As enzimas diidroorotato desidrogenases (DHODHs) são flavo-enzimas que catalisam a oxidação do diidroorotato em orotato na quarta etapa da biossíntese de novo de nucleotídeos de pirimidina. Durante a rápida proliferação celular em mamíferos, a via de salvação de pirimidinas é insuficiente para suprir deficiências na síntese de nucleotídeos. Além disso, certos parasitas não possuem a via de salvação e contam somente com a biossíntese de novo para a produção de nucleotídeos. Por esta razão, DHODH se tornou um excelente alvo na busca por inibidores que interrompam a síntese de nucleotídeos. As enzimas DHODHs de E. coli (EcDHODH) e de X. fastidiosa (XfDHODH) são membros da classe 2 das DHODHs e encontram-se associadas à membrana citoplasmática através de uma extensão em seu N-terminal, enquanto que DHODH de T. cruzi (TcDHODH), membro da classe 1 de DHODHs, é uma proteína citosólica. Neste trabalho, usamos uma combinação de metodologias de biologia molecular e bioquímica com técnicas espectroscópicas para obter informações estruturais e funcionais acerca da enzima DHODH. Assim, Ressonância Paramagnética Eletrônica (RPE) associada à marcação de spin sítio dirigida (SDSL) e simulação espectral foram empregadas para estudar a interação da EcDHODH com modelos de membrana. Mudanças na dinâmica estrutural das vesículas induzidas pela enzima foram monitoradas via marcadores de spin localizados em diferentes posições ao longo da cadeia acil de fosfolipídios. Além disso, técnicas de DNA recombinante e mutações sítio dirigidas foram utilizadas para produzir mutantes de EcDHODH no qual um sondas paramagnéticas foram seletivamente ligadas em resíduos localizados na extensão N-terminal da proteína para experimentos subseqüentes de RPE-SDSL. Esses são os primeiros experimentos de marcação de spin sítio dirigida realizados no Brasil e com os quais monitoramos a dinâmica experimentada na região do N-terminal. Além disso, várias tentativas foram feitas para se expressar e purificar a enzima XfDHODH e a estabilidade estrutural da enzima TcDHODH na presença de um de seus inibidores naturais, o orotato, foi monitorada através de experimentos de Dicroísmo Circular (CD). / Dihydroorotate dehydrogenases (DHODHs) are flavin-containing enzymes which catalyse the conversion of (S)-dihydroorotate to orotate, in the fourth step of the de novo biosynthesis of pyrimidine nucleotides. In rapidly proliferating mammalian cells, pyrimidine salvage pathway is insufficient to overcome deficiencies for nucleotide synthesis. Moreover certain parasites lack salvage enzymes, relying solely on the de novo pathway to produce nucleotides. Thus, DHODH has turned out an excellent target to the development of inhibitors that block nucleotide biosynthesis. E. coli DHODH (EcDHODH) and X. fastidiosa DHODH (XfDHODH) are class 2 DHODHs found associated to cytosolic membranes through an N-terminal extension, whereas T. cruzi DHODH (TcDHODH) is a class 1 DHODH localizated in the cytoplasm. In the present work, we used a combination of molecular biology and biochemical methodologies with spectroscopic techniques to obtain structural and functional information on DHODH. On one hand, Electronic Paramagnetic Resonance (EPR) associated with Site-directed Spin Labeling (SDSL) and spectral simulation were employed to study the interaction of EcDHODH with vesicles. Changes in vesicle dynamic structure induced by the enzyme were monitored via spin labels located at different positions along the phospholipid acyl chain and via spin labels located at enzyme specific positions. On the other hand, DNA techniques and site-directed mutagenesis were used to produce mutants of EcDHODH where a nitroxide spin probe was selectively attached to some residues located at the protein N-terminal extension for subsequent EPR-SDSL experiments. These are the first site-directed spin labeling experiments performed in Brazil and the spectra allowed us to monitor dynamics experienced by those residues at the EcDHODH N-terminal domain. Furthermore, molecular biology and biochemical assays were employed with the objective of expressing and purifying XfDHODH and Circular Dichroism (CD) was utilized to probe the structural stability of TcDHODH in the presence of its natural inhibitor (orotate).

