Post-translationale Modifikationen (PTMs) sind wesentlich für die Regulierung von zellulären Mechanismen. Um diese Prozesse besser zu verstehen, ist es essentiell Methoden für deren Erforschung zu entwickeln. In dieser Arbeit wurden zwei chemoproteomische Ansätze entwickelt, um die PTMs, Proteinphosphorylierung und Proteinpyrophosphorylierung zu untersuchen. Die Proteom-weite Erforschung von Proteinphosphorylierungen beruht gewöhnlich auf der LC-MS/MS-Analyse von enzymatisch verdauten Proteomen und da die Phosphorylierung von niedriger Abundanz ist, wird ein Phosphopeptid-Anreicherungsschritt benötigt. Die Identifizierung von bestimmten Phosphopeptiden ist allerdings abhängig von der gewählten Anreicherungsmethode. Die Entwicklung von neuen Prozeduren ist daher bedeutsam, um neue Phosphorylierungsstellen zu identifizieren. Im ersten Projekt wurde eine milde und selektive Phosphopeptid-Anreicherungsmethode entwickelt und optimiert. Die Methode zeigte die Fähigkeit Phosphopeptide anzureichern und somit das Potential, das Repertoire der vorherigen Methoden zu erweitern, um neue Phosphorylierungsstellen zu identifizieren. Proteinpyrophosphorylierung ist eine unlängst identifizierte PTM, die nicht-enzymatisch an Proteine angefügt wird und es ist nur wenig ist über ihre Funktion bekannt. Vorherige Studien wiesen darauf hin, dass diese Modifikation enzymatisch entfernt wird, allerdings sind die verantwortlichen Enzyme („Proteinpyrophosphatasen“) nicht bekannt. Hier wurde eine Peptidaffinitätsmethode entwickelt, um potentielle Pyrophosphatasen und weitere interagierende Proteine aus humanen Zellen zu identifizieren. Damit wurden 6 Phosphatasen als potentielle Pyrophosphatase-Kandidaten identifiziert und weitere interagierende Proteine gaben Aufschlüsse über die Funktion der Proteinpyrophosphorylierung. Dadurch wurde das Potential der Methode aufgezeigt, interagierende Proteine der Proteinpyrophosphorylierung zu identifizieren, um die zelluläre Rolle zu verstehen. / Post-translational modifications (PTMs) are crucial for the regulation of cellular mechanisms. To better understand these processes, the development of chemical tools to investigate them is of high importance. In this thesis, two chemoproteomics approaches were established to investigate the PTMs protein phosphorylation and protein pyrophosphorylation. The proteome-wide study of protein phosphorylation usually relies on LC-MS/MS analysis of enzymatically digested proteomes, requiring a phosphopeptide enrichment step, due to the low abundance of phosphorylation. However, the identification of certain sets of phosphopeptides is dependend on the choice of enrichment method. Therefore, the development of new workflows is important to identify new phosphorylation sites. In the first project, a mild and selective phosphopeptide enrichment method was developed and optimized. The method was able to enrich phosphopeptides and therefore, showed the potential to complement the repertoire of current methods to identify new phosphorylation sites. Protein pyrophosphorylation is a recently discovered PTM, which is non-enzymatically attached to proteins and there is only sparse knowledge about the function. Previous studies have indicated the enzymatic removal of this modification, but the responsible enzymes (‘protein pyrophosphatases’) are unknown. Here, a peptide affinity capture method was developed to identify potential pyrophosphatases and further interacting proteins from human cells. Therewith, 6 phosphatases were identified as potential pyrophosphatase candidates and further interacting proteins gave insights into the function and mechanisms of protein pyrophosphorylation. Thereby, the potential of this method was demonstrated to identify interacting proteins of protein pyrophosphorylation to understand the cellular role.

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]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/114825

In Parkinson's disease, aggregates of α-synuclein within Lewy bodies and Lewy neurites represent neuropathological hallmarks. However, the cellular and molecular mechanisms triggering oligomeric and fibrillary α-synuclein aggregation are not fully understood. Recent evidence indicates that oxidative stress induced by metal ions and post-translational modifications such as phosphorylation, ubiquitination, nitration, glycation, and SUMOylation affect α-synuclein conformation along with its aggregation propensity and neurotoxic profiles. In addition, proteolytic cleavage of α-synuclein by specific proteases results in the formation of a broad spectrum of fragments with consecutively altered and not fully understood physiological and/or pathological properties. In the present review, we summarize the current knowledge on proteolytical α-synuclein cleavage by neurosin, calpain-1, cathepsin D, and matrix metalloproteinase-3 in health and disease. We also shed light on the contribution of the same enzymes to proteolytical processing of pathogenic proteins in Alzheimer's disease and report potential cross-disease mechanisms of pathogenic protein aggregation.

