF factors were identified as allergenic. Results of neutralization of citotoxic, hemolytic and myotoxic activities showed the efficacy of antivenom that had satisfactory results to be tested in clinical assay

Caenorhabditis elegans possui quatro genes de kpcs (kex2/subtilisin-like proprotein convertases): kpc-1, kpc-2/egl-3, kpc-3/aex-5, kpc-4/bli-4. Em C. elegans, dois dos quatro polipeptídeos de vitelogenina encontrados dentro dos ovócitos, YP115 e YP88, se originam a partir de um precursor polipeptídico (VIT-6) clivado pós-traducionalmente após o motivo RGKR. Nematóides transgênicos foram produzidos com construções repórteres transcricionais de GFP. Foi verificada expressão de kpc-1 tanto em neurônios quanto em células musculares e intestinais. Esses dados, aliados aos dados da literatura para os outros genes kpc de C. elegans, sugerem o envolvimento de KPC-1 no processamento de VIT-6, que é secretada por células intestinais. Ensaios de Western-blot compararam o processamento de VIT-6 em nematóides selvagens, mutantes e knock-down por RNAi para os diferentes genes kpc. A análise de nematóides mutantes e knock-down por RNAi combinado para os outros três genes de convertase de C. elegans confirmou a redundância da atividade dessas enzimas no processamento de VIT-6. / Four kpc genes are found in the Caenorhabditis elegans genome: (kex2/subtilisin-like proprotein convertases): kpc-1, kpc-2/egl-3, kpc-3/aex-5, kpc-4/bli-4. Two of the four vitellogenin polypeptides, YP115 and YP88, originate from a precursor, VIT-6. VIT-6 is cleaved post-translationally after the RGKR motif. Transgenic worms carrying GFP transcription reporter constructs were produced. Expression of kpc-1 has been localized to neurons as well as muscular and intestinal cells. These data, together with the ones available from the literature for the other kpc genes, suggest the involvement of KPC-1 in the processing of VIT-6, which is secreted from intestinal cells. Western-blot analysis compared the pattern of VIT-6 processing in wild-type, mutants and RNAi-treated worms for the other kpcs. Analysis of worms treated by combined RNAi confirmed the redundancy of KPCs in VIT-6 processing.

En influençant le degré de compaction de la chromatine ainsi que ses interactions avec différents partenaires protéiques, les modifications post-traductionnelles des histones sont impliquées dans la régulation de l’expression des gènes. Avec les différents variants d’histones incorporés dans la chromatine, ces modifications dynamiques et sensibles à l’environnement sont constitutives du code histone. Ce travail présente une approche globale de criblage baptisée approche histonomique, visant à révéler une perturbation épigénétique à l’échelle des histones. Cette approche originale offre une comparaison rapide et fiable des abondances relatives des variants d’histones et de leurs modifications post-traductionnelles dans des cellules humaines en une seule analyse LC-MS. Comme preuve de concept, des cellules BeWo issues de choriocarcinome humain ont été exposées au butyrate de sodium, un inhibiteur non spécifique d’histones désacétylases. Les histones extraites des échantillons témoins ou traités au butyrate de sodium à 1 ou 2,5 mM ont été analysées par chromatographie liquide ultra performante couplée à un spectromètre de masse de type Q-TOF. Les analyses statistiques multivariées ont permis de discriminer les échantillons témoins des échantillons traités sur la base des différences de degrés d’acétylation observés sur plusieurs formes d’histones. La même approche a ensuite été appliquée à des cellules exposées au B[a]P à 1 μM et a révélé deux principaux marqueurs caractéristiques d’un remodelage de la chromatine induit par les effets génotoxiques duB[a]P. En résumé, cette approche histonomique globale pourrait se révéler être un outil complémentaire très utile pour explorer une potentielle perturbation du code histone lors d’exposition à des xénobiotiques