Les protéines amyloïdes sont impliquées dans de nombreux processus pathologiques de maladies qui restent souvent incurables. Ces protéines solubles dans leur forme native, s’auto- assemblent pour former des oligomères, des fibrilles, des fibres et enfin des agrégats riches en feuillets β. C’est ce processus délétère qui est le point commun entre ces maladies amyloïdes. La protéine amyloïde la plus décrite est le peptide Aβ suspecté de jouer un rôle primordial dans la maladie d’Alzheimer. Récemment, les petits oligomères du peptide Aβ1-42 formés lors des étapes précoces de ce processus ont été décrit comme étant les plus toxiques.Au cours de cette thèse, nous avons donc développé deux méthodes pour pouvoir évaluer l’activité des composés synthétisés sur les étapes précoces de l’oligomérisation et une pour étudier l’affinité du peptide Aβ1-42 pour son ligand. Nous avons également conçu et synthétisé des peptidomimétiques comme ligands du peptide Aβ1-42 capables ainsi de perturber les interactions protéine-protéine du processus d’agrégation du peptide Aβ1-42. L’évaluation de ces composés ainsi que de différents ligands synthétisés au laboratoire a permis une intéressante étude sur la relation entre la structure des composés évalués et leurs activités sur les étapes cruciales du processus d’oligomérisation du peptide Aβ1-42. Des études de viabilité cellulaire, de RMN et de Docking sont en cours pour améliorer notre compréhension du mode d’action de ces composés et du processus d’oligomérisation du peptide Aβ1-42. / Amyloid proteins are involved in many pathological processes of diseases that are often incurable. These soluble proteins in their native form self-assemble to form oligomers, fibrils, fibers and finally aggregates rich in β-sheets. It is this deleterious process which is the common point between these amyloid diseases. The most described amyloid protein is the Aß peptide suspected of playing a key role in Alzheimer's disease. Recently, small peptide Aβ1-42 oligomers formed during the early stages of this process have appeared to be the most toxic species.In this thesis, we have developed two methods to evaluate the activity of different synthesized compounds on the early steps of oligomerization and one method to study the affinity of Aβ1-42 peptide for its ligand. We have also designed and synthesized peptidomimetics as ligands of Aβ1-42 peptide which are able to disrupt protein-protein interactions involved in the aggregation process of Aβ1-42 peptide. Evaluation of these compounds as well as other ligands synthesized in the laboratory allowed an interesting study on the relationship between the structure of the tested compounds and their activities on the critical steps of the oligomerization process of Aβ1-42 peptide. Cell viability studies, NMR and docking are underway to improve our understanding of the mode of action of these compounds and of the oligomerization process of Aβ1-42 peptide.

A Esclerose Lateral Amiotrófica (ELA) é uma doença progressiva e fatal causada pela degeneração seletiva dos neurônios motores do cérebro e medula. Dos casos familiares de ELA (fELA), 20% são causados por mutações pontuais no gene da sod1. O ácido docosahexaenoico (C22:6, n-3, DHA) é um ácido graxo altamente insaturado, sendo um dos principais ácidos graxos da massa cinzenta do cérebro. Estudos têm correlacionado mutações de SOD1 com a formação de agregados que poderiam ser induzidos por ácidos graxos insaturados. O objetivo deste estudo foi avaliar os efeitos e mecanismos do DHA e de seus hidroperóxidos (DHAOOH) na agregação de SOD1 in vitro. As análises de dicroísmo circular (CD) mostraram mudanças na estrutura secundária de ambas as proteínas apo-SOD1WT e G93A promovidas pelo DHA, resultando em aumento de superfície hidrofóbica e formação de estruturas do tipo beta-amilóide, como mostrado pelos ensaios do bis- ANS e Tioflavina, respectivamente. Estas mudanças resultam na formação de agregados amorfos como observado por microscopia eletrônica de varredura (MEV). Espécies de alto peso molecular foram observadas nas incubações do DHA com as formas apo da SOD1 por SDS-PAGE sob condições não redutoras e também por cromatografia de exclusão por tamanho. A formação dos agregados mostrou-se dependente de resíduos de Cys na sua forma desprotonada, visto que agregados não foram observados na presença de beta-mercaptoetanol e sua formação foi inibida na presença de bloqueador de tióis e em pH ácido. Além disso, análises por cromatografia de exclusão mostraram que a agregação é dependente da insaturação e conformação cis dos ácidos graxos. Comparativamente ao DHA, os hidroperóxidos do DHA