Parkinson’s disease (PD) is a progressive neurodegenerative disorder that is neuropathologically characterized by degeneration of dopaminergic neurons of the substantia nigra (SN) and formation of Lewy bodies and Lewy neurites composed of aggregated α-synuclein. Proteolysis of α-synuclein by matrix metalloproteinases was shown to facilitate its aggregation and to affect cell viability. One of the proteolysed fragments, Gln79-α-synuclein, possesses a glutamine residue at its N-terminus. We argue that glutaminyl cyclase (QC) may catalyze the pyroglutamate (pGlu)79-α-synuclein formation and, thereby, contribute to enhanced aggregation and compromised degradation of α-synuclein in human synucleinopathies. Here, the kinetic characteristics of Gln79-α-synuclein conversion into the pGlu-form by QC are shown using enzymatic assays and mass spectrometry. Thioflavin T assays and electron microscopy demonstrated a decreased potential of pGlu79-α-synuclein to form fibrils. However, size exclusion chromatography and cell viability assays revealed an increased propensity of pGlu79- α-synuclein to form oligomeric aggregates with high neurotoxicity. In brains of wild-type mice, QC and α-synuclein were co-expressed by dopaminergic SN neurons. Using a specific antibody against the pGlu-modified neo-epitope of α-synuclein, pGlu79-α-synuclein aggregates were detected in association with QC in brains of two transgenic mouse lines with human α-synuclein overexpression. In human brain samples of PD and dementia with Lewy body subjects, pGlu79-α-synuclein was shown to be present in SN neurons, in a number of Lewy bodies and in dystrophic neurites. Importantly, there was a spatial co-occurrence of pGlu79-α-synuclein with the enzyme QC in the human SN complex and a defined association of QC with neuropathological structures. We conclude that QC catalyzes the formation of oligomer-prone pGlu79-α-synuclein in human synucleinopathies, which may—in analogy to pGlu-Aβ peptides in Alzheimer’s disease—act as a seed for pathogenic protein aggregation.

La déubiquitinase BRCA-associated protein 1 (BAP1) est un suppresseur de tumeurs chez l’homme, dont l’activité enzymatique est inactivée dans une variété de cancers. Les modèles actuels proposent que BAP1 forme un complexe de plusieurs méga daltons avec des protéines associées à la chromatine, et que ces protéines et les modifications post-traductionnelles (PTM) facilitent sa fonction de suppresseur de tumeurs en agissant sur la chromatine. Il a été démon-tré que les facteurs de transcription FOXK1 et FOXK2 recrutent BAP1 pour cibler des gènes et réguler la transcription. Cependant, comment FOXK1/2 sont régulés dans le complexe BAP1 reste inconnu. FOXK1/2 sont récemment apparus comme des régulateurs clés du métabolisme et de la prolifération cellulaire dans des conditions normales et de stress. Les observations dans les can-cers humains suggèrent que FOXK1, et non FOXK2, possède des propriétés oncogéniques. En effet, une expression élevée de FOXK1 est associée à une prolifération accrue, ainsi qu’à une invasion et des métastases plus importantes. Cependant, les mécanismes moléculaires exacts de la dérégulation de FOXK1 restent méconnus. Nos analyses sur la survie des patients montrent qu’une forte expression du transcrit de FOXK1 diminue significativement la survie par rapport aux patients présentant de faibles taux du transcrit de FOXK1, ce qui n’est pas observé avec FOXK2. Pour mieux comprendre les fonctions de FOXK1 et FOXK2, nous avons surexprimé ses protéines dans des fibroblastes humains normaux. Cela nous a conduits à découvrir que les pro-priétés oncogéniques de FOXK1 agissent en partie par l’induction de la voie des E2Fs. Contrai-rement à FOXK2, la surexpression de FOXK1 dans les fibroblastes humains normaux favorise la prolifération cellulaire et retarde l’induction de la sénescence. Nous avons également constaté que lorsque la surexpression de FOXK1 était combinée à d’autres oncogènes, sa capacité à transformer les fibroblastes était significativement augmentée. Ces résultats suggèrent que FOXK1 et FOXK2 jouent différents rôles dans les cellules et qu’un mécanisme peut réguler leur activité différemment. De manière intéressante, nous avons découvert que FOXK1, et non FOXK2, est modifié par O-GlcNAcylation, une modification post-traductionnelle unique connue pour être étroite-ment régulée par les fluctuations du métabolisme cellulaire. Par conséquent, nous avons émis l’hypothèse que la O-GlcNAcylation est un mécanisme important de régulation de l’activité transcriptionnelle de FOXK1. L’identification des sites modifiés sur FOXK1 nous a permis de créer des mutants déficients en O-GlcNAcylation de ce facteur. Alors