Les canaux ioniques ASIC (Acid-Sensing Ion Channel), largement exprimés dans le système nerveux central et périphérique, sont activés par une acidification extracellulaire. Ils sont impliqués dans un nombre croissant de fonctions physiologiques ou pathologiques telles que la transmission synaptique, la plasticité synaptique, l'apprentissage, la mémoire, la peur, la dépression, l’épilepsie et la neuro-dégénérescence, ainsi que la nociception et la mécanosensibilité. Ils sont des cibles thérapeutiques d’intérêt dans le traitement de la douleur ou de troubles neurologiques. Les toxines peuvent modifier le fonctionnement des canaux ioniques et sont par conséquent utiles pour l’étude de leurs structures et de leurs fonctions biologiques. Des médicaments élaborés à partir de toxines issues de venins ont également permis le traitement de nombreuses pathologies. Mon travail de thèse comporte deux axes. D’une part, la mise en place de nouvelles stratégies de collectes de venins avec la mise au point d’un nouvel extracteur électronique. Et d’autre part, l’identification du site de liaison de la mambalgine sur les canaux ASIC1a, la description de son mécanisme d’action ainsi que la caractérisation des résidus clefs de cette toxine. En se basant sur une nouvelle approche pharmacologique tirant partie des mécanismes d’action des toxines et des petites molécules nous avons pu établir un modèle plus global du mécanisme de régulation des ASIC par les protons. Ce travail ouvre des perspectives pour le développement de formes optimisées de la mambalgine et apporte de nouvelles informations sur les mécanismes d’activation et d’inactivation des canaux ASIC / The Acid-Sensing Ion Channels (ASIC) are widely expressed in the central and peripheral nervous system and are activated by extracellular acidification. They are involved in increasing number of physiological or pathological functions such as synaptic transmission, synaptic plasticity, learning, memory, fear, depression, epilepsy and neurodegeneration, as well as nociception and mechanical sensitivity. They represent interesting therapeutic targets for the treatment of pain and neurological disorders. Several toxins are able to modify the gating of ion channels and are useful for studying their structures and biological functions. Moreover, drugs developed from toxins venoms have allowed the discovery of new treatments for many pathologies. My thesis work has two axes. First, the development of a new electronic extractor has allowed the implementation of new strategies to collect venoms. On the other hand, we were able to identify the binding site of the mambalgine toxin on ASIC1a, to describe its mechanism of action and to characterize the key residues of this toxin. Based on a new pharmacological approach using action mechanisms of toxins and small molecules, we were able to establish a global model of the pH-dependent gating of ASIC channels. This work opens some new perspectives for the development of optimized forms of mambalgine and provides new information on the gating of ASIC

Der Komplex aus Kohlenmonoxid-Dehydrogenase und Acetyl-CoA-Synthase (CODH/ACS Komplex) des thermophilen Bakteriums Carboxydothermus hydrogenoformans katalysiert die Fixierung von CO2 in Acetyl-CoA und ist damit ein potenzieller Katalysator für die Erzeugung erneuerbarer Kraftstoffe aus CO2. Die Katalyse erfolgt an zwei verschiedenen Stellen: CO2 wird am Cluster C in der CODH-Untereinheit zu CO reduziert, das dann durch einen Tunnel innerhalb des Proteins zum Cluster A in der ACS-Untereinheit wandert, wo es mit einer Methylgruppe und CoA zu Acetyl-CoA reagiert. Die Art und Weise, wie die beiden katalytischen Aktivitäten zusammenwirken, sind noch unklar. Um hier mehr Licht ins Dunkel zu bringen, verfolgte diese Arbeit drei Ziele: die Bestimmung der Struktur des CODH/ACS-Komplexes von C. hydrogenoformans, die Untersuchung der CO2-Reduktionsaktivität von CODHasen und die Analyse der Rolle des internen Tunnels im CODH/ACS-Komplex. Die Struktur des CODH/ACS-Komplexes von C. hydrogenoformans wurde durch Röntgenkristallographie mit einer Auflösung von 2,04 Å bestimmt. Die CO2-Reduktion am Cluster C wurde kinetisch untersucht. Es zeigte sich, dass die CO2-Reduktion durch einen Ping-Pong-Mechanismus mit zwei Reaktionsstellen erfolgen könnte, der in früheren Studien vorgeschlagen wurde, aber auch durch andere Mechanismen. Um eine Struktur-Funktionsbeziehung für CODHs zu ermitteln, wurde die CO2-Reduktionsaktivität für drei CODHasen von C. hydrogenoformans untersucht, deren Strukturen bekannt sind: CODH-II, CODH-IV, und der CODH/ACS-Komplex. Das Tunnelsystem im CODH/ACS-Komplex ist viel enger als in den anderen CODHs und könnte somit der Grund für die vergleichsweise geringe Aktivität des CODH/ACS-Komplexes sein. Dies wurde auch durch die Manipulation und Analyse des internen Tunnels des CODH/ACS-Komplexes unterstützt. Die Ergebnisse deuten darauf hin, dass der Hauptzweck des Tunnels im CODH/ACS-Komplex die Kompartimentierung von CO und nicht der schnelle Substrattransport ist. / The complex of carbon monoxide dehydrogenase and acetyl-CoA synthase (CODH/ACS complex) of the thermophilic bacterium Carboxydothermus hydrogenoformans catalyses the fixation of CO2 into acetyl-CoA and is thus a potential catalyst for the production of renewable fuels from CO2. Catalysis occurs at two different sites: CO2 is reduced to CO at cluster C in the CODH subunit, which then travels through a tunnel within the protein to cluster A in the ACS subunit, where it reacts with a methyl group and CoA to form acetyl-CoA. The way in which the two catalytic activities interact is still unclear. To shed more light on this, this work pursued three goals: to determine the structure of the CODH/ACS complex of C. hydrogenoformans, to investigate the CO2 reduction activity of CODHases and to analyse the role of the internal tunnel in the CODH/ACS complex. The structure of the CODH/ACS complex of C. hydrogenoformans was determined by X-ray crystallography at 2.04 Å resolution. The CO2 reduction at cluster C was investigated kinetically. It was found that CO2 reduction could occur by a two-site ping-pong mechanism proposed in previous studies, but also by other mechanisms. To establish a structure-function relationship for CODHs, CO2 reduction activity was investigated for three CODHases of C. hydrogenoformans whose structures are known: CODH-II, CODH-IV, and the CODH/ACS complex. The tunnel system in the CODH/ACS complex is much narrower than in the other CODHs and could thus be the reason for the comparatively low activity of the CODH/ACS complex. This was also supported by the manipulation and analysis of the internal tunnel of the CODH/ACS complex. The results suggest that the main purpose of the tunnel in the CODH/ACS complex is to compartmentalise CO and not to rapidly transport substrate.

No description available.

A esquistossomose é a mais importante das doenças helmínticas humanas em termos de morbidez e mortalidade. A proteína Sm14 de Schistosoma mansoni, que pertence à família de proteínas ligadoras de ácidos graxos (fatty acid-binding proteins, FABPs) (Moser et al., 1991), mostrou um bom nível de proteção (65%) contra a esquistossomose em animais experimentais (Tendler et al., 1996). No presente trabalho foram desenvolvidos sistemas de expressão que possibilitará a produção da proteína Sm14 em larga escala em E.coli. Com o intuito de conhecer a estrutura do gene da proteína Sm14, foi clonado um fragmento de DNA genômico de S. mansoni que contém a seqüência codificante da proteína Sm14. Como os outros membros da família gênica das FABP, o gene para a proteína Sm14 contém quatro \"exons\" separados por três \"introns\" de 674, 585 e 42 bp. Esta é a primeira descrição da estrutura gênica de um membro das FABP correspondente a um helminto. A Sm14 é uma proteína que pode ser potencialmente usada como vacina. Estudamos a existência de polimorfismo em duas linhagens de S. mansoni endêmicas do Brasil: LE e BH. Para a análise de polimorfismo, a ORF correspondente à proteína Sm14 foi amplificada por RT-PCR do RNA total de vermes adultos de S. mansoni. Os produtos de amplificação independentes foram clonados no vetor pGEM-T e seqüenciados. As análises de seqüências mostraram duas isoformas principais para a proteína Sm14: Sm14-M20, com seqüência idêntica a proteína Sm14 previamente reportada para a linhagem de Puerto Rico de S. mansoni (Moser et AL., 1991), e Sm14-T20, onde o códon da Met20 (ATG) mudou para o códon de Thr (ACG) (polimorfismo M20T). Dois clones mostraram uma deleção de seqüência de aminoácidos correspondente ao \"exon\" 3 inteiro (clones ΔExon3), gerada por \"splicing\" alternativo. As outras trocas observadas acontecem em posições onde os aminoácidos são menos conservados e estão representados apenas por um único clone que podem ter sido obtidas por mutagênese na PCR. A metionina correspondente à posição 20 na Sm14 é altamente conservada nas FABP dos mais diversos organismos,e não se tem nenhuma outra proteína com treonina nesta posição. Para o estudo da estrutura e função destas isoformas, os cDNAs correspondentes foram subclonados no vetor pAE (desenvolvido no nosso laboratório), assim como o mutante M20A (Sm14-A20) construído para efeitos de comparação. A estabilidade e estrutura das proteínas recombinantes purificadas foram caracterizadas por dicroísmo circular (CD). A comparação da estrutura e termoestabilidade mostrou que as formas Sm14-T20 e Sm14-A20 são menos termoestáveis do que a Sm14-M20 (um ΔTm de aproximadamente 10°C). Porém, todas as formas de Sm14 foram capazes de ligar o DAUDA [ácido 11-(dansylamino) undecanoico] com a mesma afinidade. Para poder diferenciar as propriedades de ligação de ácidos graxos pelas isoformas, experiências de competição do deslocamento do DAUDA por ácidos graxos naturais, foram realizadas. A partir destes dados podemos assumir que a forma Sm14-M20 liga melhor todos os ácidos graxos naturais testados do que a forma Sm14-T20. Porém esta forma mantém a capacidade de ligar ácidos graxos, ao contrario do mutante Sm14-A20. Pode-se deduzir como resultado destas experiências que a proteína Sm14-M20 é mais estável e liga com maior afinidade os ácidos graxos naturais do que a forma Sm14-T20. Pelo visto, a proteína Sm14-T20 tem menos estrutura-β, porém, mantém a capacidade de ligar moléculas hidrofóbicas. Ainda é desconhecido o papel funcional do polimorfismo da proteína Sm14 no metabolismo dos vermes de S. mansoni. Problemas de estabilidade da proteína Sm14 recombinante, durante seu transporte e armazenamento, comprometem sua viabilidade como vacina. Com o intuito de melhorar a estabilidade desta proteína, foi feita uma mutagênese no único resíduo de cisteína presente na Sm14 na posição 62. Este resíduo é responsável pela formação de dímeros, o que é relacionado a estabilização da perda de estrutura-β e precipitação da proteína. Esta cisteína foi trocada por serina (C62S) e por valina (C62V) por mutagênese sítio dirigida, resultando nas proteínas Sm14-M20S62 e Sm14-M20V62. As formas mutantes não apresentaram maior termoestabilidade, mas a renaturação após o aquecimento a 80°C atingiu quase 100%, diferentemente das proteínas com Cys62. As proteínas com o resíduo de cisteina trocado foram as únicas formas que conservaram a estrutura de β-barril após 3 meses de armazenamento a 4°C, como mostram as análises de dicroísmo circular, sendo a forma mais estável a proteína Sm14-M20V62. Após estes estudos, a isoforma Sm14-M20 