environnementaux. / While acting upon chromatin compaction, histone post-translational modifications (PTMs) are involved in modulating gene expression through histone–DNA affinity and protein–protein interactions. These dynamic and environment-sensitive modifications are constitutive of the histone code that reflects the transient transcriptional state of the chromatin. Here we describe a global screening approach for revealing epigenetic disruption at the histone level. This original approach enables fast and reliable relative abundance comparison of histone PTMs and variants in human cells within a single LC-MS experiment. As a proof of concept, we exposed BeWo human choriocarcinoma cells to sodium butyrate (SB), a universal histone deacetylase (HDAC) inhibitor. Histone acide xtracts equally representing 3 distinct classes, Control, 1 mM and 2.5 mM SB, we reanalyzed using ultra-performance liquid chromatography coupled with a hybrid quadrupoletime-of-flight mass spectrometer (UPLC-QTOF-MS). Multivariate statistics allowed us to discriminate control from treated samples based on differences in the acetylation level of several histone forms. We then applied the same procedure to cells treated with 1 μMB[a]P and suceeded in revealing two markers of chromatin remodeling in relation withgenotoxic properties of B[a]P. Indeed, this untargeted histonomic approach could be auseful exploratory tool in many cases of environmental xenobiotic exposure when histone code disruption is suspected.

The 2OG oxygenases comprise a superfamily of ferrous iron dependent dioxygenases with multiple biological roles, including in hypoxia sensing, transcriptional control, and splicing control. It was recently proposed that 2OG oxygenases catalyse the hydroxylation of ribosomal proteins in prokaryotes (ycfD) and in humans (NO66 and MINA53), raising the possibility that 2OG oxygenases also control translation. The work described in this thesis concerned investigations on the biochemical and functional aspects of prokaryotic and mammalian ribosomal protein hydroxylases (ROX) in vitro and in cells. An efficient chromatographic system linked to mass spectrometric analysis (LC-MS) was developed for studying the masses of individual ribosomal proteins (>90% coverage of ribosomal proteome) to ±1 Da accuracy. It was demonstrated that ycfD catalyses the hydroxylation of R81 on L16 in E. coli, in a manner dependent on atmospheric oxygen levels. YcfD deletion results in growth phenotype at low temperatures and in minimal medium, and in decreased global translation rates in minimal medium; ycfD deletion does not affect translational accuracy and ribosome assembly. Furthermore, ycfD-deletion results in increased sensitivity to the antibiotics chloramphenicol and lincomycin. Consistent with a 2OG-oxygenase mediated mechanism of antibiotic resistance, chloramphenicol sensitivity of the E. coli wild-type strain could be increased by inhibiting the activity of ycfD by removing co-factors required for catalytic activity (Fe(II) and O2), and, at least in part, by using a ycfD inhibitor, IOX1, which inhibits ycfD with IC₅₀ of 38 μM in vitro. The therapeutic potential of a post-translational modification mediating antibiotic resistance provides an opportunity for medicinal targeting of ribosome-modifying enzymes, for example ycfD, which may be more ‘druggable’ than the ribosome itself. In co-treatment with an existing antibiotic, such as chloramphenicol, a small molecule inhibitor would achieve a potentiated antibiotic effect. Structural aspects of ROX hydroxylation were pursued by characterising a thermophilic ROX-substrate complex; a ycfD homologue was identified in the thermophilic bacterium Rhodothermus marinus and shown to be a thermophilic 2OG oxygenase ycfD_RM, acting on R82 of ribosomal protein L16_RM. The activity of ycfD_RM in cells was limited at high growth temperature and oxygen solubility was demonstrated as a likely limiting factor of