que la perte de la O-GlcNAcylation n’impacte pas sur le recrutement de FOXK1 à la chromatine, nous avons décou-vert que la O-GlcNAcylation régule les propriétés oncogéniques de FOXK1. En effet, l’absence de la O-GlcNAcylation de FOXK1 diminue la capacité proliférative des cellules ainsi que la crois-sance tumorale. De plus, nos analyses de génomiques nous ont permis de mettre en évidence que la O-GlcNAcylation régule le recrutement de BAP1 sur la chromatine. La diminution de la O-GlcNAcylation sur FOXK1 entraîne une réduction du recrutement de BAP1, ce qui est associé à une augmentation des niveaux de H2AK119Ub, une marque de répression génique ciblée par la déubiquitinase BAP1, ainsi qu’à une diminution de la marque d’activation H3K4me1. Nous proposons un modèle dans lequel la O-GlcNAcylation régule les fonctions biolo-giques de FOXK1 et promeut la croissance tumorale en pilotant les propriétés oncogéniques de ce facteur. Nos analyses suggèrent que la O-GlcNAcylation de FOXK1 est importante pour le bon fonctionnement des complexes sur la chromatine. Comprendre comment FOXK1 et FOXK2 régu-lent BAP1 pourrait nous aider à mieux définir les fonctions de suppression tumorale de BAP1 et comment la dérégulation des facteurs de transcription contribue au développement du cancer. / The deubiquitinase BAP1 (BRCA-associated protein 1) is a tumor suppressor in humans, whose enzymatic activity is inactivated in a variety of cancers. Current models suggest that BAP1 forms mega dalton complex with chromatin-associated proteins, and that these proteins and post-translational modifications (PTMs) facilitate its tumor suppressor function by acting on chromatin. It has been shown that the transcription factors FOXK1 and FOXK2 recruit BAP1 to target genes and regulate transcription. However, how FOXK1/2 are regulated within the BAP1 complex remains unknown. FOXK1/2 have recently emerged as key regulators of metabolism and cell proliferation under normal and stress conditions. Observations in human cancers suggest that FOXK1, and not FOXK2, has oncogenic properties. Indeed, high expression of FOXK1 is associated with increased proliferation, as well as greater invasion and metastasis. However, the exact molecular mechanisms of FOXK1 deregulation remain unknown. Our analyses of patient survival show that high expression of FOXK1 transcript significantly reduces survival compared to patients with low levels of FOXK1 transcript, which is not observed with FOXK2. To better understand the functions of FOXK1 and FOXK2, we overexpressed their proteins in normal human fibroblasts. This led us to discover that the oncogenic properties of FOXK1 act in part by inducing the E2F pathway. Unlike FOXK2, overexpression of FOXK1 in normal human fibroblasts promotes cell proliferation and delays the induction of senescence. We also found that when overexpression of FOXK1 was combined with other oncogenes, its ability to transform fibroblasts was significantly increased. These results suggest that FOXK1 and FOXK2 play different roles in cells and that a mechanism may regulate their activity differently. Interestingly, we discovered that FOXK1, and not FOXK2, is modified by O-GlcNAcylation, a unique post-translational modification known to be tightly regulated by fluctuations in cellular metabolism. Consequently, we hypothesized that O-GlcNAcylation is an important mechanism for regulating the transcriptional activity of FOXK1. Identifying the modified sites on FOXK1 allowed us to create mutants deficient in O-GlcNAcylation of this factor. While the loss of O-GlcNAcylation does not affect the recruitment of FOXK1 to chromatin, we discovered that O-GlcNAcylation regulates the oncogenic properties of FOXK1. Indeed, the absence of O-GlcNAcylation of FOXK1 reduces the proliferative capacity of cells as well as tumor growth. Moreover, our genomic analyses have allowed us to highlight that O-GlcNAcylation regulates the recruitment of BAP1 to chromatin. Loss of FOXK1 O-GlcNAcylation leads to a reduction of BAP1 recruitment to chromatin, which is associated with an increase in the levels of H2AK119Ub, a gene repression mark targeted by the deubiquitinase BAP1, as well as a decrease in the activation mark H3K4me1. We propose a model in which O-GlcNAcylation regulates the biological functions of FOXK1 and promotes tumor growth by driving the oncogenic properties of this factor. Our analyses suggest that O-GlcNAcylation of FOXK1 is important for the proper functioning of complexes on chromatin. Understanding how FOXK1 and FOXK2 regulate BAP1 could help us better define the tumor-suppressing functions of BAP1 and how the deregulation of transcription factors contributes to cancer development.

