In eukaryotes, the first step of interpreting the genetic information is the transcription of DNA into RNA. For protein-coding genes, such transcription is carried out by RNA polymerase II. A special domain of RNA polymerase II, called the C-terminal domain (CTD), functions as a master controller for the transcription process by providing a platform to recruit regulatory proteins to nascent mRNA (Chapter 1-2). The modifications and conformational states of the CTD, termed the 'CTD code', represent a critical regulatory checkpoint for transcription. The CTD, found only in eukaryotes, consists of 26--52 tandem heptapeptide repeats with the consensus sequence, Tyr₁Ser₂Pro₃Thr₄Ser₅Pro₆Ser₇. Phosphorylation of the serines and prolyl isomerization of the prolines represent two major regulatory mechanisms of the CTD. Interestingly, the phosphorylation sites are typically close to prolines, thus the conformation of the adjacent proline could impact the specificity of the corresponding kinases and phosphatases. Understanding how those modifying enzymes recognize and regulate the CTD is important for expanding our knowledge on the transcription regulation and deciphering the 'CTD code'. During my PhD study, I studied the function of CTD phosphatases and prolyl isomerase in the CTD regulation using Scp1, Ssu72 and Pin1 as model regulators. Scp1 and Ssu72 are both Ser5 phosphatases. However, Ssu72 is an essential protein and regulates the global transcription while Scp1 epigenetically silences the expression of specific neuronal genes. Pin1 is a highly conserved phosphorylation-specific prolyl isomerase that recognizes the phospho-Ser/Thr-Pro motif within the CTD as one of its primary substrates in vivo. Among these enzymes, Scp1 is the focal point of this dissertation, as it was studied from different angles, such as enzymatic mechanism (Chapter 3 describes the capture of phospho-aspartyl intermediate of Scp1 as a direct evidence for the proposed two-step mechanism), specific inhibition (Chapter 4 describes the identification and characterization of the first specific inhibitor of Scp1), and its non-active-site contact with the CTD (Chapter 5 describes the structural basis of this contact). These studies are of great importance towards understanding the molecular mechanism of the dephosphorylation process of the CTD by Scp1. / text

Nous avons caractérisé cinq protéines bovines qui sont potentiellement impliquées dans la reproduction.Un travail de clonage a été initié qui permettra à terme de purifier les GPA2 et GPB5 recombinantes puis naturelles pour étudier leurs structures. GPA2 et GPB5 sont considérés comme les ancêtres moléculaires des sous-unités α et β des hormones glycoprotéiques. Nous avons montré la relative fragilité thermique de la structure quaternaire de la FSH bovine par rapport aux FSH ovine et humaine et nous avons étudié les propriétés enzymatiques de la PDI (Protein Disulfide Isomerase) en préalable à l’étude de l’activité PDI de GPA2/GPB5. Nous avons aussi purifié la phosphatidyl-ethanolamine-binding protein (PEBP) et l’ubiquitine testiculaires par chromatographie hydrophobe à très haute concentration de sulfate d’ammonium. A partir de la PEBP purifiée, on a produit des anticorps spécifiques chez le lapin qui nous ont permis d’être les premiers à développer un dosage ELISA fiable pour cette protéine. / We characterized five bovine proteins that are potentially involved in reproduction. We started with the cloning of gpa2 and gpb5 cDNAs in order to eventually purify recombinant and natural GPA2 and GPB5 to study their possible quaternary structure. GPA2 and GPB5 are the evolutionary ancestors of Glycoprotein hormones α and β subunits respectively. Meanwhile, we have shown the relative quaternary structure fragility of bovine FSH compared to human and sheep FSH. We also studied the effect of endocrine disruptors on PDI (Protein Disulfide Isomerase) before addressing GPA2/GPB5 PDI activity of GPA2/GPB5 once purified.We succeeded to purify the phosphatidyl-ethanolamine-binding protein (PEBP) and ubiquitin from bovine testis by hydrophobic interaction chromatography at very high ammonium sulfate concentration and we produced specific antibodies (anti-PEBP) in rabbits that allowed us to be the first to develop a reliable Elisa assay for this protein.

Made available in DSpace on 2016-06-02T19:56:52Z (GMT). No. of bitstreams: 1 5322.pdf: 3668179 bytes, checksum: e3612c159bebd3f0a5be1640868316ae (MD5) Previous issue date: 2013-03-21 / Financiadora de Estudos e Projetos / The conversion of the hemicellulosic fraction in ethanol is a factor that impacts on the economic viability of the second generation ethanol production process from sugar cane bagasse. Hemicellulose from bagasse is a heteropolymer constituted by pentoses and glucose, being xylose the predominant sugar (~ 21 %). Among the available technological alternatives for ethanol production from xylose, SIF process (Simultaneous Isomerization and Fermentation), consisting of xylose conversion to xylulose by glucose isomerase (GI) enzyme and xylulose fermentation by the yeast S. cerevisiae, is considered a promising alternative. The main objectives of the present work were: i) evaluate the performance of different S. cerevisiae strains towards xylulose intake and ethanol productivity; ii) assess the influence of cultivation conditions (temperature, oxygen availability and initial xylose concentration) upon ethanol and xylitol production by the selected strains; iii) define the operation conditions for the continuous SIF process, using a system of fixed bed reactors associated in series. Preliminary experiments were conducted in 50 mL flasks, containing 4 g of pellets with a load of 20 % of immobilized GI, co-immobilized with yeast (load of 10 %) in alginate gel. For the screening of yeasts showing better performance on ethanol production from xylose, two commercial baker´s yeast strains (Itaiquara® e Fleischmann®), three industrial strains (BG-1, CAT-1 e PE-2) and one lab strain (CEN.PK113-7D) were evaluated. These experiments were performed at 35 oC, using a medium composed by xylose (60 g/L), urea (5 g/L), CaCl2 (1.9g/L) and several salts, at initial pH of 5.6. Additional SIF studies were carried out with the selected yeasts Itaiquara®, BG-1 or CEN.PK113-7D under different temperature conditions (40 oC), aeration (15 mL flasks) and initial xylose concentration (130 g/L) for comparison with the results obtained at the standard conditions. For SIF cultures, samples were withdrawal and the concentrations of reducing sugars were determined by DNS method while xylose, xylulose, ethanol and by-products (xylitol, glycerol etc) concentrations were assessed by liquid chromatography. Cell viability was also measured at the beginning and end of the experiment. When comparing the different yeasts, Itaiquara® strain presented the best performance, reaching ethanol concentrations of 22.4 g/L, with a productivity of 2.1 g/Lh. The conversion of xylose was similar for all studied industrial strains as well as among the baker s yeast and lab strains. Concerning the group of additional experiments, at 40 oC, a decrease of viability and ethanol selectivity was observed for Itaiquara®, whereas productivity and selectivity for CEN.PK113-7D. was improved. For the studies conducted under semianaerobic conditions, the yeast BG-1 showed an increase in selectivity and yield. However, the reaction time increased to app. 45 hours. On the other hand, the performance of strain Itaiquara® was not altered by the lower level of oxygen tested. In the experiment with 120 g/L of xylose, more than 40 g/L of ethanol was obtained in 24 hours of cultivation. Thus, we conclude that the SIF process proposed in the present work is a viable alternative for the production of ethanol from xylose or lignocellulosic residues. For the operation of the continuous system composed by fixed bed reactors associated in series, the recommended conditions include the Itaiquara® yeast with a temperature no higher than 35 oC, keeping the total residence time around 10 hours for a feeding supply containing 60 g/L of xylose. / A conversão da fração hemicelulósica da biomassa em etanol é um dos fatores que impactam a viabilidade econômica do processo de produção de etanol de segunda geração a partir do bagaço de cana-de-açúcar. A hemicelulose do bagaço é um heteropolímero constituído por pentoses e glicose, sendo a xilose o açúcar predominante (~ 21 %). Dentre as diversas alternativas tecnológicas para a produção de etanol a partir de xilose, o processo SIF (Simultânea Isomerização e Fermentação), consistindo na isomerização da xilose em xilulose pela enzima glicose-isomerase (GI) e na fermentação da xilulose pela levedura S. cerevisiae, é considerado uma alternativa promissora. Os principais objetivos do presente trabalho foram: i) avaliar o desempenho de diferentes cepas de S. cerevisiae em termos de assimilação de xilulose e produtividade em etanol; ii) estudar a influência das condições de cultivo (disponibilidade de oxigênio, temperatura e da concentração inicial de xilose) na produção de etanol e xilitol pelas cepas selecionadas; iii) definir as condições de operação para um processo SIF contínuo em sistema de reatores de leito fixo associados em série. Os experimentos