tiveram um efeito menor na agregação de SOD1, porém revelaram a propriedade de induzir a dimerização covalente de SOD1. No geral, os dados mostram que o DHA induz a agregação de SOD1, através de um processo envolvendo a exposição de superfícies hidrofóbicas, formação de pontes dissulfeto e também de possíveis cross-links envolvendo reações do tipo \"ene-tiol\". / ALS is a progressive and fatal disease caused by selective degeneration of motor neurons in the brain and spinal cord. Twenty percent of familial ALS (fALS) cases are caused mainly by point mutations in the sod1 gene. Docosahexaenoic acid (C22:6, n-3, DHA) is a highly unsaturated fatty acid, wich is one of the main fatty acids in the cerebral gray matter. Studies have linked SOD1 mutations to the formation of aggregates that could be induced by unsaturated fatty acids. The aim of this study was to evaluate the effect of DHA on aggregation of SOD1 fALS mutants in vitro and its mechanisms. CD analysis shows changes in the secondary structure of both apo-SOD1WT and G93A promoted by DHA resulting in an increase in the surface hydrophobicity and formation of structures such as beta amyloid, which was also confirmed by bis-ANS assay and Thioflavin, respectively. These changes enhance the interaction of SOD1 and DHA, leading to amorphous aggregates as revealed by FESEM. Incubation of DHA with apo-SOD1 forms results in high-molecular weight species as detected by SDS-PAGE analyses under non-reducing conditions and also by size exclusion chromatography. This appears to require Cys residues in their thiolate forms because high aggregates are not observed under reducing conditions and also by size exclusion chromatography or at acidic pH. Also, size-exclusion chromatography indicates that the mutant apo-SOD1 aggregation is dependent on the unsaturation and cis-conformation of fatty acids. Compared to the DHA, DHAOOH had a minor effect on SOD1 aggregation, however revealed the ability to induce covalent dimerization of SOD1. Overall, the data suggest a mechanism of DHA aggregation, by a process involving exposure to hydrophobic surfaces, formation of disulfide bonds and also for possible cross-links involving reactions such \"thiol-ene\".

Os receptores tireoidianos (TRs) são proteínas envolvidas em várias funções fisiológicas importantes para os organismos, pois são potentes reguladores do desenvolvimento, divisão e diferenciação celular, metabolismo e homeostase. Eles são responsáveis pela regulação da transcrição de genes-alvo específicos, mediando efeitos pleiotrópicos de hormônios lipofílicos nas células. Na ausência de ligantes essas proteínas estão complexadas a correpressores, impedindo a transcrição de genes por elas regulados. Por outro lado, a presença do ligante induz à transcrição através da ligação a elementos responsivos do DNA e coativadores. Nesse trabalho alguns aspectos do TR foram evidenciados, permintindo-se um melhor conhecimento acerca do funcionamento e estrutura desse receptor. Os experimentos de oligomerização revelaram a presença dos tetrâmeros do TR, os quais estavam restritos ao Receptor X Retinóico, sugerindo mecanismos novos na regulação do receptor. Os ensaios de raios-X a baixos ângulos resultaram nos primeiros modelos estruturais de baixa resolução de construções maiores do TR, demonstrando o correto posicionamento de seus domínios em sua estrutura geral, o que forneceu informações importantes sobre sua estrutura geral. Os experimentos de fluorescência avaliaram a ligação desses receptores a diversos elementos responsivos, em termos de constantes de dissociação e seletividade para cada um deles. E, por fim, os experimentos de troca de hidrogênio por deutério revelaram a movimentação que ocorre no domínio de ligação do ligante do receptor antes e após a adição do ligante T3. Esses resultados ampliam um pouco mais os conhecimentos sobre os mecanismos de ação e sobre a estrutura quaternária dos TR, promovendo um melhor entendimento dos conceitos básicos envolvidos na atuação dessas macromoléculas, as quais estão inseridas em redes complexas de regulação e interação com outras proteínas. / The thyroid receptors (TRs) are proteins, which are involved in diverse and important physiological functions in the organisms, since they are regulators of development, cell divison and differentiation, metabolism and homeostasis. They are responsible by the regulation of specific gene transcription, through pleiotropic effects of lipophilic hormones in the cells. In the absence of the ligand these proteins are complexed to correpressors and block the transcription of genes that are regulated by them On the other hand, in