[ES] La cristalografía de rayos X es una potente técnica para la resolución de la estructura atómica de macromoléculas. La información generada, tiene gran impacto sobre diferentes campos relacionados con la investigación básica y aplicada, como son la biomedicina y diseño de fármacos, al igual que en el desarrollo de aplicaciones nanotecnológicas y biotecnológicas. Esta Tesis se centra en determinadas problemáticas actuales y en las proteínas involucradas en las mismas (TryR, eEF1A2 y CBDP35), siendo éstas sujeto de desarrollo biotecnológico en los campos de la biomedicina, farmacia y de la industria alimentaria, en el que la cristalografía de rayos X juega un papel crucial para dilucidar sus estructuras atómicas y funciones. En consideración a la biomedicina y diseño de fármacos, hemos resuelto la estructura de la Tripanotión reductasa (TryR) de Leishmania infantum en complejo con potentes inhibidores de su actividad oxidorreductasa, con potencial de desarrollo como fármacos. Así, se ha caracterizado la unión y mecanismo de acción de éstos inhibidores. TryR es una reconocida diana farmacológica para el tratamiento de la enfermedad de Chagas, la Tripanosomiasis Humana Africana y la leishmaniosis, ya que desempeña un papel crucial y esencial en el metabolismo redox de los parásitos de la familia Trypanosomatidae. Además, se han analizado los parámetros de cristalización y difracción de novedosos inhibidores de la dimerización de TryR, cuyo diseño racional se basa en la unión a la interfaz de dimerización de la misma. La oncoproteína eEF1A2, involucrada en múltiples funciones celulares y sujeto de numerosas modificaciones post-traduccionales, se une al fármaco anticancerígeno plitidepsina. La cristalografía de rayos X, combinada con experimentos de espectrometría de masas, se han utilizado como herramientas para identificar nuevas modificaciones post-traduccionales y características estructurales en eEF1A2:GDP. Una modificación única, la adición de etanolamina fosfoglicerol (EPG) a aminoácidos conservados (Glu301 y Glu374 en mamíferos), se ha observado aquí por primera vez. El análisis estructural de estos hallazgos facilita la comprensión de las múltiples funciones y regulaciones de eEF1A2. La adquisición de una muestra conformacionalmente homogénea de eEF1A2:GTP, necesaria para la unión a la plitidepsina, ha sido evaluada en ensayos de cristalización del complejo terciario de eEF1A2: GTP: plitidepsina. Con respecto al dominio de unión a la pared celular de la endolisina PlyP35 codificada por el fago P35 de Listeria monocytogenes (CBDP35), hemos resuelto la estructura cristalina de CBDP35 en un complejo con ácido teicoico natural de L. monocytogenes serovar 1/2a. Esta estructura es el primer módulo de unión a la pared celular en complejo con ácidos teicoicos jamás dilucidado. El análisis estructural reveló los principales determinantes para la unión de la pared celular bacteriana, en particular, el mecanismo molecular del reconocimiento de N-acetil-d-glucosamina, una decoración de carácter glicosídico en ácidos teicoicos de serovares patógenos de L. monocytogenes. Estos hallazgos arrojan luz sobre el desarrollo biotecnológico de nuevas herramientas en la industria alimentaria y las terapias derivadas de fagos para detectar y tratar infecciones bacterianas. / [CA] La cristal·lografia de raig X és una potent tècnica per a la resolució de l'estructura atòmica de macromolècules. La informació generada té gran impacte sobre diferents camps relacionats amb la investigació bàsica i aplicada, com són la biomedicina i disseny de fàrmacs, igual que en el desenvolupament d'aplicacions nanotecnológiques i biotecnològiques. Aquesta Tesi es centra en determinades problemàtiques actuals i en les proteïnes involucrades en les mateixes (TryR, eEF1A2 i CBDP35), sent estes subjecte de desenvolupament biotecnològic en els camps de la biomedicina, farmàcia i de la indústria alimentària, en el que la cristal·lografia de raig X juga un paper crucial per a dilucidar les seues estructures atòmiques i funcions. En consideració a la biomedicina i disseny de fàrmacs, hem resolt l'estructura de la Tripanotión reductasa (TryR) de Leishmania infantum en complex amb potents inhibidors de la seua activitat oxidorreductasa, amb potencial de desenrotllament com a fàrmacs. Així, s'ha caracteritzat la unió i mecanisme d'acció d'estos inhibidors. TryR és una reconeguda diana farmacològica per al tractament de la malaltia de Chagas, la Tripanosomiasi Humana Africana i la leishmaniosi, ja que exerceix un paper crucial i essencial en el metabolisme redox dels paràsits de la família Trypanosomatidae. A més, s'han analitzat els paràmetres de cristal·lització i difracció de nous inhibidors de la dimerizació de TryR, el disseny racional dels quals es basa en la unió a la interfície de dimerización de la mateixa. L'oncoproteína eEF1A2, involucrada en múltiples funcions cel·lulars i subjecte de nombroses modificacions posttraduccionals, s'unieix al fàrmac anticancerigen plitidepsina. La cristal·lografia de raig X, combinada amb experiments d'espectrometria de masses, s'han utilitzat com a ferramentes per a identificar noves modificacions posttraduccionals i característiques estructurals en eEF1A2:GDP. Una modificació única, l'addició d'etanolamina fosfoglicerol (EPG) a aminoàcids conservats (Glu301 i Glu374 en mamífers), s'ha observat ací per primera vegada. L'anàlisi estructural d'estes troballes facilita la comprensió de les múltiples funcions i regulacions d'eEF1A2. L'adquisició d'una mostra conformacionalmente homogènia d'eEF1A2:GTP, necessària per a la unió a la plitidepsina, ha sigut avaluada en assajos de cristal·lització del complex terciari d'eEF1A2: GTP: plitidepsina. Respecte al domini d'unió a la paret cel·lular de l'endolisina PlyP35 codificada pel fago P35 de Listeria monocytogenes (CBDP35), hem resolt l'estructura cristal·lina de CBDP35 en un complex amb àcid teicoico natural de L. monocytogenes serovar 1/2a. Esta estructura és el primer mòdul d'unió a la paret cel·lular en complex amb àcids teicoicos mai dilucidat. L'anàlisi estructural va revelar els principals determinants per a la unió de la paret cel·lular bacteriana, en particular, el mecanisme molecular del reconeixement de N-acetil-d-glucosamina, una decoració de caràcter glicosídico en àcids teicoicos de serovares patògens de L. monocytogenes. Estes troballes fan llum sobre el desenrotllament biotecnològic de noves