Le clan « arrestine » est une superfamille de protéines adaptatrices regroupant les arrestines conventionnelles (β-arrestines et arrestines visuelles), bien décrites pour leur rôle dans la régulation des récepteurs membranaires couplés aux protéines G hétéro-trimériques et la signalisation associée et des protéines de type arrestin-like identifiées plus récemment, qui semblent partager des fonctions communes dans la régulation et le trafic de cargos membranaires. Ce travail de thèse a porté sur l'étude de la protéine arrestin-like AdcA de Dictyostelium discoideum dans l'objectif d'en définir le rôle fonctionnel. En plus de son module arrestine, caractéristique de cette famille de protéines, AdcA possède un domaine FYVE qui permet son association à la voie endocytaire, un domaine C-terminal riche en tyrosines et un domaine N-terminal riche en histidines impliqué dans son oligomérisation. Mes travaux ont permis d'établir que la protéine AdcA répond à des stress de natures diverses dont l'hyper-osmolarité, par une multi-phosphorylation massive, notamment au niveau du domaine N-terminal. La sensibilité d'AdcA à la dépolymérisation du réseau d'actine suggère un lien fonctionnel entre le cytosquelette d'actine et la cascade de signalisation menant à la phosphorylation de la protéine. En conditions de stress modéré, la phosphorylation d'AdcA est transitoire et sa déphosphorylation, en partie dépendante du facteur de transcription STATc, corrèle avec l'adaptation des cellules aux conditions de stress. Cette modification post-traductionnelle transitoire pourrait permettre de contrôler l'activité d'AdcA et d'optimiser la réponse des cellules au stress. Parallèlement, la caractérisation fonctionnelle d'un partenaire d'AdcA, la protéine FrmC, a été entreprise. FrmC est une nouvelle protéine, encore non caractérisée, présentant plusieurs domaines fonctionnels dont un domaine FERM capable de lier l'actine in vitro et un domaine à motifs LRR. Mes travaux ont notamment mis en évidence que FrmC est recrutée à la membrane plasmique et joue un rôle dans l'adhésion cellulaire. Par ailleurs, l'absence de FrmC affecte l'adaptation des cellules et la réponse d'AdcA en situation de stress hyper-osmotique. / The arrestin clan represents a large group of adaptor proteins which includes the canonical arrestins - β-arrestins and visual arrestins – well described for their role in the regulation of membrane receptors coupled to heterotrimeric G-proteins and associated signaling as well as arrestin-like proteins identified more recently that seem to share functions in the regulation and trafficking of membrane cargoes. This work focused on the study of the arrestin-like protein AdcA of Dictyostelium discoideum, to determine the functional role of this atypical member. In addition to the arrestin module common to all the members of the arrestin family, AdcA harbors a FYVE domain responsible for its association to the endocytic pathway, a C-terminal tyrosine-rich domain and an N-terminal extension rich in histidine residues mediating its oligomerization. I have established that AdcA responds to a variety of stresses such as hyperosmolarity by a massive multi-phosphorylation of the protein. Sensitivity of AdcA to changes in F-actin polymerization status suggests a link between the signaling cascade leading to AdcA phosphorylation and the actin cytoskeleton. In conditions of moderate stress, AdcA response is transient and its dephosphorylation depends on the transcription factor STATc and correlates with cell adaptation to the stress conditions. This post-translational modification of AdcA could modulate its activity and optimitize the cell response to stress. In parallel, the functional characterization of a partner of AdcA, the protein FrmC, has been undertaken. This so-far uncharacterized protein presents a multimodular structure with a FERM domain able to bind F-actin in vitro and several leucine-rich repeats (LRR). I have shown that FrmC is recruited to the plasma membrane and is involved in cell-substrate adhesion. In addition, disruption of FrmC affects cell adaptation and AdcA response in conditions of hyperosmotic stress.