ycfD_RM activity, thus identifiying a potential 2OG oxygenase oxygen sensor in prokaryotes. A crystal structure of ycfD_RM in complex with L16RM substrate fragment was determined to 3.0 Å resolution. Structural analyses suggested that ycfD_RM contains 30% more hydrophobic interactions and 100% more salt-bridge interactions than ycfD_EC, suggesting that these interactions are important for thermal stabilisation of ycfD_RM. The structures reveal key interactions required for binding of ribosomal proteins. Substantial structural changes were observed in the presence of the substrate fragment, which implies induced-fit binding of the L16_RM substrate. The work has informed further structural studies on the evolutionarily related human ROX, NO66 and MINA53, for which substrate structures have been obtained since the completion of the work. The LC-MS analysis of ribosomal proteins was extended to mouse and human cells to demonstrate that the human ROX homologue of ycfD, MINA53, hydroxylates the 60S ribosomal protein rpL27a in cells. It was demonstrated that rpL27a hydroxylation is widespread and found in all mouse organs analysed, as well as in cancer cell lines and in clinical cancer tissues. A partial or complete reduction of rpL27a hydroxylation was observed in a number of clinically identified MINA53 mutations from the COSMIC database of cancer mutations. Structural analysis suggested that mutations occur more frequently at structurally important regions of MINA53, including the βIV-βV insert in the core fold of MINA53. The identification of inhibiting clinical mutations suggests that rpL27a hydroxylation level could be used as a cancer mark, and in the future for selective inhibition by ribosomal antibiotics. The work presented in this thesis demonstrates that it is possible to selectively inhibit modified ribosomes; an inhibitor of unhydroxylated rpL27a could therefore, at least in principle, be active against the sub-set of tumours with inactivating mutation(s) of MINA53, but not normal tissue. Future work should therefore focus on identifying a selective inhibitor of unhydroxylated eukaryotic ribosomes which could be applied for treatment of cancers harbouring deactivating MINA53 mutations. The same approach could be applied to other ribosome modifications (to rRNA, ribosomal proteins, and ribosome-associate factors) that are different in cancer compared to normal cells.

Our understanding of protein lysine methyltransferases and their substrates remains limited despite their importance as regulators of the proteome. The SMYD (SET and MYND domain) methyltransferase family plays pivotal roles in various cellular processes, including transcriptional regulation and embryonic development. Among them, SMYD2 is associated with oesophageal squamous cell carcinoma, bladder cancer and leukemia as well as with embryonic development. Initially identified as a histone methyltransferase, SMYD2 was later reported to methylate p53, the retinoblastoma protein pRb and the estrogen receptor ERalpha and to regulate their activity. Our proteomic and biochemical analyses demonstrated that SMYD2 also methylates the molecular chaperone HSP90 on K209 and K615. We also showed that HSP90 methylation is regulated by HSP90 co-chaperones, pH, and the demethylase LSD1. Further methyltransferase assays demonstrated that SMYD2 methylates lysine K* in proteins which include the sequence [LFM]-₁-K*-[AFYMSHRK]+₁-[LYK]+₂. This motif allowed us to show that SMYD2 methylates the transcriptional co-repressor SIN3B, the RNA helicase DHX15 and the myogenic transcription factors SIX1 and SIX2. Finally, muscle cell models suggest that SMYD2 methyltransferase activity plays a role in preventing premature myogenic differentiation of proliferating myoblasts by repressing muscle-specific genes. Our work thus shows that SMYD2 methyltransferase activity targets a broad array of substrates in vitro and in situ and is regulated by intricate mechanisms.