the presence of the ligand transcription is induced through the binding of the receptors to DNA response elements and coactivators. New findings about TR described in this study helped to improve the understanding of the function and structure of the receptor. This was accomplished by: oligomerization experiments which showed the presence of TR tetramers, a quarternary structure described before only for the Retinoid Receptor X, and suggested new regulation mechanisms for the receptors; the small angle X-ray scattering assays which resulted in the first low resolution structural models of bigger constructions of TR, showing the correct position of TR domains and providing important information about the global TR structure; the anisotropy fluorescence experiments which evaluated the binding of these receptors to diverse response elements, in terms of dissociation constants and selectivity for each one of the HREs tested; and finally, the hydrogen/deuterium experiments which revealed the ligand binding domain mobility before and after the ligand addition. In summary, we can say that these results all together extended the knowledge about the TR action mechanisms and its quarternary strucuture, providing better understanding of the basic concepts involved in these macromolecules behavior, which are inserted into a complex network of regulation and interaction with other proteins.

UDP-glucose pyrophosphorylase (UGPase) is an important enzyme of carbohydrate metabolism in all living organisms. The main aim of this thesis was to investigate the function and regulation of plant UGP genes as well as the UGPase proteins. Both in vivo and in vitro approaches were used, including the use of transgenic plants deficient in UGPase activity, and using purified proteins and their mutants to elucidate the structure/ function properties of UGPase. In both Arabidopsis and aspen, there are two highly similar UGP genes being actively transcribed, but not to the same extent. For both species, the UGP genes could be classified into two categories: a “house-keeping” gene and a subsidiary gene, with the former functioning universally in all the tissues to support the normal growth, whereas the latter usually expressed at a lower level in most of the organs/tissues tested. Besides, the two UGP genes were also found being differentially regulated under abiotic stress conditions, e.g. low temperature. By investigating the Arabidopsis T-DNA insertion mutants, which respectively have one or both of the UGP genes knocked out, we noticed that as little as 10% of the remaining UGPase activity could still support normal growth and development under controlled conditions, with little or no changes in carbohydrate contents, including soluble sugars (e.g. sucrose), starch and cell wall polysaccharides. Those plants, however, had a significantly decreased fitness under field conditions, i.e. the plants most deficient in UGPase activity produced up to 50% less seeds than in wt. Therefore, we concluded that UGPase is not a rate-limiting enzyme in carbohydrate synthesis pathways, but still is essential in viability of Arabidopsis plants. In order to characterize two Arabidopsis UGPase isozymes, both proteins were heterologously overexpressed in prokaryotic cells and purified by affinity chromatography. The two isozymes showed little differences in physical and biochemical properties, including substrate specificity, Km values with substrates in both directions of the reaction, molecular masses, isoelectric point (pI), and equilibrium constant. On the other hand, possibilities of distinct post-translational regulatory mechanisms were indicated, based on amino acid (aa) motif analyses, and on 3D analyses of derived crystal structures of the two proteins. We used the heterologous bacterial system also to overexpress barley UGPase and several of its mutants, both single mutants and those with whole domains/ exons deleted. As a result, we have identified several aa residues/ protein domains that may be essential for structural integrity and catalytic/ substrate-binding properties of the protein. For instance, we found that the last exon of UGPase (8 aa at the end of C-terminus) was important for the protein ability to oligomerize and that Lys-260 and the second-to-last exon were essential for pyrophosphate (but not UDP-glucose) binding. The data emphasized the critical role of central part of the active site (so called NB-loop) in catalysis, but also pointed out to the role of N-terminus in catalysis and oligomerization, but not substrate binding, and that of C-terminus in both catalysis/substrate binding and oligomerization.