com a mutação C62V (Sm14-M20V62) mostrou-se como a melhor alternativa ao antígeno Sm14-T20 usado até agora como modelo de vacina experimental para S. mansoni. Esta indicação deve ser confirmada em ensaios de imunização e posterior desafio com cercárias de S. mansoni. / The schistosomiasis is the most important human helmintic disease in terms of morbidity and mortality. The Sm14 protein of Schistosoma mansoni belongs to the family of fatty acid-binding proteins (FABPs) (Moser et aI. , 1991) and showed a good protection level as vaccine antigen against the schistosomiasis in experimental animals (Tendler et al., 1996). In the present work were developed systems for the expression of Sm14 protein that will facilitate its large scale production in E.coli.. In order to know the gene structure of the Sm14 protein, we amplified by PCR a genomic DNA fragment of S. mansoni that contains the coding sequence for the Sm14 protein. As the other members of the FABP family, the Sm14 gene contains four exons separated by three introns of 674,585 and 42 bp, respectively. This is the first detailed description of the genomic structure for a member of FABPs corresponding to a helmint. We also studied the existence of polymorphisms within two Brazilian endemic strains of S.mansoni: LE and BH. For the polymorphism analysis, the ORF corresponding to the Sm14 protein was amplified by RT-PCR from total RNA of S. mansoni adult worms. The independent amplified products were cloned into pGEM-T vector and sequenced. The sequence analyses showed two main isoforms: Sm14-M20, with identical sequence to that previously reported Sm14 protein from the Puerto Rican strain of S. mansoni (Moser et al., 1991), and Sm14-T20, where the codon for Met20 (ATG) was changed for the Thr codon (ACG) (M20T polymorphism). Two clones showed the same amino acid sequence deletion corresponding to the whole third exon (ΔExon3 clones), generated by alternative splicing. The other observed changes occurred in positions where the amino acids were less conserved and were just represented by only one clone that could be obtained by PCR mutagenesis. The methionine corresponding to the position 20 in Sm14 is highly conserved among FABPs and no other related protein has threonin in this position. To study the structure and function of these amino acid in the isoforms, the corresponding cDNAs were subcloned in to the pAE vector (developed in our laboratory), as well as the mutant M20A (Sm14-A20). The stability and structure of the purified recombinant proteins were characterized by circular dicroism (CD). The comparison of their structure and thermo stability showed that the forms Sm14-T20 and Sm14-A20 are less thermostable than Sm14-M20 (ΔTm around 10ºC). However, all of the Sm14 forms were capable to bind the DAUDA [11- (dansylamine) undecanoic acid] with similar affinities. To differentiate the fatty acid binding properties of Sm14 isoforms, displacement experiments of DAUDA with natural fatty acid were performed. From these data we can assume that the Sm14-M20 form binds better than the Sm14-T20 and Sm14-A20 forms of all natural fatty acid assayed. This suggests that the Sm14-20 protein is most stable and binds better the natural fatty acids than the Sm14-T20 form. Although the Sm14-T20 protein has less structure, it maintains the capacity to bind fatty acids. It is still unknown the functional role of this Sm14 protein polymorphism in the metabolism of S. mansoni worms. Stability problems of the recombinant Sm14 protein during its transport and storage, could hamper its use as vaccine. With the aim to improve the stability of this protein, it was made a mutagenese at the unique cysteine residue present in Sm14 at the position 62. This residue is responsible for the dimer formation and is related the loss of the terciary structure and precipitation of the protein. This cysteine was changed by serine (C62S) and for valine (C62V) by site directed mutagenesis, resulting in the proteins Sm14-M20S62 and Sm14-M20V62. The mutant forms did not present a higher thermal stability but the renaturation after heating at 80°C almost reached 100%, in contrast to Sm14 proteins with Cys62. These mutants conserved the β-barrel structure after 3 months of storage at 4°C, in contrast to proteins with Cys62, as shown by circular dicroism analyses. After these studies, the Sm14-M20 isoform with the C62V mutation (Sm14-M20V62) was considered the best alternative to the antigen Sm14-T20 used up to now as the model for an experimental vaccine for S. mansoni. This indication should be confirmed by immunization and posterior challenge with S. mansoni cercaria.