Les gènes Hox codent des facteurs de transcription à homéodomain (HD). Bien que ce dernier reconnaisse des séquences similaires in vitro, les protéines Hox achèvent des fonctions hautement spécifiques in vivo. Des séquences protéiques en dehors de l’HD influencent la spécificité d’action des protéines Hox par le recrutement de cofacteurs, dont le mieux caractérisé est Extradenticle (Exd) chez la drosophile. Des travaux récents au sein de notre équipe ont démontré la contribution fonctionnelle de trois motifs de AbdA, aussi bien dans des fonctions Exd-dépendantes qu’à des fonctions Exd-indépendantes. Mon travail de thèse a porté sur la caractérisation de la contribution des motifs protéiques de AbdA dans la sélection puis dans la régulation des gènes cibles en utilisant une approche combinée ChIPseq/RNAseq, dans un contexte Exd-indépendant. Le code ADN identifié nous a renseigné sur la présence d’inputs transcriptionnels additionnels. Ces derniers correspondant à des facteurs de transcription déjà connus, leur présence dans un complexe protéique avec AbdA a été démontrée par des analyses de spectrométrie de masse. Un second volet de mon travail de thèse a été l’identification de modifications post-traductionnelles pouvant rendre compte d’un mécanisme de régulation de l’activité des protéines Hox. Des analyses prédictives in silico, confirmées par des approches biochimiques et des analyses in vivo ont démontré la SUMOylation de AbdA. Ces résultats préliminaires posent les bases pour des travaux futures qui auront pour objectif d’identifier les résidus d’AbdA SUMOylés et d’élucider le rôle de cette modification dans la régulation de l’activité de la protéine AbdA. / Hox genes encode homeodomain-containing transcription factors (HD). Although the HD binds to similar DNA sequences in vitro, Hox proteins display a high functional specificity in vivo. Protein motifs outside of the HD influence Hox specificity through recruiting additional cofactors, with the best characterized being Extradenticle (Exd in Drosophila). Recent evidence from our group has uncovered the functional contribution of AbdA intrinsic motifs to AbdA Exd-dependent functions as well as AbdA Exd-independent functions. My PhD work has aimed to characterize the contribution of AbdA motifs to target gene selection and regulation using a combined approach of ChIPseq/RNAseq in an Exd-independent context. The DNA code identified provides us with new insights about additional transcriptional inputs from additional DNA-binding proteins lying in the vicinity of AbdA recognition sites. Mass spectrometry analysis establishes the occurrence of these additional DNA binding proteins in a multi-protein complex with AbdA. Deciphering the involvement of post-translational modifications in the regulation of Hox protein activity was another aspect of my PhD work. In silico predictive analysis, followed by biochemical approaches and in vivo assays reveal the potential for SUMOylation of AbdA as a potentially important regulatory component of AbdA activity. These preliminary results set the bases for further work aimed at identifying SUMOylated residues on AbdA and the functional relevance of such post-translational modification on AbdA activity regulation.

Ces vingt dernières années, les réactions d'insertion d'atomes ou de fragments moléculaires dans des liaisons C-H peu réactives ont fait l'objet de nombreuses études sans que les mécanismes de ces réactions aient pu être établis. Les enzymes de la superfamille « Radical-SAM » catalysent l'activation de leur substrat en utilisant un centre [4Fe-4S] et le co-substrat S-adénosylméthionine (SAM). Les enzymes d'insertion radicalaire constituent un sous-groupe de cette famille et contiennent un second centre fer-soufre impliqué, lui, dans l'activation du deuxième substrat rendant ainsi possible la réaction d'insertion par couplage radicalaire. Le travail présenté dans cette thèse concerne deux de ces enzymes, la première, RimO, est une méthylthiotransférase (MTTase) qui catalyse l'insertion d'un groupement thiométhyle en beta du résidu D89 de la protéine ribosomale S12 (β-ms-D89-S12). La seconde TYW1 ou 4-demethylwyosine synthase catalyse l'insertion d'un groupement acétyle dérivé du pyruvate dans une liaison C-H d'un groupement N-CH3 appartenant à une guanine spécifique de certains ARNt eucaryotes. Cette réaction d'insertion