ferramentes en la indústria alimentària i les teràpies derivades de fagos per a detectar i tractar infeccions bacterianes. / [EN] X-ray crystallography is a powerful technique for atomic structure resolution of macromolecules. The information generated impacts different fields involving basic and applied research on biomedicine and drug design and the development of nanotechnology and biotechnological applications. This dissertation focuses on current problematics and the target proteins involved (TryR, eEF1A2 and CBDP35) that are in sight for biotechnological development in the biomedical, pharmaceutical and food industry fields, in which X-ray crystallography plays a crucial role in the elucidation of their atomic structures and functions. Attaining to biomedical and drug design problematics, we have solved the structure of Leishmania infantum TryR in complex with potent oxidoreductase inhibitors prone to further development as anti-trypanosomal drugs, thereby characterizing their binding and mechanism of action. This protein is a long recognized drug target for the treatment of Chagas disease, Human African Trypanosomiasis and leishmaniasis, as it plays a crucial and essential role in the redox-metabolism of the Trypanosomatidae parasites. Moreover, the crystallization and diffraction parameters of novel TryR dimerization disruptors have been assayed for inhibitors which have been rationally designed to bind the dimerization interface of TryR. The "moonlighting" oncoprotein eEF1A2 is known to be highly post-translationally modified and to bind the anticancer drug plitidepsin. X-ray crystallography, combined with mass-spectrometry experiments, have been used as tools to identify novel post-translational modifications and structural features in eEF1A2:GDP. A unique modification, namely the addition of ethanolamine phosphoglycerol (EPG) to conserved glutamic residues (Glu301 and Glu374 in mammals), has been here observed for the first time. Structural analysis of these findings facilitate the understanding of eEF1A2's multiple functions and regulations. The acquirement of a conformationally homogenous eEF1A2:GTP sample, necessary for plitidepsin binding, has been has been assayed for eEF1A2:GTP:plitidepsin complex crystallization. Regarding the cell wall binding domain of Listeria monocytogenes phage-encoded endolysin PlyP35 (CBDP35), we have solved the crystal structure of CBDP35 in complex with natural Listeria serovar 1/2a teichoic acid. This structure is the first cell wall binding module in complex with teichoic acids ever elucidated. Structural analysis revealed the main determinants for bacterial cell-wall binding, in particular, the molecular mechanism of N-acetyl-d-glucosamine recognition, a glycosidic moiety in teichoic acids of pathogenic serovars of L. monocytogenes. These findings shed light upon the biotechnological development of new tools in the food industry and phage-derived therapies to detect and treat bacterial infections. / Agradecer al Ministerio de Educación, Cultura y Deporte por haberme proporcionado el contrato FPU (FPU14/03190) que me ha permitido desarrollar esta Tesis Doctoral en el Instituto de Química-Física “Rocasolano” del Consejo Superior de Investigaciones Científicas (IQFR-CSIC), así como la financiación otorgada para poder realizar mi estancia predoctoral en el laboratorio del Prof. Hammershmidt, en Greifswald, Alemania (EST17/00751). / Carriles Linares, AÁ. (2019). STRUCTURAL BIOMEDICINE: CHARACTERIZATION OF THE STRUCTURAL BASIS IN PROTEIN-DRUG RECOGNITION IN DIFFERENT HUMAN DISEASES [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/130844

This thesis explores roles of 2-oxoglutarate-dependent (2OG) oxygenases as interfaces that modulate steps in the flow of genetic information in cells in response to oxygen availability. Chapter 1 introduces mechanistic, biochemical and physiological aspects of major subfamilies of 2OG oxygenases, and their established regulatory roles in cells. In addition, structural and functional aspects of the ribosome and the translation process are discussed, with a focus on post-translational ribosome modifications. Chapter 2 investigates histone demethylases, which mediate chromatin-dependent regulation of gene expression and provides proof-of-concept for the rational, structure-guided design of small-molecules for selective inhibition of 2OG oxygenases with roles in cancer and inflammatory disease. Chapter 3 suggests regulatory roles for ten-eleven-translocation (TET)- catalysed DNA hydroxylation; calorimetric and thermal analyses reveal a duplex-stabilizing effect of the epigenetic 5-methylcytosine mark that is reversed upon conversion to 5- hydroxymethylcytosine (also termed the ‘sixth’ DNA base), raising the possibility that 2OG oxygenase catalysis might affect transcription via biophysical effects. Chapter 4 investigates fluoride release assays as a technology to enable medicinal chemistry studies on 2OG oxygenases with roles in fat mass regulation and obesity, cancer and inflammation; studies on the ALKBH5 enzyme show that it is a hypoxically upregulated 2OG oxygenase with a substrate preference distinct from previously characterized ALKBH enzymes. Chapter 5 identifies OGFOD1 as a 2OG-dependent ribosomal protein hydroxylase. OGFOD1 catalysis is conserved from yeast to humans. OGFOD1 catalyses formation of trans-3- hydroxy-L-proline in a highly conserved loop of ribosomal protein S23 proximal to the ribosomal decoding centre, possibly to modulate the interactions of eukaryotic ribosomes with tRNA, mRNA and translation factors in an oxygen-dependent manner. OGFOD1 is the functionally most well-conserved protein-modifying 2OG oxygenase; likewise, ribosomal protein S23 hydroxylation is the most well-conserved post-translational ribosome modification in eukaryotes. Some cell lines require OGFOD1 for proliferation, and scaffolds for OGFOD1- selective inhibitors are developed for use as potential antiproliferative agents and probes for cellular function. Chapter 6 shows the development of assays to investigate whether OGFOD1 catalysis affects ribosome assembly and function, including processivity, accuracy of initiation, elongation and termination, in yeast and mammalian cell lines. Chapter 7 concludes that ribosome hydroxylation might present an additional layer of regulatory complexity by which 2OG oxygenases could enable cells to respond to fluctuating oxygen levels.