This PhD thesis is the result of the combination of experimental and computational techniques with the aim of understanding the mechanism of action of de novo cyclic decapeptides with high antimicrobial activity. By experimental techniques the influence of the replacement of the phenylalanine for tryptophan residue in their antimicrobial activity was tested and the stability in human serum was also analyzed, in order to evaluate their potential therapeutic application as antitumor agents. On the other hand, the interaction amongst the peptide BPC194 c(KKLKKFKKLQ), the best candidate from the whole library of cyclic peptides, and a model anionic membrane was simulated. The results showed a structure-function relationship derived from the stable conformation of the peptides involved in the membrane permeabilization. As a result, a rational design was performed being BPC490 the peptide with best antimicrobial activity compared with the best active peptide from the original library. / Aquesta tesi doctoral resulta de la combinació d’estudis mitjançant tècniques experimentals i computacionals amb l’objectiu d’entendre el mecanisme d’acció de "de novo" decapèptids cíclics amb elevada activitat antimicrobiana. Experimentalment, es va avaluar la influència de la substitució dels residus de fenilalanina per triptòfan en la seva activitat antimicrobiana i també la seva estabilitat sèrum humà, per tal de valorar la seva possible aplicació terapèutica envers el càncer. Per altra banda, es va simular la interacció del pèptid BPC194 c(KKLKKFKKLQ), millor candidat de la biblioteca de pèptids cíclics, amb models aniònics de bicapa lipídica. Els resultats van posar en manifest una relació estructura-funció derivada de la conformació estable dels pèptids que participen directament en la permeabilització de la membrana. Es va procedir doncs al disseny racional de nous pèptids cíclics sent el pèptid BPC490 el que va presentar una millor activitat bacteriana en comparació amb el pèptid més actiu de la llibreria original.

In der vorliegenden Arbeit wurden die molekularen Grundlagen für die Agonistspezifität des G-Protein-gekoppelten Rezeptors GPR34 untersucht. Mittels verschiedener funktioneller Versuche konnte an ausgewählten Orthologen des Rezeptors gezeigt werden, dass, im Gegensatz zu publizierten Daten, Lysophosphatidylserin (Lyso-PS) nicht der natürliche Agonist des GPR34 ist. Lediglich an einigen cyprinoiden Subtypen des GPR34 hat Lyso-PS surrogat-agonistische Effekte. Anhand eines detaillierten evolutionären Vergleichs von Orthologen konnten Bereiche des Rezeptors ermittelt werden, welche an der Ligandenbindung, und damit an der Agonistspezifität des GPR34 beteiligt sind. Durch Übertragung dieser Bereiche vom Karpfen-GPR34-Subtyp 2a auf den humanen GPR34 konnte dieser zu einem Lyso-PS-sensitiven Rezeptor modelliert werden. Weiterhin wurde Aminoethyl-Carbamoyl-ATP (EDA-ATP) als inverser Agonist an cyprinoiden Orthologen des GPR34 identifiziert. Die Erweiterung des möglichen Ligandenspektrums von Lipiden zu Nukleotidderivaten gibt Hinweise auf die Promiskuität der Bindungsstelle des GPR34. Die Ergebnisse zeigen, dass Lyso-PS nur eine zufällige Aktivität an einigen Orthologen des GPR34 hat. Mit Identifizierung eines Nichtlipides als invers-agonistischen Liganden ist die Suche nach dem natürlichen Liganden des GPR34 noch nicht abgeschlossen und sollte auf weitere chemische Entitäten ausgeweitet werden. / Lyso-PS (lyso-phosphatidylserine) has been shown to activate the G(i/o)-protein-coupled receptor GPR34. Since in vitro and in vivo studies provided controversial results in assigning lyso-PS as the endogenous agonist for GPR34, we investigated the evolutionary conservation of agonist specificity in more detail. Except for some fish GPR34 subtypes, lyso-PS has no or very weak agonistic activity at most vertebrate GPR34 orthologues investigated. Using chimaeras we identified single positions in the second extracellular loop and the transmembrane helix 5 of carp subtype 2a that, if transferred to the human orthologue, enabled lyso-PS to activate the human GPR34. Significant improvement of agonist efficacy by changing only a few positions strongly argues against the hypothesis that nature optimized GPR34 as the receptor for lyso-PS. Phylogenetic analysis revealed several positions in some fish GPR34 orthologues which are under positive selection. These structural changes may indicate functional specification of these orthologues which can explain the species- and subtype-specific pharmacology of lyso-PS. Furthermore, we identified aminoethyl-carbamoyl ATP as an antagonist of carp GPR34, indicating ligand promiscuity with non-lipid compounds. The results of the present study suggest that lyso-PS has only a random agonistic activity at some GPR34 orthologues and the search for the endogenous agonist should consider additional chemical entities.