est suivie d'une cyclisation conduisant en plusieurs étapes à la wybutosine (yW), une base tricyclique importante pour la fidélité traductionnelle de la cellule. Dans ce travail il a été montré que les deux centres de cette famille d'enzyme coopèrent pour ces réactions et contrôlent l'utilisation des différents acteurs par des mécanismes redox originaux. / Over the last twenty years, the insertion reactions of atoms or molecular fragments into poorly reactive C-H bonds have been actively investigated but the details of their mechanisms remain largely unknown. Enzymes belonging to the "Radical-SAM" superfamily catalyze the activation of their substrate using a [4Fe-4S] in conjunction with the co-substrate S-adenosylmethionine (SAM). Radical insertion enzymes are a subgroup of this family and contain a second iron-sulfur cluster involved in the activation of the second substrate allowing the insertion reaction by radical coupling to take place. The work presented in this thesis is focusing on two enzymes, the first one, RimO is a methylthiotransferase (MTTase) that catalyzes the insertion of a thiomethyl group on the beta position of D89 residue of the ribosomal protein S12 (β-ms-D89-S12). The second one, TYW1, or 4-demethylwyosine synthase, catalyzes the insertion of the acetyl moiety of pyruvate into a C-H bond of a N-methyl group of a guanine derivative in some eukaryotic and archeal tRNAs. This insertion reaction leads to the formation of a tricyclic ring and through several steps to wybutosine (yW), a hypermodified nucleotide important for the translational fidelity of the cell. In this work we demonstrate that these radical inserting enzymes utilize the two iron-sulfur clusters to cooperate and that they control the different partners of the reaction by original redox mechanisms.

El óxido nítrico (NO) es una molécula gaseosa altamente reactiva que regula el crecimiento y el desarrollo de las plantas así como sus respuestas de defensa. El NO se produce principalmente a partir de nitrito por las nitrato reductasas (NRs) en balance con las nitrito reductasas (NiRs), y es percibido a través de un mecanismo en el que está involucrada la proteólisis dirigida por la secuencia aminoterminal del grupo VII de los factores de transcripción ERF (ERFVIIs). El NO ejerce especialmente su función señalizadora al causar modificaciones postraduccionales en las proteínas y alterar su función, estructura y/o estabilidad. Por estos medios y en colaboración con distintas rutas de señalización fitohormonales, el NO es capaz de regular un amplio abanico de procesos celulares en plantas, incluyendo aquellos relacionados con la adquisición de tolerancia a la congelación. Utilizando Arabidopsis thaliana como planta modelo, en este trabajo se descubrió que el NO puede regular su propia biosíntesis, puesto que las enzimas NRs y NiRs fueron reguladas por tres factores principales: señalización inducida por nitrato y controlada por la función del factor de transcripción NIN-like protein 7 (NLP7), la proteólisis dirigida por la secuencia aminoterminal, y la degradación mediada por el proteasoma, probablemente ocasionada por modificaciones postraduccionales relacionadas con el NO. Adicionalmente, se descubrió que el factor de transcripción ERFVII RAP2.3 regula negativamente tanto la biosíntesis de NO como las respuestas que desencadena a través de un mecanismo similar a un reóstato en el que están involucradas ramas específicas relacionadas con el NO de las rutas de señalización de jasmonato y ácido abscísico. Por otro lado, una caracterización metabolómica y transcriptómica combinada de plantas mutantes nia1,2noa1-2 deficientes en NO y plantas fumigadas con NO permitió desentrañar una serie de mecanismos que están controlados por NO. En primer lugar, la percepción de NO en los hipocotilos requeriría varias hormonas para ser completada, como fue confirmado por los rastreos de acortamiento de hipocotilo por NO con mutantes relacionados con hormonas y la colección TRANSPLANTA de líneas transgénicas que expresan condicionalmente factores de transcripción de Arabidopsis. En segundo lugar, dosis elevadas de NO causan una reprogramación masiva aunque transitoria de los metabolismos primario y secundario, incluyendo la alteración del estado redox celular, la alteración de la permeabilidad de estructuras lipídicas y el recambio de proteínas y