Le long bio-polymère d'ADN est condensé à l’intérieur du noyau des cellules eukaryotes à l'aide de petites protéines appelées histones. En plus de leurs fonctions condensatrices,ces histones sont également la cible de nombreuses modifications post-traductionnelles(MPT), particulièrement au niveau de leur section N-terminale. Ces modifications réversibles font partie d’un code d’histones épi-génétique transmissible qui orchestre et module dynamiquement certains événements impliquant la chromatine, tels l’activation et la désactivation de gènes ainsi que la duplication et la réparation d’ADN. Ces modifications sont impliquées subséquemment dans la signalisation et la progression de cancers, tels que la leucémie. En conséquence, l'élucidation des modifications d’histones est importante pour comprendre leurs fonctions biologiques. Une méthodologie analytique a été mise au point en laboratoire pour isoler, détecter, et quantifier les MPT d’histones en utilisant une approche rapide à deux volets à l’aide d’outils bioinformatiques spécialisés. La méthodologie développée en laboratoire a été validée en utilisant des histones de souche sauvage ainsi que deux types d’histones mutants déficients en enzymes acétyltransferase. Des trois sources d’histones utilisées, la seule MPT qui a démontré un changement significatif est l’acétylation de l’histone H3 à lysine 56 (H3K56ac). L’expression et la stoechiométrie de cette MPT, issue de cellules de souche sauvage et de cellules mutantes, ont été déterminées avec précision et comparées. Les fonctions de balayage polyvalentes d'un instrument à trappe ionique quadrupôle linéaire hybride ont été utilisées pour améliorer la détection de protéines intactes. Le mode de balayage « enhanced multiply charged » (EMC) a été modifié pour contenir et détecter les ions de protéines intactes situées dans la trappe ionique linéaire. Ce mode de balayage nommé « targeted EMC » (tEMC) a permis de quadrupler le niveau de sensibilité (signal/interférence), et quintupler la résolution du mode de balayage conventionnel. De plus, la capacité de séparation des charges du tEMC a réduit de façon significative les effets de « space charge » dans la trappe ionique linéaire. La résolution supérieure du mode tEMC a permis de différencier plusieurs isoformes modifiées, particulièrement pour l’histone H3. L’analyse des peptides d’histones trypsiques à l’aide du mode de balayage « MRM » a permis le séquençage et la quantification de MPT avec un haut degré de précision. La seule MPT qui était sous-exprimée entre l’histone de souche sauvage et le mutant DOT1L fut la méthylation de l’histone H3 lysine 79(H3K79me1). Les effets de deux inhibiteurs d’enzymes HDAC (HDACi) sur l’expression de MPT d’histone ont été évalués en utilisant la méthodologie analytique mentionnée. Les histones extraites de cellules normales et cancéreuses ont été exposées à du Vorinostat(SAHA) ou du Entinostat (MS-275) pour une période de 24 à 72 heures. Deux histones furent principalement affectées, soit H3 et H4. Étonnamment, les mêmes effets n'ont pas été détectés lorsque les cellules normales ont été traitées avec le HDACi pour une période de 48 à 72 heures. Une méthode absolue de quantification avec une courbe d’étalonnage a été développée pour le peptide H3K56ac. Contrairement à certaines publications, nos résultats démontrent que cette MPT est présente dans les cellules mammifères avec une stoechiométrie très basse (< 0,1%) et n'est pas surexprimée de façon significative après le traitement au HDACi. / In eukaryotic cells, the lengthy DNA biopolymer is condensed into the cell nucleus with the aid of small packaging proteins called histones. In addition to their packing functions,histones are also targets of numerous post translational modifications (PTMs), especially on their N-terminus. These reversible modifications are believed to be constituents of a heritable epigenetic “histone code” that dynamically orchestrate and modulate chromatin based events such as gene activation and silencing, DNA replication and repair, and are also involved in the downstream signaling and progression of cancers, such as leukemia. Thus, the elucidation of histone PTMs is important in understanding their biological function. An analytical workflow was designed and set-up in the laboratory to isolate, detect, and quantitate histone PTM, using a two-pronged, unbiased, and rapid approach with specialized bioinformatic tools. The workflow was validated using histones from wildtype, and 2 mutants deficient in acetyltransferase activity. Between the three histone sources, the only PTM that demonstrated any change was acetylation at histone H3 lysine 56 (H3K56ac). The down-regulation and stoichiometry of this PTM was accurately assessed between wild-type and mutant cells. The versatile scan functions of a hybrid quadrupole-linear ion trap instrument were exploited to enhance the detection of intact histone proteins. The enhanced multiply charged (EMC) scan was modified in order to contain and detect intact protein ions within the linear ion trap. This targeted EMC (or tEMC) resulted in not only a 4-fold increase in signal-to-noise, but also a 5-fold increase in resolution. Furthermore, the charge separation capability of the tEMC