preliminares foram conduzidos em frascos de 50 mL contendo 4 g de pelletes com carga de 20 % de glicose isomerase imobilizada, coimobilizada com levedura (carga de 10%) em gel de alginato. Para a seleção da levedura com melhor desempenho na produção de etanol a partir de xilose, foram avaliadas duas linhagens de levedura de panificação comercial (Itaiquara® e Fleischmann®), três cepas industriais (BG-1, CAT-1 e PE-2) e uma utilizada em laboratório (CEN.PK113-7D). Esses experimentos SIF foram conduzidos a 35ºC utilizando meio composto por xilose (60 g/L), ureia (5 g/L), CaCl2 (1,9 g/L) e sais diversos, em pH inicial 5,6. Experimentos SIF complementares foram realizados com as leveduras selecionadas Itaiquara®, BG-1 ou CEN.PK113-7D em diferentes condições de temperatura (40oC), aeração (frascos de 15 mL) e concentração inicial de xilose (130 g/L) para comparação com os resultados obtidos nas condições padrão. Em todos os experimentos SIF, amostras foram retiradas para determinação da concentração de açúcares redutores (método DNS) e de xilose, xilulose, etanol e subprodutos (xilitol, glicerol etc.) por cromatografia em fase líquida. Foi também acompanhada a viabilidade celular ao longo do cultivo. Na comparação entre as diferentes leveduras, destacou-se especialmente a levedura Itaiquara®, alcançando concentrações de etanol de 22,4 g/L, com produtividade em etanol de 2,1 g/Lh. A conversão de xilose foi semelhante entre as leveduras industriais e entre as leveduras de panificação e a de laboratório. Quanto ao conjunto de experimentos complementares, na temperatura de 40ºC houve diminuição de viabilidade e seletividade em etanol para a Itaiquara® e melhora na produtividade e seletividade para a CEN.PK113-7D. Nos experimentos realizados em condições semianaeróbias, a levedura BG-1 apresentou aumento de seletividade e rendimento em etanol, porém para um tempo de reação de 45 horas, aproximadamente. Já a levedura Itaiquara® não teve seu desempenho influenciado pela menor disponibilidade de oxigênio. No experimento realizado com 130 g/L de xilose, alcançou-se mais de 40 g/L de etanol em 24 horas de cultivo. Conclui-se, assim, que o processo SIF de xilose, proposto no presente trabalho, é uma alternativa viável para a produção de etanol a partir de xilose ou de resíduos lignocelulósicos. Para a operação em sistema contínuo composto por reatores de leito fixo associados em série recomenda-se a utilização de levedura Itaiquara® e de temperatura de, no máximo, 35ºC, mantendo-se o tempo de residência total em torno de 10 horas para uma alimentação contendo 60 g/L de xilose.

Doctor of Philosophy / Department of Chemistry / Duy H. Hua / Three research projects are described in this dissertation, and they are: (i) discovery of piperidine derivatives as T-type calcium channel inhibitors for the treatment of epilepsy and neuropathic pain and as protein disulfide isomerase inhibitors for the treatment of influenza viral infection; (ii) discovery of peptide-based calcitonin gene-related peptide receptor antagonists for the treatment of inflammatory pain; and (iii) synthesis of substituted 6-(dimethylamino)-2-phenylisoindolin-1-ones for the inhibition of luciferase. T-type calcium channels are important regulators of nervous system, and upregulated T-type calcium channel activities have been found to link to various types of neurological disorders, such as epilepsy and neuropathic pain. To discover novel T-type calcium channel blockers, a series of 1,4-disubstituted piperidine derivatives were designed and synthesized. Among them, compound 1-4 was found to be a good T-type calcium channel inhibitor with an IC₅₀ of 1 nM for Ca[subscript v]3.2 inhibition. It also showed 86% suppression of seizure induced death in mice and good in vivo analgesic effects on both thermal and mechanical pain thresholds in Spared Nerve Injury rat models. Therefore 1-4 can potentially be used as a T-type calcium channel blocker in the treatment of epilepsy and neuropathic pain. Influenza is a respiratory viral infection. Since viruses rely on host cell proteins for their entry, survival and replication, development of drugs targeting host cell proteins has identified as an effective strategy in controlling viral infections. We synthesized a series of 1,4-disubstituted piperidine derivatives for the inhibition of protein disulfide isomerase enzyme and influenza. Among them, 1-29 was found to possess strong anti-influenza activity (EC₅₀ = 2.5 µM). This suggests the potential use of piperidine scaffold in designing anti-influenza drugs in future. Calcitonin gene-related peptide (CGRP) receptor antagonism has been identified as a successful approach for the treatment of inflammatory pain. Therefore, a novel class of peptide antagonists of CGRP receptor was synthesized and screened for their binding affinities to the CGRP receptor and their analgesic effects on inflammatory-induced pain in rats. Among them, peptide 2-3 showed a higher binding affinity towards