Orientador: Mário Tyago Murakami / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T10:58:02Z (GMT). No. of bitstreams: 1 Polo_CarlaCristina_M.pdf: 4286137 bytes, checksum: cf12a2b67d90255468d1906d719759a3 (MD5) Previous issue date: 2012 / Resumo: As miosinas pertencem a uma família de proteínas motoras que através da hidrólise de ATP são capazes de se movimentarem pelas fibras de actina. Sua estrutura é dividida em três domínios principais: motor, responsável pela hidrólise do ATP; regulador, envolvido na ligação de cadeias leves de calmodulina e a cauda, que tem papel essencial na mediação de interações com cargas celulares, como organelas, ácidos nucleicos e outras proteínas. O organismo modelo para insetos, Drosophila melanogaster, possui 11 miosinas pertencentes a 8 classes sendo que informações funcionais e bioquímicas são escassas, e estruturais, inexistentes. Apesar dos grandes avanços obtidos nos estudos de miosinas humanas, as miosinas de inseto ainda são pouco caracterizadas não havendo estudos comparativos, como por exemplo, para a determinação de conservação de parceiros moleculares entre os diferentes filos da classificação de Lineu. Neste contexto, foram selecionados os seguintes domínios de inseto para caracterização molecular: quinase da miosina III, cauda globular da miosina V e FERM da miosina XV. A partir do cDNA da pupa e adulto do inseto foram amplificados os fragmentos de genes de interesse e clonados em vetor de clonagem, pGEM T-easy, e posteriormente de expressão, pET28a e pET28aSUMO. Após testes em diversas condições no sistema bacteriano, as construções cauda globular da miosina V (GLOB_2 e GT-f) e domínio FERM-f da miosina XV foram obtidos na fração solúvel e com rendimento suficiente para estudos estruturais. O protocolo de expressão em larga escala assim como de purificação foram estabelecidos para cada uma dessas construções. Estudos biofísicos por aSEC, DLS, CD e SAXS foram realizados para a proteína GT-f da miosina V, mostrando a proteína monomérica com alto conteúdo de hélices-alfa corroborando as análises in silico e com as caudas globulares de miosinas ortólogas já descritas. Além disso, sua termoestabilidade foi avaliada com Tm de 46 ºC e que sua estabilidade é alterada pela adição de alguns ligantes, mas não pela força iônica como verificado para a cauda globular da miosina Va humana. A estrutura a baixa resolução da GT-f foi determinada pelos experimentos de SAXS, mostrou uma proteína monomérica com forma alongada (Dmax =100 Å). Para a construção FERM-f, apesar do sucesso alcançado no protocolo de expressão e purificação, a amostra permaneceu polidispersa e com uma estrutura random coil, inviabilizado futuros experimentos. Acredita-se que a ausência do domínio MyTH4, gerou a perda de estabilidade do domínio FERM-f e será necessária a clonagem dos dois domínios fusionados para garantir a estabilidade estrutural e funcional / Abstract: Myosins belong to a motor protein superfamily that uses the ATP hydrolysis to move along actin filaments. Its structural architecture comprises three main domains: motor, responsible for ATP hydrolysis; regulator, which binds calmodulin light chains; and tail, responsible for cellular cargoes binding. Drosophila melanogaster, an insect model, has 11 myosins belonging to 8 different myosin classes; however functional and biochemical data are scanty, and, structural, absent. Although the great advances on investigating human and yeast myosins with the elucidation of molecular partners and molecular mechanisms involved in signaling, there are no comparative studies among different phyla. Therefore, in order to gain insights into insect myosins, in silico analysis were performed and some domains like kinase (myosin III), globular tail (myosin V), and FERM (myosin XV) were selected for biophysical and structural studies. From pupa and adult cDNAs, the target genes were amplified and cloned into pGEM- T-easy, pET28a and pET28aSUMO vectors. After expressions tests, the myosin V globular tail constructs, GLOB_2 and GT-f, and myosin XV FERM-f were obtained in soluble fraction and in sufficient amounts for structural studies. aSEC, DLS, CD e SAXS biophysical studies were performed to myosin V GT-f, showing that the protein is monomeric with predominance of alpha-helix in accordance with the in silico atomic model. The stability of GT-f was assessed with a Tm of 46 ºC and some molecules are able to increase the stability, while the ionic strength has no difference like was observed for the human globular tail of myosin Va. The low resolution structure determined by SAXS experiments confirmed the monomeric state and its elongated molecular shape (Dmax = 100 Å). The FERM-f construct, although the purification and expression protocol were standardized, the sample remained polydisperse with a random coil structure, disabling other biophysical experiments. It is supposed that the absence of the MyTH4 domain in the FERM-f domain results in loss of stability, and cloning and expression of fused domains will be necessary to guarantee structural and functional stability / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular