The extracellular loops of muscarinic acetylcholine receptors are predicted to play a role in the binding and regulation of allosteric modulators. Furthermore, the sequence of the five subtypes of muscarinic receptors shows a large degree of diversity in this region. M3 receptor mutants, K523E, D518K and N132G, in which the substituted residues were those corresponding to the M1 subtype, were studied. As the amino acids in positions 518 and 523 are charged, the uncharged mutants, K523Q and D518N, were also created in order to observe any possible effect of charge. One question examined is whether these mutations changed the binding of orthosteric and allosteric ligands, generating a M1 receptor phenotype.Radioligand binding experiments revealed that one mutant, K523E, had a profound potentiating effect on the binding of prototypical modulators like gallamine, strychnine, brucine and N-chloromethylbrucine, but had minimal effects on the binding of a number of orthosteric ligands, including [3H]N-methylscopolamine ([3H]NMS) and acetylcholine (ACh). The increase in affinity was found at both the unoccupied and [3H]NMS-occupied receptors, with up to 70 fold increases in affinity being observed. Switches from negative to positive cooperativity for some strychnine-related compounds were found.At K523E, the affinities of the strychnine-related ligands were also increased up to 160 fold at the receptor-ACh complex, with up to 35 fold positive cooperativity being observed. Positive cooperativity of this magnitude is the highest that has been reported for M3 receptors.The dramatic changes in cooperativities and affinities of allosteric ligands at K523E did not result in generation of the M1 phenotype. The K523Q data suggest that the large changes in K523E result from the introduction of the negatively charged glutamate residue and not the loss of the positively charged lysine. The effect of K523E seems to be solely on the binding of allosteric ligands and the transmission of the effects of their binding to the orthosteric site.For the ligands acting at the gallamine site, all the effects of the allosteric modulators on ACh binding have been reproduced in functional studies, indicating that the allosteric modulation, seen in binding, is transmitted to the cellular response. A novel and unexpected finding is that WIN62,577 is an allosteric agonist at M3 muscarinic receptors and at K523E and N132G. The study also revealed that nanomolar concentrations of ACh may be present in assays of muscarinic receptor function and may give misleading interpretations of data. These artefacts were removed by preincubation with acetylcholinesterase, a control not previously used in functional studies of muscarinic receptors.The sensitivity of the binding of both orthosteric and allosteric ligands to the composition of the binding assay buffer has also been investigated in detail. In a phosphate buffer of low ionic strength (PB) the affinity constants of all the compounds studied, both orthosteric and allosteric, were increased, relative to a Hepes buffer of higher ionic strength, except for WIN 62,577, an allosteric ligand which binds to a different allosteric site from the prototypical modulators, and SVT-40776 a new M3 selective antagonist, indicating their different modes of binding. Cooperativities have also been switched from negative to positive by changing buffer.The two factors affecting the allosteric binding parameters of M3 receptors, PB and the mutation K523E, mutually potentiate each others effects. We have been able to obtain up to 10,000 fold changes in the affinity at the unoccupied receptor and 6400 fold increases in affinity at the ACh occupied receptor.The possible location of K523, relative to other residues on the external loops of muscarinic receptors shown to be important for the binding of allosteric ligands, has been explored using different models based on the X-ray structures of rhodopsin and the â2 adrenergic receptor.