ácidos nucleicos. Por último, se descubrió que el NO previene el desarrollo de la tolerancia a congelación bajo condiciones no estresantes de temperatura, mientras que resulta esencial para la aclimatación a frío desencadenada por bajas temperaturas que conduce a una tolerancia mejorada a congelación. El NO conseguiría esta modulación afinada de la activación de respuestas relacionadas con frío al coordinar la acumulación de diferentes metabolitos y hormonas. En conjunto, este trabajo arroja luz sobre los mecanismos mediante los cuales, al interactuar con varias rutas señalizadoras y metabólicas, el NO puede regular distintos procesos clave de la fisiología vegetal. / L'òxid nítric (NO) és una molècula gasosa altament reactiva que regula el creixement i desenvolupament de les plantes així com les seves respostes de defensa. El NO es produeix principalment a partir de nitrit per les nitrat reductases (NRs) en balanç amb les nitrit reductases (NiRs), i és percebut a traves d'un mecanisme que inclou la proteòlisi dirigida per la seqüència aminoterminal del grup VII dels factors de transcripció ERF (ERFVII). El NO exerceix la seva funció senyalitzadora majoritàriament al provocar modificacions postraduccionals en les proteïnes i alterar la seva funció, estructura i/o estabilitat. Mitjançant aquestes modificacions i en col·laboració amb distintes rutes de senyalització fitohormonals, el NO es capaç de regular un ampli espectre de processos cel·lulars en plantes, inclosos aquells relacionats amb l'adquisició de tolerància a la congelació. Emprant Arabidopsis thaliana com a planta model, en aquest treball es va descobrir que el NO regula la seva pròpia biosíntesi, donat que els enzims NRs i NiRs foren regulades per tres factors principals: senyalització induïda per nitrat i controlada per la funció del factor de transcripció NIN-like protein 7 (NLP7), la proteòlisi dirigida per la seqüència aminoterminal, i la degradació mitjançant el proteasoma, probablement a causa de modificacions postraduccionals relacionades amb el NO. A més, es va descobrir que el factor de transcripció ERFVII RAP2.3 regula negativament tant la biosíntesi de NO com les respostes que desencadena aquest a través d'un mecanisme similar a un reòstat en el que estan involucrades branques específiques de les rutes de senyalització de jasmonat i àcid abscísic relacionades amb el NO. Per altre costat, una caracterització metabolòmica i transcriptòmica combinada de plantes mutants nia1,2noa1-2 deficients en NO i plantes fumigades amb NO va permetre desentranyar una sèrie de mecanismes que estan controlats per NO. En primer lloc, la percepció de NO en els hipocòtils requeriria de varies hormones, com fou confirmat pels rastrejos d'acurtament d'hipocòtil per NO amb mutants relacionats amb hormones i la col·lecció TRANSPLANTA de línies transgèniques d'expressió condicional de factors de transcripció d'Arabidopsis. En segon lloc, dosis elevades de NO causen una reprogramació massiva, encara que transitòria, dels metabolismes primari i secundari, incloent l'alteració de l'estat redox cel·lular, canvis en la permeabilitat de estructures lipídiques i el recanvi de proteïnes i àcids nucleics. Per últim, es va descobrir que el NO prevé el desenvolupament de la tolerància a congelació en condicions no estressants de temperatura, mentre que resulta essencial per a l'aclimatació a fred induïda per baixes temperatures que condueix a una tolerància millorada a congelació. El NO aconseguiria aquesta modulació minuciosa de l'activació de les respostes relacionades amb fred al coordinar l'acumulació de diferents metabòlits i hormones. En conjunt, aquest treball clarifica els mecanismes pels quals el NO pot regular distints processos clau de la fisiologia vegetal al interactuar amb varies rutes senyalitzadores i metabòliques. / Nitric oxide (NO) is a highly reactive gaseous molecule that regulates plant growth and development as well as defense responses. NO is mainly produced from nitrite by nitrate reductases (NRs) in balance with nitrite reductases (NiRs), and is sensed through a mechanism involving the N-end rule pathway-mediated proteolysis of the group VII of ERF transcription factors (ERFVIIs). NO especially exerts its signaling function by triggering post-translational modifications in proteins and altering their function, structure and/or stability. By these means and