DivIVA is a Gram-positive cell division protein involved in chromosome segregation, midcell placement of the cell division machinery, complete septum closure, and polar growth and morphogenesis. Although well conserved across various Gram-positive species, DivIVA is believed to be relatively species specific. One similarity among DivIVA homologues is the ability to oligomerize through coiled-coil interaction into complexes comprising 10-12 monomers. To date, the importance of DivIVA oligomerization and the N-terminal coiled-coil for its proper function in bacterial cell division has not been reported. This study examined the biological significance of DivIVA oligomerization and the N-terminal coiled-coil in bacterial cell division. This research provides evidence that the N-terminal coiled-coil and oligomerization is essential for the proper biological function of DivIVA_Ef in <i>Enterococcus faecalis</i> cell division. Introduction of point mutations into chromosomal <i>divIVA</i>_Ef known to disrupt either the N-terminal coiled-coil or the two central coiled-coils, involved in oligomerization, were found to be lethal unless rescued by <i>in trans</i> expression of wild type DivIVA_Ef. Using this rescue method, the N-terminal <i>divIVA</i>_Ef mutant strain, <i>E. faecalis</i> MWMR5, and the mutant strain with partial disruption of oligomerization, <i>E. faecalis</i> MWMR10, were successfully rescued. Differential Interference Contrast (DIC) and Transmission Electron Microscopy (TEM) were utilized to determine the phenotypes of <i>divIVA</i>_Ef mutant strains <i>E. faecalis</i> MWMR5 and MWMR10. Both these strains showed asymmetrical division, loss of normal lancet shape, and irregular chains. Full disruption of oligomerization with point mutations in both central coiled-coils resulted in a dominant lethal phenotype. These results demonstrate the essentiality of the N-terminal coiled-coil and oligomerization of DivIVA_Ef for its proper biological function in <i>E. faecalis</i> cell division.<p> Previous detection of DivIVA interaction with a novel cell division protein, MLJD1, by screening a Yeast Two-Hybrid (Y2H) was weak. GST-pulldown and immunoprecipitation did indicate DivIVA_Ef interaction with MLJD1, but another in vivo assay was required to support these results. In this study I demonstrate a strong interaction, using an in vivo Bacterial Two-Hybrid (B2H) assay, between DivIVA_Ef and a fragment of MLJD1 containing two cystathionine-beta-synthase (CBS) domains. The <i>in vitro</i> and <i>in vivo</i> results thus confirm interaction between DivIVA_Ef and MLJD1.<p> Another objective of this study was to determine the localization of DivIVA and MLJD1 in <i>E. faecalis</i>. Localization of DivIVA_Ef in <i>E. faecalis</i> was found to be similar to DivIVA localization in <i>Bacillus subtilis</i> and <i>Streptococcus pneumonia</i>. DivIVA_Ef was diffused along the cell membrane and, as chromosome replication and segregation and cell division proceeded, DivIVA_Ef migrated to the cell poles and then concurrently to the division site. Intriguingly, MLJD1 was found to localize in the same pattern as DivIVA_Ef in <i>E. faecalis</i>, further implicating MLJD1 as a bacterial cell division protein.<p> Since MLJD1 has potential DNA binding capabilities a proposed model of its role in cell division has been proposed. I hypothesize that MLJD1 could be forming a bridge between DivIVA_Ef and the chromosome to aid in proper chromosomal replication and segregation. This model could explain how DivIVA_Ef is involved in chromosome replication. This model is similar to the role of RacA in sporulation in <i>B. subtilis</i> where RacA directs the chromosome during sporulation through direct interaction with DivIVA_Bs and Spo0J.<p> This study has set some important and essential ground work for developing a novel model of cell division for the elusive Gram-positive coccal bacterial strains.

As one of the mechanisms of antibiotic resistance, bacteria use several families of membrane-embedded α-helical transporters to remove cytotoxic molecules from the cell. The small multidrug resistance protein family (SMR) is one such group of drug transporters that because of their relative small size [ca. 110 residues with four transmembrane (TM) helices] must form at the minimum dimers to efflux drugs. We have used the SMR homologue Hsmr from Halobacterium salinarum to investigate the oligomerization properties of the protein family at TM helix 4. We produced point mutations along the length of the TM4 helix in the full length Hsmr protein and assayed their dimerization and functional properties via SDS-PAGE and bacterial cell growth assays. We found that Hsmr forms functionally dependent dimers via an evolutionarily conserved 90GLxLIxxGV98 small residue heptad repeat. Upon investigation of the large hydrophobic residues in this motif by substituting each large residue to Ile, Leu, Met, Phe, and Val, we determined that Hsmr efflux function relies on an optimal level of dimerization. While some substitutions led to either decreased or increased dimer and substrate-binding strength, several Ile94 and Val98 mutants were equal to wild type dimerization levels but were nonfunctional, leading to the hypothesis of a mechanistic role at TM4 in addition to the locus of dimerization. The functionally sensitive TM4 dimer represents a potential target for SMR inhibition using a synthetic TM4 peptide mimetic. Using exponential decay measurements from a real-time cellular efflux assay, we observed the efflux decay constant was decreased by up to ~60% after treatment with the TM4 peptide inhibitor compared to control peptide treatments. Our results suggest that this approach could conceivably be used to design hydrophobic peptides for disruption of key TM-TM interactions of membrane proteins, and represent a valuable route to the discovery of new therapeutics.

En este trabajo de tesis doctoral se caracteriza funcional y molecularmente el canal TRPV4 en varios modelos de células epiteliales respiratorias mostrando por primera vez la participación de este canal en la función osmoreguladora a nivel celular así como la identificación de nuevas variantes del canal. Se demuestra que la entrada de Ca2+ en respuesta a un hinchamiento hipotónico se produce a través del canal TRPV4 y es necesaria para una eficiente recuperación del volumen o RVD. Por su parte, las células epiteliales respiratorias con fenotipo de fibrosis quística no son capaces de reducir su volumen en un medio hipotónico a causa de una regulación defectuosa del canal, indicando, al mismo tiempo, que la regulación del TRPV4 por el estímulo hipotónico es dependiente de la CFTR.La caracterización de las variantes del canal TRPV4 demuestra que los dominios de ANK son determinantes moleculares claves en el proceso de oligomerización del canal. Al mismo tiempo este trabajo describe nuevos aspectos relacionados con la biogénesis del TRPV4 hasta ahora desconocidos: la oligomerización del canal tiene lugar en el RE, orgánulo donde es N-glicosilado de forma simple antes de ser transportado hacia el Golgi donde sus N-glicanos son madurados. / This thesis characterizes molecularly and funcionally the TRPV4 channel in various models of airway epithelial cells showing, for the first time, the involvement of this channel in an osmoregulatory cellular function as well as the isolation of new splice variants of this channel. It is demonstrated that the TRPV4 channel is the molecular Ca2+ pathway activated by hypotonic estimulus needed to trigger the RVD response. Furthermore, the cystic fibrosis airway epithelial cells showed an impaired RVD due to the misregulation of the TRPV4 channel, indicating that the regulation by the hypotonic stimulus is CFTR-dependent.The characterization of the new variants demonstrated that the ANK domains are key structural determinants in the oligomerization process of the TRPV4. This work also describes new aspects related to the biogenesis of this channel: oligomerization is achieved in the ER, where the TRPV4 is N-glycosilated and then transported to the Golgi where the glycans are matured.

DivIVA is a Gram-positive cell division protein involved in chromosome segregation, midcell placement of the cell division machinery, complete septum closure, and polar growth and morphogenesis. Although well conserved across various Gram-positive species, DivIVA is believed to be relatively species specific. One similarity among DivIVA homologues is the ability to oligomerize through coiled-coil interaction into complexes comprising 10-12 monomers. To date, the importance of DivIVA oligomerization and the N-terminal coiled-coil for its proper function in bacterial cell division has not been reported. This study examined the biological significance of DivIVA oligomerization and the N-terminal coiled-coil in bacterial cell division. This research provides evidence that the N-terminal coiled-coil and oligomerization is essential for the proper biological function of DivIVA_Ef in <i>Enterococcus faecalis</i> cell division. Introduction of point mutations into chromosomal <i>divIVA</i>_Ef known to disrupt either the N-terminal coiled-coil or the two central coiled-coils, involved in oligomerization, were found to be lethal unless rescued by <i>in trans</i> expression of wild type DivIVA_Ef. Using this rescue method, the N-terminal <i>divIVA</i>_Ef mutant strain, <i>E. faecalis</i> MWMR5, and the mutant strain with partial disruption of oligomerization, <i>E. faecalis</i> MWMR10, were successfully rescued. Differential Interference Contrast (DIC) and Transmission Electron Microscopy (TEM) were utilized to determine the phenotypes of <i>divIVA</i>_Ef mutant strains <i>E. faecalis</i> MWMR5 and MWMR10. Both these strains showed asymmetrical division, loss of normal lancet shape, and irregular chains. Full disruption of oligomerization with point mutations in both central coiled-coils resulted in a dominant lethal phenotype. These results demonstrate the essentiality of the N-terminal coiled-coil and oligomerization of DivIVA_Ef for its proper biological function in <i>E. faecalis</i> cell division.<p> Previous detection of DivIVA interaction with a novel cell division protein, MLJD1, by screening a Yeast Two-Hybrid (Y2H) was weak. GST-pulldown and immunoprecipitation did indicate DivIVA_Ef interaction with MLJD1, but another in vivo assay was required to support these results. In this study I demonstrate a strong interaction, using an in vivo Bacterial Two-Hybrid (B2H) assay, between DivIVA_Ef and a fragment of MLJD1 containing two cystathionine-beta-synthase (CBS) domains. The <i>in vitro</i> and <i>in vivo</i> results thus confirm interaction between DivIVA_Ef and MLJD1.<p> Another objective of this study was to determine the localization of DivIVA and MLJD1 in <i>E. faecalis</i>. Localization of DivIVA_Ef in <i>E. faecalis</i> was found to be similar to DivIVA localization in <i>Bacillus subtilis</i> and <i>Streptococcus pneumonia</i>. DivIVA_Ef was diffused along the cell membrane and, as chromosome replication and segregation and cell division proceeded, DivIVA_Ef migrated to the cell poles and then concurrently to the division site. Intriguingly, MLJD1 was found to localize in the same pattern as DivIVA_Ef in <i>E. faecalis</i>, further implicating MLJD1 as a bacterial cell division protein.<p> Since MLJD1 has potential DNA binding capabilities a proposed model of its role in cell division has been proposed. I hypothesize that MLJD1 could be forming a bridge between DivIVA_Ef and the chromosome to aid in proper chromosomal replication and segregation. This model could explain how DivIVA_Ef is involved in chromosome replication. This model is similar to the role of RacA in sporulation in <i>B. subtilis</i> where RacA directs the chromosome during sporulation through direct interaction with DivIVA_Bs and Spo0J.<p> This study has set some important and essential ground work for developing a novel model of cell division for the elusive Gram-positive coccal bacterial strains.

No description available.

Current drugs used to treat Congestive Heart Failure target the renin-angiotensin and adrenergic systems. Studies showed increased mortality rates in patients treated with a combination of these medications. Angiotensin-AT1 and ?2-Adrenergic receptors were shown to form receptor heteromers. Blockade of one receptor in the complex can affect the signal transmitted by the other; suggesting that ligand-based therapy is not as selective as we might think. Modulating receptor trafficking after synthesis might prove to be a valid therapeutic strategy. Unfortunately, little is known about receptor assembly and transport from Endoplasmic Reticulum to Plasma Membrane. The objectives of this study are to identify the proteins that participate in the assembly of AT1R-?2AR heteromer and the regulators of the anterograde trafficking of G-Protein Coupled Receptors. This thesis introduces the role of important targets in those poorly understood processes. The identification of such targets could lead to developing better drugs with fewer adverse effects.