Objectives: Three objectives of this thesis have been: (i) Mastering of the main analytical platforms used in metabolomics, (ii) Developing an untargeted metabolomic workflow, involving novel aspects of sample preparation, and data processing for metabolite identification, (iii) Implementing our untargeted metabolomic workflow to the study of human patients with Polycystic Ovary Syndrome (PCOS) and their response to drug treatment Results: In Work 1: Optimization metabolite extraction conditions for NMR analysis, followed by LC/ESI-MS by using the same sample extract with no need for solvent exchange or further pretreatment. In Work 2: Investigate the impact of different aspects of univariate statistical analysis on untargeted LC-MS based metabolomic experiments. In Work 3: Implementation of GC-MS untargeted metabolomic approach to provide new insights on the impact that obesity exerts on the metabolic derangements associated with PCOS. In Work 4: Implementation of multiplatform metabolomics approach based on NMR and LC-MS to provide new insights in PCOS disease in a cohort of young lean PCOS patients. In Work 5: Implementation of multiplatform metabolomics approach based on NMR, GC-MS and LC-MS to provide new insights on the action of drug polytherapy to PCOS disorder. Conclusion: Metabolomics can be consider as a powerful tool for the study of metabolic disorders. Furthermore, metabolite profiling has demonstrated feasibility and flexibility for revealing new mechanistic insights in metabolic disorders that are not been consider when classical analysis is used. Therefore, our metabolomic analysis have demonstrated a great potential as a useful diagnostic technique and can facilitate monitoring of both disease progression and effects of therapeutic treatment. / Objetivos: El presente trabajo tiene dos objetivos generalizables que han sido estudiados con más detalle en la presente tesis doctoral. El primero de ellos es mejorar aspectos metodológicos en el ámbito de la metabolómica y el segundo ha sido la aplicación de la metabolómica en el estudio del síndrome del ovario poliquístico (PCOS). Resultados: Del primer objetivo se han realizado dos trabajos: en el primero, la optimización de un método de extracción común para analizar muestras biológicas en dos plataformas analíticas complementarias utilizadas en metabolómica como son la resonancia magnética nuclear y la espectrometría de masas. Del segundo trabajo realizado se han obtenido unas pautas para abordar los retos que surgen del análisis de datos de metabolómica en espectrometría de masas. Del segundo objetivo también han sido realizados dos trabajos: en ambos se ha utilizado la metabolómica no dirigida para abordar el estudio del PCOS. En el primer trabajo, se ha utilizado la metabolómica para conocer el impacto que ejerce la obesidad en los trastornos metabólicos asociados al PCOS. En el segundo trabajo, se ha utilizado la metabolómica no dirigida para evaluar como afecta la aplicación de una politerapia con medicamentos al metabolismo de pacientes con PCOS. Conclusión: La metabolómica puede ser utilizada como una nueva herramienta para estudiar los trastornos metabólicos.

Els productes naturals derivats de plantes són una font abundant de compostos biològicament actius, molts dels quals han servit de base pel desenvolupament de productes nutracèutics i farmacèutics. El disseny de fàrmacs assistit per ordinador juga un paper essencial en la identificació de productes naturals bioactius, reduint els costos i esforços experimentals. La diabetis mellitus tipus 2 és considerada com "l’epidèmia del segle 21" i, en conseqüència, és un dels principals reptes en el descobriment de fàrmacs en l'actualitat. Per tant, aquesta tesi doctoral es centra en la identificació de nous compostos i extractes naturals que actuïn com a agents antidiabètics. En aquest sentit, PPARgamma i DPP-IV han estat les dianes estudiades, en les quals s’han desenvolupat i validat diferents protocols de cribatge virtual. Els compostos bioactius trobats poden ser de gran utilitat com additius en l’alimentació funcional adreçada en la prevenció o tractament de la diabetis. / Natural products derived from medicinal plants are an abundant source of biologically active compounds, many of which have formed the basis for development of nutraceuticals and pharmaceuticals compounds. Computer-aided drug design methods such as virtual screening workflows play an essential role in the identification of undescribed bioactivities of natural products by minimizing the synthetic and biological testing efforts. Type 2 diabetes mellitus (T2DM) is considered to be the “epidemic of the 21st century” and, consequently, is one of the main challenges in drug discovery today. There is a significant unmet medical need for better drugs to treat T2DM because some therapies suffer undesirable side effects. Therefore, this PhD thesis is focus on the identification of new natural compounds as antidiabetic agents. In that sense, PPARgamma and DPP-IV have shown to be appropriate targets for antidiabetic drugs, targeting them appears to have good prospects for a successful therapeutic approach in the T2DM.