A esquistossomose é a mais importante das doenças helmínticas humanas em termos de morbidez e mortalidade. A proteína Sm14 de Schistosoma mansoni, que pertence à família de proteínas ligadoras de ácidos graxos (fatty acid-binding proteins, FABPs) (Moser et al., 1991), mostrou um bom nível de proteção (65%) contra a esquistossomose em animais experimentais (Tendler et al., 1996). No presente trabalho foram desenvolvidos sistemas de expressão que possibilitará a produção da proteína Sm14 em larga escala em E.coli. Com o intuito de conhecer a estrutura do gene da proteína Sm14, foi clonado um fragmento de DNA genômico de S. mansoni que contém a seqüência codificante da proteína Sm14. Como os outros membros da família gênica das FABP, o gene para a proteína Sm14 contém quatro \"exons\" separados por três \"introns\" de 674, 585 e 42 bp. Esta é a primeira descrição da estrutura gênica de um membro das FABP correspondente a um helminto. A Sm14 é uma proteína que pode ser potencialmente usada como vacina. Estudamos a existência de polimorfismo em duas linhagens de S. mansoni endêmicas do Brasil: LE e BH. Para a análise de polimorfismo, a ORF correspondente à proteína Sm14 foi amplificada por RT-PCR do RNA total de vermes adultos de S. mansoni. Os produtos de amplificação independentes foram clonados no vetor pGEM-T e seqüenciados. As análises de seqüências mostraram duas isoformas principais para a proteína Sm14: Sm14-M20, com seqüência idêntica a proteína Sm14 previamente reportada para a linhagem de Puerto Rico de S. mansoni (Moser et AL., 1991), e Sm14-T20, onde o códon da Met20 (ATG) mudou para o códon de Thr (ACG) (polimorfismo M20T). Dois clones mostraram uma deleção de seqüência de aminoácidos correspondente ao \"exon\" 3 inteiro (clones ΔExon3), gerada por \"splicing\" alternativo. As outras trocas observadas acontecem em posições onde os aminoácidos são menos conservados e estão representados apenas por um único clone que podem ter sido obtidas por mutagênese na PCR. A metionina correspondente à posição 20 na Sm14 é altamente conservada nas FABP dos mais diversos organismos,e não se tem nenhuma outra proteína com treonina nesta posição. Para o estudo da estrutura e função destas isoformas, os cDNAs correspondentes foram subclonados no vetor pAE (desenvolvido no nosso laboratório), assim como o mutante M20A (Sm14-A20) construído para efeitos de comparação. A estabilidade e estrutura das proteínas recombinantes purificadas foram caracterizadas por dicroísmo circular (CD). A comparação da estrutura e termoestabilidade mostrou que as formas Sm14-T20 e Sm14-A20 são menos termoestáveis do que a Sm14-M20 (um ΔTm de aproximadamente 10°C). Porém, todas as formas de Sm14 foram capazes de ligar o DAUDA [ácido 11-(dansylamino) undecanoico] com a mesma afinidade. Para poder diferenciar as propriedades de ligação de ácidos graxos pelas isoformas, experiências de competição do deslocamento do DAUDA por ácidos graxos naturais, foram realizadas. A partir destes dados podemos assumir que a forma Sm14-M20 liga melhor todos os ácidos graxos naturais testados do que a forma Sm14-T20. Porém esta forma mantém a capacidade de ligar ácidos graxos, ao contrario do mutante Sm14-A20. Pode-se deduzir como resultado destas experiências que a proteína Sm14-M20 é mais estável e liga com maior afinidade os ácidos graxos naturais do que a forma Sm14-T20. Pelo visto, a proteína Sm14-T20 tem menos estrutura-β, porém, mantém a capacidade de ligar moléculas hidrofóbicas. Ainda é desconhecido o papel funcional do polimorfismo da proteína Sm14 no metabolismo dos vermes de S. mansoni. Problemas de estabilidade da proteína Sm14 recombinante, durante seu transporte e armazenamento, comprometem sua viabilidade como vacina. Com o intuito de melhorar a estabilidade desta proteína, foi feita uma mutagênese no único resíduo de cisteína presente na Sm14 na posição 62. Este resíduo é responsável pela formação de dímeros, o que é relacionado a estabilização da perda de estrutura-β e precipitação da proteína. Esta cisteína foi trocada por serina (C62S) e por valina (C62V) por mutagênese sítio dirigida, resultando nas proteínas Sm14-M20S62 e Sm14-M20V62. As formas mutantes não apresentaram maior termoestabilidade, mas a renaturação após o aquecimento a 80°C atingiu quase 100%, diferentemente das proteínas com Cys62. As proteínas com o resíduo de cisteina trocado foram as únicas formas que conservaram a estrutura de β-barril após 3 meses de armazenamento a 4°C, como mostram as análises de dicroísmo circular, sendo a forma mais estável a proteína Sm14-M20V62. Após estes estudos, a isoforma Sm14-M20 com a mutação C62V (Sm14-M20V62) mostrou-se como a melhor alternativa ao antígeno Sm14-T20 usado até agora como modelo de vacina experimental para S. mansoni. Esta indicação deve ser confirmada em ensaios de imunização e posterior desafio com cercárias de S. mansoni. / The schistosomiasis is the most important human helmintic disease in terms of morbidity and mortality. The Sm14 protein of Schistosoma mansoni belongs to the family of fatty acid-binding proteins (FABPs) (Moser et aI. , 1991) and showed a good protection level as vaccine antigen against the schistosomiasis in experimental animals (Tendler et al., 1996). In the present work were developed systems for the expression of Sm14 protein that will facilitate its large scale production in E.coli.. In order to know the gene structure of the Sm14 protein, we amplified by PCR a genomic DNA fragment of S. mansoni that contains the coding sequence for the Sm14 protein. As the other members of the FABP family, the Sm14 gene contains four exons separated by three introns of 674,585 and 42 bp, respectively. This is the first detailed description of the genomic structure for a member of