in collaboration with different phytohormone signaling pathways, NO is capable of regulating a wide array of cell processes in plants, including those related to the acquirement of freezing tolerance. By using Arabidopsis thaliana as model plant, during the development of this work it was found that NO can regulate its own biosynthesis, as NRs and NiR enzymes were regulated by three main factors: nitrate-induced signaling controlled by the function of the NIN-like protein 7 (NLP7) transcription factor, N-end rule proteolytic pathway, and proteasome-mediated degradation, likely triggered by NO-related post-translational modifications. In addition, the ERFVII transcription factor RAP2.3 was found to negatively regulate both the NO biosynthesis and their triggered responses through a rheostat-like mechanism that involves specific NO-related branches of jasmonate and abscisic acid signaling pathways. On the other hand, a combined metabolomic and transcriptomic characterization of NO-deficient nia1,2noa1-2 mutant plants and NO-fumigated plants allowed to unravel a number of mechanisms that are controlled by NO. First, NO perception in hypocotyls would require various hormones to be fulfilled as it was confirmed by NO-triggered hypocotyl shortening screenings with hormone-related mutants and the TRANSPLANTA collection of transgenic lines conditionally expressing Arabidopsis transcription factors. Second, high NO doses caused a massive but transient reprogramming of primary and secondary metabolism, including alteration of the cellular redox status, alteration of the permeability of lipidic structures or turnover of proteins and nucleic acids. Lastly, NO was found to prevent the development of freezing tolerance under non-stress temperature conditions, while being essential for the low temperature stress-triggered cold acclimation that leads to enhanced freezing tolerance. NO would achieve this fine-tuned modulation of the activation of the cold-related responses by coordinating the accumulation of different metabolites and hormones. Altogether, this work sheds light on the mechanisms by which, by interacting with various signaling and metabolic pathways, NO can regulate several key processes of plant physiology. / Costa Broseta, Á. (2018). Regulation of the nitric oxide synthesis and signaling by posttranslational modifications and N-end rule pathway-mediated proteolysis in Arabidopsis thaliana [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/114825 / TESIS

Histone post-translational modifications (PTMs), as the histone code assumes, are related with regulation of gene transcription, an important mechanism of cells in the differentiation process. Many PTMs are simultaneously present in histone proteins, and changes in the PTM stoichiometric ratios can have several effects, like changes in the chromatin structure leading to a transcriptionally active or repressive state. Significant progresses were made to map variations of histone PTMs by mass spectrometry (MS), and although many protocols were developed there are still some drawbacks. Incomplete and side reactions were identified, which can directly affect the quantification of histone PTMs, because both (incomplete and side reactions) can be misinterpreted as endogenous histone post translational modifications. Therefore, a protocol for derivatization of histones with no noticeable undesired reactions (<10%) was required. In this thesis a new chemical modification methodology is presented, which allows the improvement of sequence coverage by acylation with propionic anhydride of lysine residues and N-terminal (free ε- and α- amino groups) and trypsin digestion. more than 95% of complete reaction was achieved with the new derivatization methodology. This strategy (chemical derivatization of histones), in combination with bottom-up MS approach, allows the quantification of lysine methylation (Kme) and acetylation (Kac) in histones from Saccharomyces cerevisiae (S.cerevisiae), mouse embryonic stem cells (mESCs) and human cell lines. The results showed histone H3 PTM pattern as the most variable profile regarding histone Kme and Kac across the three different organisms and experimental conditions. Therefore, it was concluded that quantification of H3 PTM pattern can be used to examine changes in chromatin states when cells are subjected to any kind of perturbation.