dramatically reduced space charge effects within the linear ion trap. The superior resolution of the tEMC mode allowed for the discimination of many modified histone isoforms, especially for histone H3. Using the bottom-up strategy with multiple reaction monitoring (MRM), histone peptides were quantified and sequenced with a high degree of precision. The only PTM that was down-regulated between wild-type and DOT1L mutant histones was methylation at histone H3 lysine 79 (H3K79me1). The effects of two clinically relevant small molecule HDAC inhibitors (HDACi) on histone PTMs patterns were assessed using the analytical workflow developed. Histones derived from both normal and cancer cells were exposed to either Vorinostat (SAHA) or Entinostat (MS-275) over a 24- to 72 hour period. The two core histones primarily affected were H3 and H4. Surprisingly, the same effects were not observed when normal cells were treated with three doses of SAHA at 24-hour intervals over a 72-hour period. An absolute quantitation method using a calibration curve was developed for H3K56ac. In opposition to other published literature, our findings demonstrate that this PTM is present in very low stoichiometry (< 0.1%) in mammalian cells, and exhibits no significant up-regulation in different cell lines treated with several types of HDACi.

L’adaptation des cellules à leur environnement externe repose sur la transduction adéquate de signaux régulés par une pléthore d'événements moléculaires. Parmi ces événements moléculaires, les modifications post-traductionnelles (MPT) de protéines aident à intégrer, à traduire et à organiser de façon spatiotemporelle ces signaux pour que les cellules puissent réagir aux stimuli externes. Parmi les modifications post-traductionnelles, les petites protéines de la famille de l’Ubiquitine (Ublps, Ubiquitin-like proteins) jouent un rôle majeur dans presque toutes les voies de signalisation. Cette thèse rapporte des études fonctionnelles et structurales des interactions covalentes et non covalentes entre SUMO (Small Ubiquitin related MOdifier), un membre de la famille des Ublps, et trois protéines d'échafaudage, TIF1beta, le corépresseur universel des protéines KRAB-multidoigt de zinc, PIAS1, une ligase E3 pour SUMO et PML, un suppresseur de tumeur. La première étude rapporte l'identification et la caractérisation biochimique des sites de SUMOylation de TIF1beta. Nous avons déterminé que la modification covalente de six résidus lysine par SUMO est essentielle à l’activité de répression de la transcription induit par TIF1beta. En outre, nous présentons des évidences indiquant que la SUMOylation de TIF1 exige non seulement sa capacité à homo-oligomériser, mais est aussi positivement régulée par son interaction avec le domaine KRAB des protéines à doigts de zinc. Partant de ce constat, nous postulons que les protéines KRAB-multidoigt de zinc recrutent leur corépresseur TIF1betaà des gènes cibles, mais aussi accentuent son activité répressive grâce à l'augmentation de sa SUMOylation. Notre seconde étude révèle qu’en plus de réprimer la transcription en tant que MPT covalente, SUMO joue aussi un rôle important dans la répression en tant que partenaire non covalent d’interactions protéine-protéine. Nous avons montré que SUMO interagit simultanément avec deux enzymes de la machinerie de SUMOylation, l’unique enzyme de conjugaison E2, UBC9, et la ligase E3 PIAS1 au sein d’un complexe ternaire répresseur. En outre, nous révélons que la formation du complexe ternaire PIAS1:SUMO:UBC9 est modulée par le niveau de phosphorylation de résidus sérine juxtaposés à un motif d’interaction avec SUMO (SIM) dans PIAS1. Ainsi, SUMO agit comme un adaptateur spécifique qui stabilise les interactions UBC9 E2: E3 PIAS1. Partant de ce constat, nous proposons que les enzymes E2 et E3 des autres systèmes Ublps exploitent des mécanismes similaires dans le cadre de leur fonction Enfin, notre troisième étude explore la régulation des interactions non covalentes de SUMO par la phosphorylation. En utilisant une combinaison d'études in vivo et in vitro, nous démontrons que l'interaction entre SUMO1 et PML est régi par la phosphorylation dépendant de CK2 sur quatre résidus sérine de PML. Les structures cristallographiques des complexes PML-SIM:SUMO1 révèlent que les phospho-sérines de PML contactent des résidus de la région basique de SUMO1. Sachant que la kinase CK2 peut être induite par des kinases activables par le stress, ces résultats suggèrent que les interactions non-covalentes avec SUMO sont modulées par le stress cellulaire. Sur la base de cette constatation, nous postulons que des événements analogues affectent des protéines contenant des séquences SIM ciblées par CK2. En résumé, cette étude révèle qu’en plus de son rôle de MPT, SUMO peut fonctionner comme un adaptateur permettant des interactions spécifiques entre protéines tel que pour les enzymes E3 et E2. / Cell adaption to the external environment relies on proper signal transduction that is orchestrated by a plethora of molecular events. Among these molecular events, post-translational modifications (PTMs) of proteins help to spatiotemporally integrate, translate and dispatch signals so cells can respond to external stimuli. Among these post-translational modifications, the Ubiquitin-like proteins (Ublps) play a major role in almost all signaling pathways. This thesis reports functional and structural studies of the covalent and non-covalent interactions between the Small Ubiquitin-related MOdifier (SUMO), a member of the Ublps family, and three scaffold proteins, TIF1beta, the corepressor of KRAB-Multifinger proteins, PIAS1, a SUMO E3 ligase and the Promyleocytic leukemia (PML) tumor suppressor protein. The first study reports the identification and the biochemical characterization of TIF1betaSUMOylation sites. We mapped six SUMOylation sites in TIF1beta and determined that the covalent modification of these sites by SUMO is essential for its transcriptional repression activity. In addition, we present evidence indicating that SUMOylation of TIF1beta requires not only its ability to homo-oligomerize, but is positively regulated through its interaction with KRAB domains found in zinc-finger proteins. Based on this finding, we postulate that these KRAB domain containing multifinger proteins not only recruit TIF1beta co-repressor to target genes but also increase its repressive activity through enhancement of its SUMOylation. The work in the second study reveals that in addition to suppressing transcription as a covalent PTM, SUMO plays an important role in repression as a non-covalent protein-protein interaction partner. We determine that SUMO can form a repressive complex by simultaneously forming non-covalent interactions with UBC9 and PIAS1, the E2 and E3 enzymes in the SUMOylation system. In addition, we report that the formation of the PIAS1:SUMO:UBC9 ternary complex is modulated by the phosphorylation of serine residues juxtaposed to a SUMO-Interacting Motif (SIM) found in PIAS1. Thus SUMO acts as a specific adaptor that stabilizes UBC9 E2: PIAS1 E3 interactions. Based on this finding, we propose that the E2 and E3 enzymes from other Ublps systems exploit similar mechanisms as part of their function Finally, our third study explores the regulation of SUMO non-covalent interactions by phosphorylation. Using a combination of in vivo and in vitro studies we demonstrate that the interaction between SUMO1 and PML is governed by CK2-dependent phosphorylation of four serine residues in PML. Crystal structures of PML-SIM:SUMO1 complexes reveal that these PML phospho-serine specifically contact SUMO1 basic patch residues. Since CK2 kinase is induced by stress activated kinases pathways, this indicates that SUMO non-covalent interactions are regulated by cellular stress. Based on this finding, we postulated that analogous events influence other CK2-targeted SIM-containing proteins. In summary, this study reveals that in addition to its well described function as PTM, SUMO can function as an adaptor enabling specific proteins interactions such as functional E3:E2 enzymes pairs.

T-lymphocytes are an essential cell type of the adaptive immune system. Due to their importance in immune responses and disorders, the molecular mechanisms leading to T-lymphocyte activation have been the subject of extensive research which has translated into important therapeutic developments. Early signalling events involving tyrosine phosphorylation are well characterised. However, later events involving other post-translational modifications are less well understood. Several studies have provided evidence suggesting a role for protein arginine methylation in T-lymphocyte activation. Arginine methylation is an essential post-translational modification in mammals and yet has not been extensively studied. No large scale analysis of arginine methylation sites has been performed. To gain insight into the role of protein arginine methylation in T-lymphocyte activation, the aims of this work were to: 1. Establish whether levels of arginine methylation are altered during Tlymphocyte activation 2. Use mass spectrometry based proteomics to identify arginine methylated proteins in the T-lymphocyte proteome 3. Further characterise an arginine methylated protein important to Tlymphocyte activation Arginine methylation was found to be induced after long term (>20 hours) stimulation of primary T-lymphocytes. Large increases in the main protein arginine methyltransferase, PRMT1, were also observed. Enrichment and labelling methods were developed to detect arginine methylated peptides from T-lymphocytes by mass spectrometry. This resulted in the identification of 265 unique arginine methylation sites in 141 proteins. 204 of the methylation sites were novel and 103 of the proteins had not previously been described as arginine methylated. Individual arginine methylation sites were characterised before and after activation of T-lymphocytes, with some sites showing significant changes in abundance. Among the novel arginine methylated proteins discovered were Dynamin II, WASp and WIPF1. These proteins are involved in re-organisation of the actin cytoskeleton at the immunological synapse formed between a Tlymphocyte and an antigen presenting cell. The functional consequences of the arginine methylation sites inWASp were characterised. WASp is essential for T-lymphocyte activation and some initial evidence showed that one of the arginine methylation sites is important for WASp activation.