the CGRP receptor than previously reported peptide antagonists and exhibited analgesic effects up to 2 h in both Aδ and c-fiber pain tests. Therefore 2-3 indicates its potential use as a CGRP receptor antagonist in the treatment of inflammatory pain. Firefly luciferase is commonly used as a reporter in cells expressing a luciferase gene or its enzymatic activity under the control of a promoter of interest to assess its transcriptional activity. It has been found that some molecules such as molecules with carboxylic acid moiety can directly inhibit luciferase activity in cells. However, it is suggested that carboxylic acid moiety of the compounds may also be associated with side reactions in cells. Therefore, to study whether carboxylic acid moiety causes side effects, we designed two probe molecules, 3-1 and 3-2. Synthesis of probe molecule 3-2 is discussed. Synthesis of probe molecule 3-1 and further investigation of its luciferase inhibition will therefore be useful to understand the toxicity of carboxylic acid containing drugs in future.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2014-12-15T16:53:37Z No. of bitstreams: 2 Dissertação - Luiz Augusto Pereira - 2004.pdf: 6566857 bytes, checksum: 144c5557e8a138c6a21c528ad0262aa8 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2014-12-15T17:01:19Z (GMT) No. of bitstreams: 2 Dissertação - Luiz Augusto Pereira - 2004.pdf: 6566857 bytes, checksum: 144c5557e8a138c6a21c528ad0262aa8 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-12-15T17:01:19Z (GMT). No. of bitstreams: 2 Dissertação - Luiz Augusto Pereira - 2004.pdf: 6566857 bytes, checksum: 144c5557e8a138c6a21c528ad0262aa8 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2004-05-03 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / An antigen of Paracoccidioides brasiliensis (Pb) was gel isolated and characterized. Endoproteinase Lys-C digested peptides of the purified protein which presented, molecular mass of 29-kDa and pI of 5.8, were subjected to sequence analysis of its amino acids. Search at databases comparing the sequence of amino acids from the three peptides of the native protein, revealed strong homology to triosephosphate isomerase (TPI: E.C. 5.3.1.1) from several sources. The complete cDNA and gene encoding PbTPI were obtained and both contained an open reading frame predicted to encode a 249 amino acid protein that presented all the peptides characterized in the native PbTPI. The Pbtpi gene contained 6 exons interrupted by 5 introns. Analysis performed with the deduced PbTPI suggested its usefulness in providing phylogenetic relatedness, as well as evidenced the correlation between the phylogeny provided by the deduced proteins and introns positions in the cognate genes. The immunological reactivity of PbTPI was examined. The complete coding cDNA of PbTPI was over expressed in an Escherichia coli host to produce high levels of recombinant fusion protein with glutathione S-transferase (GST) that has been purified by affinity chromatography. The purified recombinant TPI was recognized by sera of patients with confirmed Paracoccidioidomycosis and not by sera of healthy individuals. Thus, recombinant PbTPI can be a valuable addition to the still small arsenal of P. brasiliensis immunoreactive proteins, which could be tested for incorporation in assays for serodiagnosis of the disease. / Um antígeno de Paracoccidioides brasiliensis (Pb) foi isolado do gel e caracterizado. Os peptídeos digeridos por Endoproteinase Lys-C da proteína purificada, que apresentou uma massa molecular de 29-kDA e pI de 5.8, foram submetidos à análise da seqüência de aminoácidos. Uma busca em bancos de dados de seqüências de aminoácidos comparados com os três peptídeos da proteína nativa revelou forte homologia com triose fosfato isomerase (TPI: E.C. 5.3.1.1) de vários organismos. O cDNA e o gene completos que codificam para PbTPI foram obtidos, e ambos contém uma provável ORF que codifica para uma proteína com 249 aminoácidos que apresenta todos os peptídeos caracterizados na PbTPI nativa. O gene Pbtpi apresentou 6 exons interrompidos por 5 introns. Análises realizadas com a PbTPI deduzida sugerem sua utilidade em prover relações filogenéticas, como também evidenciou a correlação entre a filogenia gerada pelas proteínas deduzidas e as posições dos introns nos genes cognatos. A reatividade imunológica da PbTPI foi examinada. O cDNA completo que codifica para PbTPI foi altamente expresso no hospedeiro Escherichia coli, produzindo altos níveis de uma proteína recombinante fundida a glutathione S-transferase (GST), que foi purificada por cromatografia de afinidade. A TPI recombinante purificada foi reconhecida por soros de pacientes com paracoccidioidomicose confirmada e não por soros de indivíduos saudáveis. Assim, PbTPI recombinante pode ser uma adição valiosa para o pequeno arsenal de proteínas imunoreativas de P. brasiliensis, que poderiam ser testadas por incorporação em ensaios de sorodiagnóstico da doença.