FABPs corresponding to a helmint. We also studied the existence of polymorphisms within two Brazilian endemic strains of S.mansoni: LE and BH. For the polymorphism analysis, the ORF corresponding to the Sm14 protein was amplified by RT-PCR from total RNA of S. mansoni adult worms. The independent amplified products were cloned into pGEM-T vector and sequenced. The sequence analyses showed two main isoforms: Sm14-M20, with identical sequence to that previously reported Sm14 protein from the Puerto Rican strain of S. mansoni (Moser et al., 1991), and Sm14-T20, where the codon for Met20 (ATG) was changed for the Thr codon (ACG) (M20T polymorphism). Two clones showed the same amino acid sequence deletion corresponding to the whole third exon (ΔExon3 clones), generated by alternative splicing. The other observed changes occurred in positions where the amino acids were less conserved and were just represented by only one clone that could be obtained by PCR mutagenesis. The methionine corresponding to the position 20 in Sm14 is highly conserved among FABPs and no other related protein has threonin in this position. To study the structure and function of these amino acid in the isoforms, the corresponding cDNAs were subcloned in to the pAE vector (developed in our laboratory), as well as the mutant M20A (Sm14-A20). The stability and structure of the purified recombinant proteins were characterized by circular dicroism (CD). The comparison of their structure and thermo stability showed that the forms Sm14-T20 and Sm14-A20 are less thermostable than Sm14-M20 (ΔTm around 10ºC). However, all of the Sm14 forms were capable to bind the DAUDA [11- (dansylamine) undecanoic acid] with similar affinities. To differentiate the fatty acid binding properties of Sm14 isoforms, displacement experiments of DAUDA with natural fatty acid were performed. From these data we can assume that the Sm14-M20 form binds better than the Sm14-T20 and Sm14-A20 forms of all natural fatty acid assayed. This suggests that the Sm14-20 protein is most stable and binds better the natural fatty acids than the Sm14-T20 form. Although the Sm14-T20 protein has less structure, it maintains the capacity to bind fatty acids. It is still unknown the functional role of this Sm14 protein polymorphism in the metabolism of S. mansoni worms. Stability problems of the recombinant Sm14 protein during its transport and storage, could hamper its use as vaccine. With the aim to improve the stability of this protein, it was made a mutagenese at the unique cysteine residue present in Sm14 at the position 62. This residue is responsible for the dimer formation and is related the loss of the terciary structure and precipitation of the protein. This cysteine was changed by serine (C62S) and for valine (C62V) by site directed mutagenesis, resulting in the proteins Sm14-M20S62 and Sm14-M20V62. The mutant forms did not present a higher thermal stability but the renaturation after heating at 80°C almost reached 100%, in contrast to Sm14 proteins with Cys62. These mutants conserved the β-barrel structure after 3 months of storage at 4°C, in contrast to proteins with Cys62, as shown by circular dicroism analyses. After these studies, the Sm14-M20 isoform with the C62V mutation (Sm14-M20V62) was considered the best alternative to the antigen Sm14-T20 used up to now as the model for an experimental vaccine for S. mansoni. This indication should be confirmed by immunization and posterior challenge with S. mansoni cercaria.

La dihydrofolate réductase humaine (DHFRh) est une enzyme essentielle à la prolifération cellulaire, ce qui en fait une cible de choix pour le traitement de différents cancers. À cet effet, plusieurs inhibiteurs spécifiques de la DHFRh, les antifolates, ont été mis au point : le méthotrexate (MTX) et le pemetrexed (PMTX) en sont de bons exemples. Malgré l’efficacité clinique certaine de ces antifolates, le développement de nouveaux traitements s’avère nécessaire afin de réduire les effets secondaires liés à leur utilisation. Enfin, dans l’optique d’orienter la synthèse de nouveaux composés inhibiteurs des DHFRh, une meilleure connaissance des interactions entre les antifolates et leur enzyme cible est primordiale. À l’aide de l’évolution dirigée, il a été possible d’identifier des mutants de la DHFRh pour lesquels l’affinité envers des antifolates cliniquement actifs se voyait modifiée. La mutagenèse dite ¬¬de saturation a été utilisée afin de générer des banques de mutants présentant une diversité génétique au niveau des résidus du site actif de l’enzyme d’intérêt. De plus, une nouvelle méthode de criblage a été mise au point, laquelle s’est avérée efficace pour départager les mutations ayant entrainé une résistance aux antifolates et/ou un maintient de l’activité enzymatique envers son substrat natif, soient les phénotypes d’activité. La méthode de criblage consiste dans un premier temps en une sélection bactérienne à haut débit, puis dans un second temps en un criblage sur plaques permettant d’identifier les meilleurs candidats. Plusieurs mutants actifs de la DHFRh, résistants aux antifolates, ont ainsi pu être identifiés et caractérisés lors d’études de cinétique enzymatique (kcat et IC50). Sur la base de ces résultats cinétiques, de la modélisation moléculaire et des données structurales de la littérature, une étude structure-activité a été effectuée. En regardant quelles mutations ont les effets les plus significatif sur la liaison, nous avons commencé à construire un carte moléculaire des contacts impliqués dans la liaison des ligands. Enfin, des connaissances supplémentaires sur les propriétés spécifiques de liaison ont put être acquises en variant l’inhibiteur testé, permettant ainsi une meilleure compréhension du phénomène de discrimination du ligand. / Human dihydrofolate reductase (hDHFR) is an essential enzyme for cellular proliferation and it has long been the target of antifolate drugs for the treatment of various types of cancer. Despite the clinical effectiveness of current antifolate treatments, new drugs are required to reduce the side-effects associated with their use. An essential requirement for design of new antifolates is a better understanding of how these drugs interact with their targets. We applied directed evolution to identify mutant hDHFR variants with modified binding to some clinically relevant antifolates. A saturation mutagenesis approach was used to create genetic diversity at active-site residues of hDHFR and a new, efficient screening strategy was developed to identify the amino acids that preserved native activity and/or conferred antifolate resistance. The screening method consists in a high-throughput first-tier bacterial selection coupled with a second-tier in vitro assay that allows for rapid detection of the best variants among the leads, according to user-defined parameters. Many active, antifolate-resistant mutants of hDHFR were identified. Moreover, the approach has proven efficient in rapidly assessing kinetic (kcat) and inhibition parameters of the hDHFR variants (IC50). Structure-function relationship analysis based on kinetic investigation, available structural and functional data as well as modeling were performed. By monitoring which mutations have the greatest effect on binding, we have begun to build a molecular picture of the contacts involved in drug binding. By varying the drugs we test against, we gain a better understanding of the specific binding properties that determine ligand discrimination.

La dihydrofolate réductase humaine (DHFRh) est une enzyme essentielle à la prolifération cellulaire, ce qui en fait une cible de choix pour le traitement de différents cancers. À cet effet, plusieurs inhibiteurs spécifiques de la DHFRh, les antifolates, ont été mis au point : le méthotrexate (MTX) et le pemetrexed (PMTX) en sont de bons exemples. Malgré l’efficacité clinique certaine de ces antifolates, le développement de nouveaux traitements s’avère nécessaire afin de réduire les effets secondaires liés à leur utilisation. Enfin, dans l’optique d’orienter la synthèse de nouveaux composés inhibiteurs des DHFRh, une meilleure connaissance des interactions entre les antifolates et leur enzyme cible est primordiale. À l’aide de l’évolution dirigée, il a été possible d’identifier des mutants de la DHFRh pour lesquels l’affinité envers des antifolates cliniquement actifs se voyait modifiée. La mutagenèse dite ¬¬de saturation a été utilisée afin de générer des banques de mutants présentant une diversité génétique au niveau des résidus du site actif de l’enzyme d’intérêt. De plus, une nouvelle méthode de criblage a été mise au point, laquelle s’est avérée efficace pour départager les mutations ayant entrainé une résistance aux antifolates et/ou un maintient de l’activité enzymatique envers son substrat natif, soient les phénotypes d’activité. La méthode de criblage consiste dans un premier temps en une sélection bactérienne à haut débit, puis dans un second temps en un criblage sur plaques permettant d’identifier les meilleurs candidats. Plusieurs mutants actifs de la DHFRh, résistants aux antifolates, ont ainsi pu être identifiés et caractérisés lors d’études de cinétique enzymatique (kcat et IC50). Sur la base de ces résultats cinétiques, de la modélisation moléculaire et des données structurales de la littérature, une étude structure-activité a été effectuée. En regardant quelles mutations ont les effets les plus significatif sur la liaison, nous avons commencé à construire un carte moléculaire des contacts impliqués dans la liaison des ligands. Enfin, des connaissances supplémentaires sur les propriétés spécifiques de liaison ont put être acquises en variant l’inhibiteur testé, permettant ainsi une meilleure compréhension du phénomène de discrimination du ligand. / Human dihydrofolate reductase (hDHFR) is an essential enzyme for cellular proliferation and it has long been the target of antifolate drugs for the treatment of various types of cancer. Despite the clinical effectiveness of current antifolate treatments, new drugs are required to reduce the side-effects associated with their use. An essential requirement for design of new antifolates is a better understanding of how these drugs interact with their targets. We applied directed evolution to identify mutant hDHFR variants with modified binding to some clinically relevant antifolates. A saturation mutagenesis approach was used to create genetic diversity at active-site residues of hDHFR and a new, efficient screening strategy was developed to identify the amino acids that preserved native activity and/or conferred antifolate resistance. The screening method consists in a high-throughput first-tier bacterial selection coupled with a second-tier in vitro assay that allows for rapid detection of the best variants among the leads, according to user-defined parameters. Many active, antifolate-resistant mutants of hDHFR were identified. Moreover, the approach has proven efficient in rapidly assessing kinetic (kcat) and inhibition parameters of the hDHFR variants (IC50). Structure-function relationship analysis based on kinetic investigation, available structural and functional data as well as modeling were performed. By monitoring which mutations have the greatest effect on binding, we have begun to build a molecular picture of the contacts involved in drug binding. By varying the drugs we test against, we gain a better understanding of the specific binding properties that determine ligand discrimination.