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.

Src homology 3 (SH3) domains are one family of the peptide recognition modules (PRMs), which bind peptides rich in proline or positively charged residues in the target proteins, and play important assembly or regulatory functions in dynamic eukaryotic cellular processes, especially in signal transduction and endocytosis. SH3 domains are conserved from yeast to human, and improper SH3 domain mediated protein-protein interaction (PPI) leads to defects in cellular function and may even result in disease states. Since commonly used large-scale PPI mapping strategies employed full-length proteins or random protein fragments as screening probes and did not identify the particular PPIs mediated by the SH3 domains, I employed a combined experimental and computational strategy to address this problem. I used yeast two-hybrid (Y2H) as my major experimental tool, as well as individual SH3 domains as baits, to map SH3 domain mediated PPI networks, “SH3 domain interactomes”. One of my important contributions has been the improvement for Y2H technology. First, I generated a pair of Y2H host strains that improved the efficiency of high-throughput Y2H screening and validated their usage. These strains were employed in my own research and also were adopted by other researchers in their large-scale PPI network mapping projects. Second, in collaboration with Nicolas Thierry-Mieg, I developed a novel smart-pooling method, Shifted Transversal Design (STD) pooling, and validated its application in large-scale Y2H. STD pooling was proven to be superior among currently available methods for obtaining large-scale PPI maps with higher coverage, high sensitivity and high specificity. I mapped the SH3 domain interactomes for both budding yeast Saccharomyces cerevisiae and nematode worm Caenorhabditis elegans, which contain 27 and 84 SH3 domains, respectively. Comparison of these two SH3 interactomes revealed that the role of the SH3 domain is conserved at a functional but not a structural level, playing a major role in the assembly of an endocytosis network from yeast to worm. Moreover, the worm SH3 domains are additionally involved in metazoan-specific functions such as neurogenesis and vulval development. These results provide valuable insights for our understanding of two important evolutionary processes from single cellular eukaryotes to animals: the functional expansion of the SH3 domains into new cellular modules, as well as the conservation and evolution of some cellular modules at the molecular level, particularly the endocytosis module.

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.

Η μυο-ειδική πρωτεΐνη δεσμίνη, αποτελεί μέλος των πρωτεϊνών του κυτταροσκελετού των ενδιαμέσων ινιδίων και εκφράζεται στους λείους και τους γραμμωτούς μυς. Στους συσταλτούς μύες, το πλέγμα του κυτταροσκελετού της Δεσμίνης περιβάλλει τους Ζ-δίσκους διασυνδέοντάς τους, ενώ παράλληλα συνδέει μεταξύ τους τις συσταλτές περιοχές της μυικής ίνας με την σαρκοπλασματική μεμβράνη, με διάφορα οργανίδια και με τον πυρήνα. Για να προσδιορίσουμε τους ακριβείς μηχανισμούς δράσης της δεσμίνης χρησιμοποιήσαμε το σύστημα υβριδισμού των ζυμών – yeast two hybrid screen system – προκειμένου να ανιχνεύσουμε πρωτεΐνες που αλληλεπιδρούν με τη δεσμίνη. Χρησιμοποιήσαμε ως «δόλωμα» αλληλουχίες των άκρων του μορίου της δεσμίνης του αμινο-τελικού και το καρβόξυ-τελικού. Μελετώντας τις πρωτεΐνες που προέκυψαν, διαπιστώσαμε ότι το αμινο-τελικό άκρο της δεσμίνης αλληλεπιδρά με διάφορες μιτοχονδριακές πρωτεΐνες. Με το ίδιο σύστημα αποκαλύψαμε αλληλεπιδράσεις της δεσμίνης με λυοσωματικές πρωτεΐνες όπως η καθεψίνη D και η προσαποσίνη οι οποίες αλληλεπιδρούν με το αμινοτελικό άκρο της δεσμίνης. Η καθεψίνη D είναι μια λυοσωματική πρωτεάση, που οδηγείται και ωριμάζει πλήρως στα λυοσώματα ενώ η προσαποσίνη είναι ένα πρόδρομο λυοσωματικό μόριο με πρωτεόλυση του οποίου, εντός του λυοσώματος, προκύπτουν οι σαποσίνες Α έως D. Η καθεψίνη D αποτελεί δείκτη καταστάσεων αυτοφαγία και τελευταία φαίνεται ότι επεμβαίνει σε φαινόμενα απόπτωσης επάγωντάς την κατά περίπτωση. Η αλληλεπίδραση της δεσμίνης με την καθεψίνη D επιβεβαιώθηκε και με βιοχημικές τεχνικές (in vitro) όπως η συνεργιστική ανοσοκαθίζηση /ανοσοκατακρήμνιση (co-immuno-precipitation) και η τεχνική GST pull-down. Μετά και από αυτές τις in vitro αποδείξεις, φαίνεται πως μάλλον συμβαίνει ευθεία αλληλεπίδραση μεταξύ της δεσμίνης και της καθεψίνης D. Γι’ αυτό, και με βάση όσα είναι γνωστά για την καθεψίνη D, προτείνουμε μια νέα λειτουργία του κυτταροσκελετού της δεσμίνης πιθανόν στην μετακίνησης και τη δημιουργία των λυοσωμάτων αλλά και έναν νέο ρυθμιστικό ίσως ρόλο της, σε διαδικασίες αυτοφαγίας και απόπτωσης, μέσω της πρόσδεσής της με σημαντικά μόρια ρυθμιστές τέτοιων διαδικασιών.................... / Desmin is the muscle - specific member of the intermediate filament family of cytoskeletal proteins, expressed both in striated and smooth muscle tissues. In mature striated muscle fibers, the desmin filament lattice surrounds the Z-discs, interconnects them to each other and links the entire contractile apparatus to the sarcolemmal cytoskeleton, cytoplasmic organelles and the nucleus. In order to identify the exact mechanisms of desmin’s action, we performed a yeast two-hybrid screen for desmin-interacting proteins. For this purpose, we used as baits the two non helical terminal regions of the desmin molecule, the amino (head)- and the carboxy (tail)- terminal domain. We have found that the head domain of desmin potentially interacts with two new groups of proteins, mitochondria and lysosome related. Specifically, in the second category, we have revealed an association of the head domain of desmin with Cathepsin D (one of the lysosomal proteinases) and prosaposin (a single precursor which gives rise to Saposins A-D by proteolytic cleavage in lysosomes and is also referred to as sphigolipid activator proteins). In addition to its targeting to lysosomes, Cathepsin D is also involved in apoptosis and autophagy processes. This protein interaction result has been retested. The interaction between cathepsin D and desmin has also been further confirmed both with reverse yeast transformation as well as biochemical assays such as co-immunoprecipitation and GST pull down assay. The above described strong evidence of direct interaction between desmin and cathepsin D, has allowed us to propose a novel function of desmin IFs in lysosomal trafficking and/or as a new regulator of autophagy and apoptotic cell death.

No description available.

3’-end modification by polyadenylation is a ubiquitous feature of almost all eukaryotic mRNA species and is catalyzed by a consortium of enzymes, the polyadenylation factors. Poly(A) polymerase (PAP), the enzyme catalyzing the addition of adenosine residues during the polyadenylation stage, exists in four isoforms within Arabidopsis. In silico and yeast two-hybrid studies showed that PAP1 has unique expression and interaction pattern in Arabidopsis, suggesting non-canonical functions of PAP1. Its exclusive interaction with PAP4 has not been reported in other living systems until now and hints at a difference in polyadenylation in plants with respect to mammals and yeast. Cleavage Stimulation Factor (CstF), a heterotrimeric complex of the polyadenylation factors CstF50, CstF64 and CstF77, plays a role largely in cleavage of pre-mRNA. This study highlights some aspects of the Arabidopsis homologs of CstF64 and CstF77, central to various cellular processes other than nuclear polyadenylation. In silico studies showed an elevated expression of CstF64 in the pollen while that of CstF77 remained fairly low. Yeast two-hybrid assays indicated a novel kind of interaction of CstF64 with Fip1(V). It is also speculated from sub-cellular localization techniques by agroinfiltration in tobacco leaves that CstF64 localizes in the cytoplasm and CstF77 in the nucleus, as found for the orthologs of CstF77 in other systems.

Thesis (M.Sc.). / Written for the Dept. of Biochemistry. Title from title page of PDF (viewed 2008/12/09). Includes bibliographical references.

Made available in DSpace on 2015-03-26T13:42:19Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1192795 bytes, checksum: 39105ae81e1f922a4d76be541c837833 (MD5) Previous issue date: 2006-09-19 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The Sw-5 protein codified by the tomato Sw-5 gene confers broad spectrum tospovirus resistance. This protein contains a coil-coiled aminoterminal domain, a central nucleotide binding site domain and a leucine-rich repeat carboxi-terminal domain. These domains suggest that Sw-5 recognizes directly or indirectly virus elicitors and it participates in a signal transduction pathway that leads to the death of infected cells and activation of plant defense responses. The objective of this work was to identifiy and characterize genes encoding proteins that interacts physically with Sw-5 in the yeast two hybrid system. The screening of 5x107 Nicotiana benthamina cDNA clones, using the yeast mating strategy, did not yield any protein capable to interact with Sw-5. Analyzing 1,8 x 105 clones by co-transformation strategy, it was possible identify a cDNA that encodes an chloroplast ferredoxin I (Nb-Fd1) capable to interact with Sw-5. This protein contains a domain and a signature of 2Fe-2S proteins. Virus-induced silencing of the Nb-Fd1 gene leads to a grater number of local necrotic lesions and apical lesions in resistance plants challenged with tospovirus showing that this gene is important for tomato tospovirus resistance. This phenotype indicates that Nb-Fd1 is possibly involved in cellular redox state alteration, reactive oxygen species accumulation and programmed cell death activation processes mediated by Sw-5. / A proteína Sw-5 codificada pelo gene Sw-5 de tomateiro confere resistência de amplo espectro a tospovírus. Essa proteína contém um domínio amino-terminal coil-coiled, um domíno central de ligação a nucleotídeos fosfatados e uma região carboxi-terminal com repetições ricas em leucina. Esses domínios sugerem que a proteína Sw-5 reconhece direta ou indiretamente elicitores produzidos pelo vírus e participa de uma cadeia de transdução de sinais que leva à morte das células inicialmente infectadas pelos tospovírus e, ou, ativação de respostas de defesa da planta. Este trabalho teve como objetivo a identificação e a caracterização molecular de genes que codificam proteínas capazes de interagir fisicamente com a proteína codificada pelo gene de resistência Sw-5 por meio da utilização do sistema duplo-híbrido de leveduras. A triagem 5x107 clones de cDNAs de Nicotiana benthamina por meio da obtenção de diplóides, não revelou nenhum cDNA que codifica proteína capaz de interagir com a proteína Sw-5. A análise de 1,8 x 105 clones pelo processo de co-transformação resultou na identificação de um cDNA que codifica uma proteína ferredoxina I de cloroplasto (Nb-Fd1), capaz de interagir com a proteína Sw-5. Essa proteína contém um domínio e uma assinatura característicos de proteínas 2Fe-2S. Resultados preliminares mostraram que o silenciamento do gene que codifica Nb-Fd1 resulta em uma maior quantidade de lesões necróticas locais e lesões no ápice das plantas resistentes desafiadas com tospovírus evidenciando a não contenção do vírus no sítio de infecção. Esse fenótipo demonstra um papel de Nb-Fd1 na resistência a tospovírus. Assim, é possível que esta proteína participe do processo de alteração do potencial redox da célula, acúmulo de espécies reativas de oxigênio e ativação de morte celular programada que caracterizam a resposta de hipersensibilidade mediada pelo gene Sw-5.