Les dues proteïnes estudiades en aquest treball (ECP o RNasa 3 i RNasa 1ΔN7) pertanyen a la superfamília de la RNasa A i resulten d'especial interès per la seva potencial aplicació en la teràpia i/o diagnòstic del càncer.A més de la seva capacitat ribonucleolítica, l'ECP presenta d'altres activitats, com l'antibacteriana, l'helmintotòxica o la citotòxica contra cèl·lules i teixits de mamífers. Per la RNasa 1 de tipus pancreàtic expressada per les cèl·lules endotelials humanes també s'ha proposat un paper defensiu. La RNasa 1ΔN7, en canvi, no presenta aquest tipus d'accions biològiques, si bé cal destacar la menor afinitat que exhibeix enfront el seu inhibidor específic en relació a d'altres membres de la família.Tant l'ECP com la RNasa 1ΔN7 s'han cristal·litzat emprant la tècnica de la difusió de vapor en gotes penjants, i s'han determinat les seves estructures tridimensionals (3D) mitjançant el mètode del reemplaçament molecular. Per l'afinament de les estructures s'han usat dades fins a 1,75 i 1,90 Å respectivament.Ambdòs molècules exhibeixen el plegament típic  +  que caracteritza a tots els membres de la superfamília de la RNasa A. Tanmateix, les diferències que mostren en comparació amb l'estructura d'altres RNases permeten explicar, d'una banda, la baixa activitat ribonucleolítica d'aquests enzims i, de l'altra, les seves peculiaritats funcionals. / The proteins studied in this work (ECP or RNase 3 and RNase 1ΔN7) belong to the RNasa A superfamily and their potencial aplication in the therapy and diagnosis of cancer is the base of the present study.Besides its ribonuclease activity, ECP is bactericidal, helminthotoxic and cytotoxic to mammalian cells and tissues. Human endothelial cells expres a pancreatic-type ribonuclease (RNase 1) also involved in defensive functions. In contrast, RNasa 1ΔN7 does not present this kind of special activities, but shows a reduced affinity for the specific ribonuclease inhibitor in comparison with other members of the family.The recombinant proteins have been crystallized by the vapour diffusion method from hanging drops and their crystal structures have been determined by molecular replacement. The refinement of ECP and RNase 1ΔN7 structures has been performed using X-ray diffraction data up to 1,75 and 1,90 Å respectively.Both molecules display the  +  folding topology typical for members of the RNase A superfamily. However, there are some differences that provide an explanation for their low ribonucleolytic activity and, on the other hand, may explain their other particular properties.

Investigation of protein-ligand interactions has been a long-standing application for molecular dynamics (MD) simulations given its importance to drug design. However, relevant timescales for biomolecular motions are orders of magnitude longer than the commonly accessed simulation times. Adequate sampling of biomolecular phase-space has therefore been a major challenge in computational modeling that has limited its applicability. The primary objective for this thesis has been the brute-force simulation of costly protein-ligand binding modeling experiments on a large computing infrastructure. We have built and developed GPUGRID: a peta-scale distributed computing infrastructure for high-throughput MD simulations. We have used GPUGRID for the calculation of protein-ligand binding free energies as well as for the reconstruction of binding processes through unguided ligand binding simulations. The promising results presented herein, may have set the grounds for future applications of high-throughput MD simulations to drug discovery programs.

A part de la secreció convencional, en la qual les proteïnes són transportades a través del reticle endoplasmàtic (RE) i de l'aparell de Golgi, s'han descrit, per a diferents proteïnes i en organismes diversos, rutes de secreció no-convencional que circumval·len l'aparell de Golgi. No obstant, aquests mecanismes de secreció no estan ben entesos. Se sap que per a la secreció no-convencional de la proteïna d'unió a acil-CoA Acb1 a Saccharomyces cerevisiae són necessàries diverses proteïnes, com ara l'ortòleg de GRASP Grh1, proteïnes relacionades amb l'autofàgia, proteïnes involucra-des en la fusió de membranes amb els endosomes, membres de la maquinària ESCRT, i la t-SNARE de la membrana plasmàtica Sso1. La manera per la qual totes aquestes proteïnes cooperen per a dur a terme la secreció d'Acb1 és encara una incògnita. Els resultats de les nostres investigacions indiquen que sota inani-ció de nutrients, condició que indueix la secreció no-convencional d'Acb1, Grh1 es concentra en estructures membranoses prop dels llocs de sortida del RE, de manera dependent de fosfatidilinositol-3-cinasa. Aquestes membranes, en forma de tassa, estan enriqui-des en PI(3)P i contenen la proteïna d'ESCRT-I Vps23 així com les proteïnes d'autofàgia Atg8 i Atg9, reunint així diverses proteïnes necessàries per a la secreció d'Acb1. Hem batejat aquestes estructures CUPS (per les sigles en anglès de compartiment per a la secreció no-convencional de proteïnes), basant-nos en la seva forma i el seu contingut. Proposem que els CUPS són l'estació de sortida per a la formació d'intermediaris vesiculars dedicats a la secreció no-convencionals que contenen Acb1. / Besides conventional secretion, in which proteins are transported through the endoplasmic reticulum (ER) and the Golgi complex, unconventional secretion routes bypassing the Golgi complex have been described for different proteins in several organisms. How-ever, the mechanisms of their release remain poorly understood. It was reported that the unconventional secretion of the acyl-CoA binding protein Acb1 from Saccharomyces cerevisiae requires a diverse group of proteins including the GRASP ortholog Grh1, autophagy-related proteins, proteins involved in fusion of membranes with endosomes, members of the ESCRT-machinery, and the plasma membrane t-SNARE Sso1. How these proteins work together for Acb1 secretion remains elusive. Our findings indicate that upon nutrient starvation, the condition known to induce unconventional secretion of Acb1, Grh1 is concentrated in a phosphatidylinositol 3-kinase-dependent manner to unique membrane structures near the ER exit sites. These membranes –shaped like cups– are enriched in PI(3)P and contain the ESCRT-I protein Vps23 as well as the autophagy-related proteins Atg8 and Atg9 thereby bringing together different proteins required for Acb1 secretion. We have named these structures CUPS (Compartment for Unconventional Protein Secretion), based on their shape and content. CUPS, we propose, are the starting point for the formation of Acb1-containing vesicular intermediates dedicated for unconventional secretion.

The calcineurin-NFATc signaling pathway is involved in many aspects of the development and function of the immune, neural, skeletal, cardiovascular and muscular system of vertebrates. Regulators of Calcineurin (RCAN) proteins constitute a family of endogenous regulators of calcineurin, which play an important role in the modulation of the calcineurin-NFATc pathway. Here, we identifiy a novel protein kinase CK2 dependent mechanism by which the CIC motif of RCAN proteins modulate the final signaling output of the pathway. Moreover, we show that the functional CIC motif of RCANs responsible for Cn-NFATc regulation is sufficient to inhibit tumor progression producing a strong anti-angiogenic phenotype in an orthotopic human breast cancer mouse model. Therefore, a CIC-derived peptide has potent immunosuppressive and antitumoral activities by itself. Finally, the results here presented provide important insights for the rational design of potent and specific NFATc inhibitory drugs, which could be of use in autoimmune diseases and cancer. / La vía de señalización calcineurina-NFATc desempeña diversos roles en el desarrollo y function en el sistema, immune, nervioso, esqueletico, cardiovascular y muscular de veretebrados. Las proteínas RCAN (Regulators of Calcineurin), constitutyen una familia de reguladores endógenos de la calcineurina (Cn) que juegan un papel importante en la modulación de dicha vía. En el presente trabajo, hemos identificado un nuevo mecanismo de regulación, dependiente de la proteína quinasa CK2, por el cual el motivo CIC de las RCAN regula la vía Cn-NFATc. Además, demostramos que el motivo CIC de las RCAN responsable de la regulación de la vía Cn-NFATc, es suficiente para inhibir la progresión tumoral, produciendo un fuerte efecto angiogénico en un modelo ortotópico murino de tumor de mama. Por tanto, un péptido derivado del motivo CIC posee actividad inmunosupresora y antitumoral por sí mismo. Finalmente, este trabajo proporciona nuevos conocimientos que pueden ser de aplicación en el desarrollo racional de nuevos fármacos, especificos y potentes inhibidores de NFATc con posibles aplicaciones en la terapia immunosupresora y del cáncer.

Ribonucleases are promising candidates for use in anticancer therapy. There are different ways to endow an RNase with antitumor properties. Targeting the enzyme to the cell nucleus is a potential approach. PE5 is a variant of HP-RNase that it’s cytotoxic because it contains a nuclear localization signal. It has been shown that this sequence allows its interaction with α-importin. An alternative strategy to endow an RNase with cytotoxic activity is its dimerization. An HP-RNase variant, PM8, which forms a dimer through domain swapping, was produced in solution. This variant dimerized through a two-steps mechanism of domain swapping. To prove his model several variants of PM8 were produced in which the side chains of residues involved were removed. The variation of flexibility of the resulting hinge peptides and the effect of these changes on the ability of the variants to swap domains and to dimerize was determined / Les ribonucleases són potencials agents anticancerosos. Hi ha diferents camins per aconseguir que una RNasa adquireixi propietats antitumorals. La direccionalització al nucli de la cèl•lula és una opció potencial. PE5 és una variant de l’HP-RNasa que és citotòxica perquè conté una seqüència de localització nuclear. Es demostra que aquesta seqüència permet la interacció de PE5 amb la importina-α. Una estratègia alternativa per dotar una RNasa d’activitat citotòxica és l’adquisició d’estructura quaternària. Es va obtenir en solució una variant de l’HP-RNasa, PM8, la qual forma un dímer per intercanvi de dominis. Aquesta variant dimeritza a través d’un mecanisme en dues etapes. Per comprovar aquest model se’n van obtenir variants en les quals es van eliminar cadenes laterals de residus implicats. Es va determinar el canvi de flexibilitat dels pèptids xarnera resultants, així com l’efecte dels canvis en la capacitat de les variants per dimeritzar i intercanviar els dominis

Sequence alignment is one of the basic methods to compare biological sequences and the cornerstone of a wide range of different analyses. Due to this privileged position at the beginning of many studies its accuracy is of great importance, in fact, each result based on an alignment is depending on the alignment quality. This has been confirmed in several recent papers investigating the effect of alignment methods on phylogenetic reconstruction and the estimation of positive selection. In this thesis, I present several projects dedicated to the problem of developing more accurate multiple sequence alignments and how to evaluate them. I addressed the problem of structural protein alignment evaluation, the accurate structural alignment of RNA sequences and the alignment of large sequence data sets. / El alineamiento es uno de los métodos básicos en la comparación de secuencias biológicas, y a menudo el primer pasó en análisis posteriores. Por su posición privilegiada al principio de muchos estudios, la calidad del alineamiento es de gran importancia, de hecho cada resultado basado en un alineamiento depende en gran medida de la calidad de ´este. Este hecho se ha confirmado en diversos artículos recientes, en los cuales se ha investigado los efectos de la elección del método de alineamiento en la reconstrucción filogenética y la estimación de la selección positiva. En esta tesis, presento varios proyectos enfocados en la implementación de mejoras tanto en los métodos de alineamiento múltiple de secuencias como en la evaluación de estos. Concretamente, he tratado problemas como la evaluación de alineamientos estructurales de proteínas, la construcción de alineamientos estructurales y precisos de ARN y también el alineamiento de grandes conjuntos de secuencias.

La monoubiquitinación de la histona H2A cumple un rol esencial en la represión génica. Diversas enzimas desubiquitinantes (DUBs) han sido identificadas, que remueven específicamente la ubiquitina de H2A en distintos contextos celulares. Sin embargo poco se sabe sobre la regulación de esta actividad enzimática y de la interrelación con la maquinaria de ubiquitinación. En el presente trabajo hemos estudiado USP21, una DUB específica de H2A, que modula la iniciación de la transcripción. Hemos identificado BEND3 como interactor del USP21 nuclear. BEND3 se distingue por la presencia de cuatro dominios BEN, está localizado en la heterocromatina y actúa como represor de la transcripción. Hemos validado y delimitado la interacción entre USP21 y BEND3 y hemos observado que BEND3 está polyubiquitinado. La estabilidad proteica de BEND3 y su localización subcelular fueron independientes de la actividad catalítica de USP21 y los niveles de H2Aub no fueron influenciados por BEND3. Finalmente, el análisis de la expresión génica en células madre embrionarias de ratón revela la expresión diferencial de genes involucrados en crecimiento, proliferación celular y ciclo celular. / Monoubiquitination of histone H2A fulfills an essential role in gene repression. Several deubiquitinating enzymes (DUBs) have been identified that specifically remove ubiquitin in different cellular contexts. However, the regulation of their enzymatic activity as well as their interplay with the ubiquitination machinery are not well understood. We studied the H2A specific deubiquitinase USP21, which modulates transcriptional initiation. We identified BEND3 as interactor of nuclear USP21. BEND3 is characterized by a quadruple repeat of BEN domains, is localized to heterochromatin, and acts as a transcriptional repressor. We validated and mapped the interaction between USP21 and BEND3 and found that BEND3 is polyubiquitinated. BEND3 protein stability and subcellular localization were independent of the catalytic activity of USP21 protein, and H2Aub levels were not influenced by BEND3. Finally microarray gene expression analysis in mouse embryonic stem cells depleted for BEND3 revealed the differential expression of genes involved in cellular growth proliferation and cell cycle amongst others.