Plantas transgênicas constituem interessantes alternativas para a produção de compostos com elevado valor agregado como polímeros industriais, proteínas farmacológicas e lipídios nutracêuticos. Como candidata à biofábrica, a cana-de-açúcar (Saccharum spp.) apresenta características agronômicas favoráveis, além de versatilidade de matéria-prima, que é empregada em fins tradicionais, como produção de álcool e açúcar, e até mesmo para geração de energia ou como forragem para alimentação de gado. Em nutrição animal, uma molécula bastante estudada é o ácido linoleico conjugado (CLA), que tem atividades anticarcinogênica, antidiabética, antiaterosclerose e moduladora do sistema imune e metabolismo de lipídios. O isômero t10,c12- CLA tem sido particularmente promissor na agropecuária por conta de inibição da síntese de gordura do leite e na melhoria do desempenho reprodutivo de vacas. Com o objetivo de avaliar a cana-de-açúcar como sistema biotecnológico para produção de CLA, o gene da isomerase do ácido linoleico de Propionibacterium acnes (pai) foi isolado e clonado, previamente caracterizado em Escherichia coli e, em seguida, utilizado na biolística de calos embriogênicos para regeneração de plantas transgênicas. No sistema procariótico, foi induzida a expressão de pai e a proteína heteróloga foi verificada como uma banda de 50 kDa, porém não houve alteração evidente do perfil de ácidos graxos da bactéria. Na cotransformação de cana-de-açúcar, o vetor pHA9, que contém o gene de seleção da neomicina fosfotransferase (nptII), foi bombardeado com uma das duas construções desenvolvidas com o promotor da poliubiquitina 1 de milho (pUbi1) dirigindo a expressão de pai adicionado ou não de sequência sinal da proteína de reparo de DNA recA para direcionamento para cloroplasto. Das cultivares CTC2 e IACSP9303046, foram obtidas eficiências de transformações de 2,2% e 1,7-7,1%, respectivamente. Um total de 156 plântulas foram regeneradas após regime de seleção com geneticina. Em seguida, 115 plântulas transgênicas foram identificadas por PCR para nptII e, entre essas, 29 e 48 foram PCRpositivas para pai e rec-pai, respectivamente. De 50 plantas aclimatizadas, 12 foram analisadas por Southern blot para nptII e 4 para pai, confirmando-se a transformação genética. Para comprovar a expressão de mRNA de pai, 27 plantas foram averiguadas por RT-PCR e RT-qPCR e indicaram produção de transcritos estudados. A análise de expressão das proteínas de folha por Western blot em 21 plantas selecionadas não produziu resultados conclusivos quanto à detecção de PAI. Essas mesmas foram analisadas por Cromatografia Gasosa, mas não foi detectado acúmulo de CLA. Embora seja possível a introdução e expressão do gene pai em cana-de-açúcar, é necessário avaliar em maior detalhe os fatores que possam afetar positivamente a produção de CLA, como tipo de tecido, compartimentos subcelulares para direcionamento proteico ou coexpressão de fosfolipases. / Transgenic plants are attractive alternatives for the production of high value compounds as industrial polymers, pharmacological proteins and nutraceutical lipids. As a candidate for biofactory, sugarcane presents favorable agronomic characteristics and versatility of raw material which is used for traditional purposes such as ethanol and sugar production and even for power generation or as forage feeding of cattle. In animal nutrition, a widely studied molecule is the conjugated linoleic acid (CLA), which has anticarcinogenic, antidiabetic, antiatherosclerosis and immune system and metabolism of lipids modulator activities. The isomer t10,c12-CLA has been particularly promising in agriculture because of inhibition of milk fat synthesis and improvement of the reproductive performance of cows. Aiming to evaluate the sugarcane as a system for biotechnological production of CLA, the linoleic acid isomerase gene from Propionibacterium acnes (pai) was isolated and cloned, previously characterized in Escherichia coli and then used for biolistic of embryogenic calli for regenerating transgenic plants. In the prokaryotic system, the expression of pai was induced and the heterologous protein was verified as a band of 50 kDa, but there was no obvious change in fatty acid profile of the bacterium. In cotransformation of sugarcane, pHA9 vector containing the selection gene neomycin phosphotransferase (nptII) was bombarded with one of the two constructions developed with the promoter of maize polyubiquitin 1 (pUbi1) driving the expression of pai, either added or not with the signal sequence of DNA repair protein recA for targeting to chloroplast. Cultivars CTC2 and IACSP93-3046 were obtained with transformation efficiencies of 2.2% and 1.7 a 7.1%, respectively. A total of 156 plantlets were regenerated after selection with geneticin regimen. After that, 115 transgenic plantlets were identified by PCR for nptII and among then, 29 and 48 were PCR-positive for pai and rec-pai, respectively. Of 50 acclimatized plants, 12 were analyzed by Southern blot for nptII and 4 for pai, confirming the genetic transformation. In order to prove the expression of pai mRNA, 27 plants were verified by RT-PCR and RT-qPCR and indicated the production of the studied transcripts. Expression analysis of leaf proteins by Western blot in 21 plants selected produced no conclusive results regarding the detection of PAI. Those were analyzed by gas chromatography and no accumulation of CLA was detected. Although it was possible to introduce and express the pai gene in sugarcane, it is necessary to evaluate in more detail the factors that may positively affect the production of CLA, as tissue type, subcellular compartments for protein targeting or coexpression of phospholipases.

Abstract Enzymes are potentially superior as catalysts for many industrial chemical processes because of their high specificity, selectivity, minimum energy requirement and environmental friendliness. However, many challenges remain in order to exploit fully the potential of industrial enzymes. The qualities which are needed are catalytic proficiency, availability in high quantities, low price, low product inhibition, and high activity and stability under process conditions. Directed evolution and rational design are the most common strategies to produce enzymes with the desired properties. The TIM barrel is the most frequent and most versatile fold among naturally occurring enzymes. In all known TIM barrel enzymes, the catalytically active residues are located at one end of the barrel structure, while residues maintaining the stability of the fold are found on the opposite end of the barrel. This special architecture of the TIM barrel proteins makes it possible to change catalytic activity of the protein without compromising its stability, which is a perfect start for protein engineering studies. In this research project, a monomeric triosephosphate isomerase (TIM) variant with an engineered binding groove (A-TIM) was created by using a rational design approach. The major aims of this work were (i) to find novel binders and (ii) characterize the new, bigger binding groove using X-ray crystallographic methods. These studies have discovered that monomeric A-TIM can bind compounds completely different from the natural substrate. Studies on three different classes of binder molecules are reported: (i) true substrate analogues of wild type TIM, (ii) substrate analogues that have an extended hydrophobic tail, and (iii) more extended, phosphate containing substrate analogues. In addition to this, the A-TIM active site was shown to be competent. In general these studies illustrate the importance of protein crystallography for characterizing the binding properties of enzyme variants being studied in enzyme discovery projects. / Tiivistelmä Entsyymit voivat toimia ylivoimaisina katalyytteinä monissa kemianteollisuuden prosesseissa johtuen niiden hyvästä spesifisyydestä, valikoimiskyvystä, alhaisesta energiantarpeesta ja ympäristöystävällisyydestä. Näistä ominaisuuksista huolimatta entsyymien kaikkien mahdollisuuksien hyödyntämisen esteenä on monia haasteita. Tarvittavia ominaisuuksia ovat katalyyttinen tehokkuus, saatavuus suurina määrinä, alhainen hinta, alhainen tuoteinhibitio sekä korkea aktiivisuus ja stabiilisuus prosessiolosuhteissa. TIM-tynnyrirakenne on yleisin ja monipuolisin proteiinien laskostumisrakenne luonnossa esiintyvissä entsyymeissä. Tässä rakenteessa katalyyttisesti aktiiviset aminohappotähteet ovat sijoittuneet tynnyrirakenteen toiselle puolelle, kun taas stabiilisuuden kannalta tärkeät aminohappotähteet ovat sijoittuneet kokonaan toiselle puolelle. Tämä erityinen rakenne antaa mahdollisuuden muokata proteiinin katalyyttistä aktiivisuutta vaikuttamatta haitallisesti sen stabiilisuuteen. Tämä on täydellinen lähtökohta proteiininmuokkaukselle. Tässä tutkimusprojektissa käytettiin ns. järkiperäistä suunnittelua monomeerisen trioosifosfaatti-isomeraasivariantin (A-TIM) luomisessa. Tämän tutkimustyön pääasialliset tavoitteet olivat (i) uusien sitoutujien löytäminen ja (ii) uuden, suuremman sitoutumistaskun ominaisuuksien määrittäminen röntgenkristallografisilla menetelmillä. Tässä tutkimuksessa havaittiin, että A-TIM kykenee sitomaan yhdisteitä, jotka ovat täysin erilaisia luonnolliseen substraattiin verrattuna. Tässä tutkimuksessa kuvaillaan kolmenlaisia sitoutujia: (i) todelliset villityypin entsyymin substraattianalogit, (ii) substraattianalogit, joihin on liitetty hydrofobinen hiilivetyketju ja (iii) villityypin substraattia suuremmat sokerifosfaatit. Tämän lisäksi A-TIM:n aktiivisen keskuksen todistettiin olevan toimintakykyinen. Yleisellä tasolla tämä tutkimus osoittaa röntgenkristallografisten menetelmien tärkeyden entsyymienmuokkausprojekteissa, joissa entsyymivarianttien ominaisuuksien määritys on tärkeää.

The redox state in the cell plays a major role in determining vital functions and its major imbalance can lead to severe cell injury or death. Redox active proteins and cytokines involved in this process includes thioredoxin (Trx), protein disulfide isomerase (PDI), and tumor necrosis factor (TNF) superfamilies. Trx is a multipotent protein and key regulator of cellular redox balance operating in synergy with Trx reductase and NADPH (the Trx system). Trx has gene regulatory activity of several transcription factors. It also controls in a fascinating way redox-sensitive “on-off” decisions for apoptotic or hypertrophic pathways. Trx protects against H2O2 and TNFmediated cytotoxicity, a pathway in which TNF receptor-binding generates ROS. TNF is an autocrine growth factor and survival factor in vitro and in vivo for B-type of chronic lymphocytic leukemia (B-CLL) cells. The overall aim of this study was to investigate the importance of redox active proteins and cytokines in inflammation and cancer. We focused on: i) the role of Trx, TrxR, and selenium in carcinogenesis and in resistant cancer cells. ii) the importance of Trx in cancer cells and the redox regulation of TNF and its receptors TNFR1 and TNFR2. iii) the potential role of Trx as a key regulator in cellular redox balance, in the pathogenesis of cardiac dysfunction; its relationship to stress response parameters. iv) whether unmutated CLL (UCLL) responses to PKC and ROS pathways were different from mutated CLL (M-CLL) responses. Our results demonstrate pronounced selective selenium-mediated apoptosis in therapy resistant cells and suggest that redox regulation through the Trx system is an important target for cancer therapy. Trx was strikingly elevated in heart failure cases compared with controls signifying an adaptive stress response that is higher the more severe the disease. TNF autocrine release was redox modulated and the TNF receptors interacted at the cell surface membrane with the redox-active PDI, which excerted a stringent redox-control of the TNFR signaling. The proliferative response as well as increase of autocrine TNF and Trx were higher in U-CLL than in M-CLL. The overall conclusion of the four papers included in this thesis is that redox-active proteins and cytokines plays an important role in control and regulation of cancer and inflammation. Furthermore, redox regulation via thioredoxin by selenium, may offer novel treatment possibilities for resistant tumors disease.

La formation de ponts disulfure constitue une modification post-traductionnelle des protéines importante pour de nombreux processus physiologiques, jouant un rôle particulier dans le repliement, la catalyse et la régulation de leur activité. Ce travail concerne l'étude des relations structure-fonction d'oxydoréductases de peuplier appartenant à deux familles de la superfamille des thiorédoxines, les glutathion peroxydases (Gpxs) et les protéine disulfure isomérases (PDIs).L'étude biochimique fine de la Gpx5 a permis de montrer que cette peroxydase réduit le peroxynitrite, propriété inconnue pour ce type de Gpx et de détailler plusieurs étapes du mécanisme catalytique (formation de l'acide sulfénique, changement structural entre formes réduites et oxydées, régénération par les Trxs). La dimérisation de la Gpx5 n'est pas requise pour son activité mais pourrait jouer un rôle dans la reconnaissance de certains substrats. Enfin, l'inactivation de la cystéine peroxydatique par suroxydation suggère que les Gpxs pourraient également avoir une fonction dans la signalisation en réponse aux peroxydes.Concernant les PDIs, suite à une analyse phylogénétique détaillée amenant à proposer une nouvelle classification en 9 classes chez les organismes photosynthétiques, la caractérisation biochimique de plusieurs isoformes présentant des organisations modulaires distinctes et appartenant à trois classes de PDIs a été entreprise. Aucune activité enzymatique typique n'a été identifiée pour la PDI-A, alors que les PDI-L1a et -M possèdent à la fois une activité oxydase et réductase. Les deux modules a de la PDI-M catalysent des réactions spécifiques, de réduction ou d'oxydation. / Protein activity and folding can be regulated by post-translational modifications that can impact on their physiological functions. One of these is the formation/reduction of disulfide bridges. The aim of the present work is to study the structure-function relationship of protein members of the thioredoxin superfamily, the protein disulfide isomerases (PDI) and the glutathione peroxidases (Gpx).A precise biochemical study has allowed us to demonstrate that this enzyme is an efficient peroxynitrite scavenger, a new finding for this type of protein and allowed investigating several steps of the Gpx5 catalytic mechanism (i.e. sulfenic acid formation, structural changes between reduce dand oxidized forms, Trx-mediated recycling). We also demonstrate that the dimer form of Gpx5 is not absolutely required for peroxide reduction but probably involved in peroxide specificity. Finally, the capability of the peroxidatic cysteine to be overoxidized brings some new clues in favor of an additional signaling function for Gpx5.Concerning PDIs, a detailed phylogenetic analysis of photosynthetic organisms allowed us to identify 9 classes of PDIs and to propose a new nomenclature that fits all these organisms. The biochemical characterization of isoforms of interest has allowed us to highlight some specificity of PDI-L1a and PDI-M in terms of reduction or oxidation reactions catalyzed. A detailed analysis of PDI-M isoform also indicates that the two Trx modules of this protein show differential oxidation or reduction capacities. We could not detect any activity for PDI-A isoforms, leaving us to wonder whether this enzyme is simply active or possesses highly specific protein partners.

A Síndrome de Marfan (SMF) é a enfermidade hereditária mais comum dentre as que afetam o sistema conjuntivo, causada por mutações da glicoproteína fibrilina-1, o principal componente estrutural das microfibrilas elásticas da matriz extracelular. As manifestações fenotípicas da SMF são sistêmicas e acometem tipicamente os sistemas ocular, esquelético e cardiovascular, este uma importante causa de morbi-mortalidade. Entretanto, não está claro como a mutação induz a doença. Estudos anteriores sugerem anomalias morfológicas do retículo endoplasmático (RE) ou retenção intracelular da fibrilina-1 nos estágios avançados da SMF. Entretanto, a contribuição do enovelamento da fibrilina-1 mutada e do estresse do RE na fisiopatologia celular da SMF não é conhecida. Proteínas mal-enoveladas podem levar à retenção intracelular e/ou aumento da degradação através da via de degradação associada ao RE (ERAD), além da indução da resposta a proteínas mal-enoveladas (UPR), ambas com potencial contribuição à fisiopatologia de doenças, incluindo a SMF. Assim, estudamos em fibroblastos embrionários isolados de camundongos (MEFs) com SMF se a fibrilina-1 mutada é reconhecida pelo controle de qualidade do RE pelo seu mal- enovelamento e induz estresse do RE por sua retenção intracelular. Demonstramos que a mutação na fibrilina-1 per se não promoveu chaperonas marcadoras de UPR ou geração de oxidantes. Além disso, não levou a uma maior sensibilização das células à indução exógena de estresse do RE, nem promoveu maior morte celular após inibição do proteassoma. Além disso, não foi observada retenção intracelular da fibrilina-1 nas células SMF, e mesmo após inibição da via secretora ou indução de estresse do RE, a inibição da secreção da fibrilina-1 foi similar nos MEFs SMF e wild-type (WT). A dissulfeto isomerase proteica (PDI), uma importante chaperona redox do RE, interage com fibrilina-1, e seu silenciamento levou a um aumento na secreção da fibrilina-1 pelos MEFs WT, mas não SMF. Além disso, o silenciamento da PDI promoveu a desorganização da matriz extracelular depositada de fibrilina-1 nos MEFs WT, enquanto nos MEFs SMF, a desorganização basal da matriz não foi adicionalmente alterada. Em paralelo, investigações in vivo mostraram que o estresse do RE não é induzido em camundongos SMF com 1 ou 3 meses de idade, apesar de manifestações fenotípicas evidentes. Entretanto, concomitante à progressão da doença, detectamos a ocorrência de estresse do RE nas aortas ascendentes dos camundongos aos 6 meses. Esta detecção foi exclusiva desta região da aorta e não ocorreu em outros órgãos afetados ou não afetados pela SMF. Assim, a manifestação do fenótipo clássico da SMF não requer uma perda da homeostase do RE diretamente induzida pela fibrilina-1 mutada. Ao contrário, esta é capaz de evadir mecanismos de controle de qualidade mediados pela PDI, sendo secretada normalmente. Assim, esta evasão do controle de qualidade pela PDI é uma condição permissiva essencial para o fenótipo da SMF. Por outro lado, o estresse do RE é uma característica evolutiva do aneurisma da aorta ascendente na SMF concomitante ao agravamento do fenótipo neste tecido / Marfan syndrome (MFS) is the most common connective tissue hereditary disease, caused by mutations in the glycoprotein fibrillin-1, the main structural component of extracellular matrix elastic microfibrils. MFS phenotypic manifestations are systemic and typically involve the ocular, skeletal and cardiovascular systems, the latter a major cause of morbidity/mortality. However, how gene mutation induxes disease is yet unclear. Previous studies suggest endoplasmic reticulum (ER) morphological abnormalities or fibrillin-1 intracellular retention in advanced MFS stages. However, the contribution of mutated fibrillin-1 folding and ER stress to MFS cellular pathophysiology is unknown. Un/misfolded proteins may associate with their intracellular retention and/or increased degradation through ER-associated degradation (ERAD), in addition to inducing the unfolded protein response (UPR), both sharing potential contributions to disease pathophysiology, including MFS. Thus, we studied in embryonic fibroblasts (MEFs) isolated from WT and MFS mice, if mutated fibrillin-1 can be recognized by ER quality control as a misfolded protein, able to induce ER stress due to its intracellular retention. We showed that fibrillin-1 mutation by itself did not promote UPR chaperone markers or oxidant generation. Moreover, it did not sensitize cells to exogenous ER stress nor affected cell survival curves after proteasome inhibition. Furthermore, no intracellular retention of fibrillin-1 was observed in MFS cells, and even after secretory pathway inhibition or ER stress induction, fibrillin-1 secretion inhibition was similar in MFS and wild-type (WT) MEFs. Protein disulfide isomerase (PDI), an important ER redox chaperone, interacts with fibrillin-1 and its silencing induced an increased fibrillin-1 secretion in WT, but not MFS MEFs. Besides, PDI silencing promoted fibrillin-1 extracellular matrix disorganization in WT MEFs, whereas in MFS MEFs, the basal matrix disorganization was not further modified. Parallel in vivo evaluations demonstrated that ER stress is also not induced in 1 and 3 month-old mice MFS, despite evident phenotypical manifestations. However, concomitant to accelerated disease progression at 6 months, ER stress was detectable in ascendant aorta, but not in other disease-affected or unaffected organs. Thus, classic MFS phenotype manifestations do not require loss of ER homeostasis directly induced by mutated fibrillin-1. Contrarily, the latter can evade a PDI-mediated quality control mechanism to be normally secreted. Therefore, evading such PDI-mediated quality control is an essential permissive condition for enabling the MFS phenotype. On the other hand, ER stress is an evolutive feature of MFS ascendant aorta aneurysm concomitant to phenotype progression in this tissue