Les polérovirus infectent de nombreuses plantes d’intérêt économique telles que la pomme de terre, la betterave à sucre et les cucurbitacées. Ces virus icosaédriques renferment un ARN simple brin et leur capside est constituée d’une protéine majeure (CP) et d’un composant mineur (RT*) localisé à la surface des virions. Ces virus sont restreints aux cellules du phloème dans lesquelles ils se multiplient et se déplacent. Les protéines CP et RT sont essentielles à la dissémination du virus par le puceron vecteur et à son mouvement dans la plante. L’objectif de cette étude a consisté à identifier dans les cellules du phloème, les protéines associées aux virions susceptibles d’intervenir dans le cycle viral en criblant une banque d’ADNc de cellules compagnes (CC) d’A. thaliana avec les protéines de structure ou des domaines protéiques du CABYV. Quatre gènes codant pour une protéine Heat Shock (HSP), la profiline 3 (PRF3) une glysosyl hydrolase ; et la protéine « Response to low sulfur 3 » ont été identifiés. Tous ces gènes candidats interagissent avec le domaine RTC-ter du CABYV et avec la protéine RT* pour la protéine HSP. En plus de ces gènes candidats, je me suis intéressée à la protéine ALY, identifiée au laboratoire, au cours du criblage d’une banque d’ADNc de puceron entier avec les deux protéines de structure du Turnip yellows virus (un autre polérovirus). Cette protéine possède quatre orthologues chez Arabidopsis susceptibles d’être impliquées dans le mécanisme de gene silencing mis en place contre le Tomato Bushy Stunt Virus. Les protéines ALY sont donc des candidats intéressants et j’ai montré une interaction entre les protéines de structure du CABYV et du TuYV et les quatre orthologues d’Arabidopsis. L’implication de ces gènes candidats n’a pas pu être confirmée à ce jour dans des mutants knock-out d’arabidopsis. Les résultats complexes obtenus pour le candidat PRF3 au cours des analyses de validation fonctionnelle, m’a conduit à étudier l’interaction entre ce candidat et le domaine RTC-ter du CABYV in planta par FLIM mais aucune interaction n’a pu être confirmée à ce jour. Tous les candidats isolés lors du criblage de la banque d’ADNc de CC interagissant avec le domaine RTC-ter du CABYV, ce travail m’a conduit à analyser le rôle dans le cycle viral de ce domaine et de la protéine RT (sous sa forme complète ou dépourvue du domaine RTC-ter), en étudiant l’accumulation de ces mutants dans les plantes et le clivage de la protéine RT. Tout d’abord, afin de localiser précisément le site de clivage de la protéine RT, des mutants ponctuels dans la zone de clivage ont été réalisés ce qui a permis de montrer que la structure secondaire de la protéine est importante pour son clivage. Puis, afin d’analyser le rôle du domaine RTC-ter dans le cycle viral, j’ai obtenu par délétion, un mutant n’exprimant plus ce domaine. Ce mutant synthétise uniquement la protéine RT tronquée, forme des particules virales semblables au virus sauvage et est transmissible par puceron. Par contre, de façon surprenante, ce mutant est incapable d’envahir les feuilles non-inoculées d’une plante. Ce résultat suggère que les deux formes de la protéine RT (complète et tronquée) sont indispensables au mouvement à longue distance du virus et nous proposons un modèle dans lequel le domaine C-terminal de la protéine RT agit en trans sur la particule virale pour promouvoir le mouvement du CABYV à longue distance. / Poleroviruses infect a wide range of cultivated plants such as potatoes, sugar beet and plants of Cucurbitaceae family. These viruses are restricted to phloem tissue where they replicate in nucleated cells and translocate over long distances through sieve elements. Polerovirus capsid is composed of the major coat protein (CP) and of a minor component referred to as the readthrough (RT*) protein and exposed at the outside of the particles. CP and RT proteins are essential for virus movement and transmission by aphids. The aim of this study is to identify phloem proteins interacting with viral proteins and potentially involved in viral cycle, by screening an A. thaliana companion cell (CC) cDNA library with structural proteins or protein domains of CABYV. Four genes encoding for a heat shock protein (HSP), a profilin (PRF3), a glycosyl hydrolase and the protein ”Response to low sulfur ” (LSU3) were identified and interact with the C-terminal part of the RT protein (RTC‑ter) and with the RT* protein for the HSP. An additional gene encoding for the protein ALY, identified in the laboratory, by screening an aphid cDNA library with structural proteins of the Turnip yellows virus (another polerovirus) was studied. This protein has four orthologues in Arabidopsis, involved in the gene silencing mechanism against Tomato Bushy Stunt Virus. Here we show that CABYV and TuYV structural proteins interact with the four orthologues of Arabidopsis. Involvement of these candidate genes was not confirmed in Arabidopsis knock-out mutants. In functional experiments, ambiguous results were obtained with PRF3 arabidopsis mutants, and this lead me to study the interaction between PRF3 protein CABYV RT c-ter domain by FLIM, but no interaction was found so far. As all candidat interact with the RTC-ter domain, we studied more precisely the role of this domain in the viral cycle and the role of the complete RT protein. We studied the in vivo RT protein processing and its consequences on systemic movement of CABYV mutants. Using a collection of point mutations introduced in the central domain of the CABYV RT protein, we approached the site of the RT processing and proposed that this process is affected by the secondary structure around the cleavage site. We also reported for the first time the generation of a polerovirus mutant able to synthesize only the RT* protein and to incorporate it into the particle. This mutant was unable to move systemically. Conversely another mutant producing a full-length RT protein impaired in correct processing and incorporating a shorter version of the RT* protein showed very weak systemic infection. These data are strongly in favor of a role of both RT proteins in efficient CABYV movement. An inefficient virus transport was still maintained in the absence of RT proteins suggesting an RT-independent movement pathway. Based on these results, we propose a model for CABYV long-distance transport in which the complete RT protein, or its C-terminal part, acts in trans on wild-type virions to promote their efficient long-distance transport.

Orientador: Nilson Ivo Tonin Zanchin / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-06T21:20:48Z (GMT). No. of bitstreams: 1 Carneiro_FlaviaRaquelGoncalves_D.pdf: 4565621 bytes, checksum: 26bdfc2a971d3837321625172c6745e8 (MD5) Previous issue date: 2006 / Resumo: O gene que codifica a proteína INT6 corresponde a um dos sítios de inserção do vírus de tumor de glândula mamária em camundongo (MMTV). Esta inserção pode levar à formação de proteínas truncadas sem a porção C-terminal e sem o domínio PCI, descrito como domínio de interação entre proteínas. Neste trabalho, realizamos 3 triagens para a identificação dos ligantes protéicos da proteína humana INT6 (hINT6) pelo método do duplo-híbrido de levedura. Embora interações específicas tenham sido identificadas, não foi possível a confirmação in vitro das novas interações isoladas. Para análises estruturais, usamos a proteína INT6 de Arabidopsis thaliana (AtINT6), visto que a proteína humana expressou em níveis muito baixos na fração solúvel do extrato de Escherichia coli. Análises de dicroísmo circular revelaram que a proteína AtINT6 é rica em estrutura do tipo a-hélice. A região que compreende os aminoácidos 172 a 415, incluindo o domínio PCI, foi identificada por proteólise parcial e espectrometria de massas como um domínio estrutural que após sua clonagem apresentou alto nível de expressão, solubilidade e estabilidade. Este trabalho envolveu também a caracterização da NY-REN-21, que foi isolada pelo duplo-híbrido para a identificação dos ligantes protéicos da proteína hINT6 e representa uma possível ortóloga para o fator de transcrição ZFP38 de camundongo. Ambas as proteínas apresentam um domínio de dimerização (SCAN), 7 domínios dedos de zinco tipo C2H2 na porção C-terminal e uma região central predita como desestruturada. A proteína NY-REN-21 se mostrou parcialmente desenovelada e sua estrutura secundária é afetada pela incubação com EDTA. Ensaios de proteólise limitada, dicroísmo circular e fluorescência confirmaram que sua região central é intrinsicamente desordenada, além de apresentar mobilidade anômala em gel de SDS-PAGE e representa uma fração flexível da proteína. Não foi possível a caracterização da região dos dedos de zinco, pois esta região se mostrou altamente instável. O domínio SCAN da NY-REN-21 é capaz de formar homodímeros e heterodímeros com a proteína SCAND1. Esta interação pode representar uma forma de regulação da atividade da proteína NY-REN-21 / Abstract: The INT6 gene was reported as a frequent genome integration site of Mouse Mammary Tumor Virus (MMTV). This integration may result in truncated forms of INT6 protein lacking its C-terminal region and the PCI domain. It has been reported that this domain is involved in protein-protein interaction. We performed 3 yeast two-hybrid screens in order to identify the human INT6 (hINT6) interaction partners. Although two previously described specific interactions were identified in these screens, it was not possible to confirm the new interactions by in vitro binding assays. The Arabidopis thalina INT6 ortholog (AtINT6) was used for structural analyses, since the hINT6 was insoluble following expression in Escherichia coli. AtINT6 showed CD spectra with high helical content. The region comprising amino acids 172 to 415 forms a compact protease resistant domain as determined by limited proteolysis and mass spectrometry analyses. This domain, comprising the PCI domain sequence, was cloned and expressed in E. coli and showed high solubility and stability. This recombinant protein has the potential to serve as a model protein for three-dimensional structure determination of the PCI domain. We also studied the NY-REN-21 protein, which was isolated as a potential hINT6 interaction partner and represents a putative ortholog of the mouse ZFP38 transcriptional factor. Both proteins are C2H2 type multifinger proteins, containing a conserved oligomerization domain (SCAN) in the N-terminal region and a predicted disordered central region. Our analyses showed that full-length NY-REN-21 is partially unfolded and its secondary structure content is affected by incubation with EDTA. The central region of NY-REN-21 shows an aberrant mobility on SDS-PAGE and is intrinsically unstructured as reveled by circular dichroism, fluorescence and limited proteolysis. The zinc finger region was not characterized because of its unstable nature. The recombinant SCAN domain of NY-REN-21 can form homodimers and heterodimers with the SCAND1 protein. This interaction may represent a novel regulatory mechanism of NY-REN-21 activity / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular