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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
21

Identificação de uma ferredoxina que interage com a proteína Sw-5 que confere resistência a tospovírus / Identification of a ferredoxin that interacts with the Sw-5 protein that confers resistance to tospovirus

Almeida, Leonardo Augusto de 19 September 2006 (has links)
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.
22

Analises estruturais e estudos das interações das proteinas INT6 e NY-REN-21 / Structural analysis and interaction studies of the INT6 and NY-REN-sa proteins

Carneiro, Flavia Raquel Gonçalves 30 May 2006 (has links)
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
23

Studium vybraných podjednotek komplexu exocyst u rostlin a jejích interaktorů v autofagické dráze / Study of selected plant exocyst subunits and its interactors in autophagy pathway.

Rácová, Denisa January 2015 (has links)
Exocyst is a binding protein complex, which is evolutionary conserved in yeast, animal and in plant cells. It has crucial role in regulation of cell morfogenesis and cell polarity. The function of the exocyst complex is binding of secretoric vesicle to the proper side on plasma membrane in penultimate step of exocytosis. This process is essecial for function and survival of cell. Another process crucial for the cell is autophagy. In plants autophagy plays important role in the responses to nutrient starvation, senescence, abiotic and biotic stress. RabG3b are small GTPases, which have positive role in autophagy. In this work I described the interaction between RabG3b and some of subunits of exocyst complex: Exo70B1, Exo70B2 and Exo84b. I also studied changes in morfogenesis of tonoplast by induction and inhibition of authophagy and induction of anthocyans synthesis in Arabidopsis thaliana.
24

Using the Yeast Two-Hybrid System to Determine the Function of Parkin E3 Ubiquitin Ligase

Nguyen, Vanessa 01 December 2014 (has links)
Parkin is a cytosolic E3 ubiquitin ligase that is recruited to the mitochondria during cellular stress and has been suggested to be involved in a variety of biological processes such as mitophagy. The recruitment of Parkin (PARK2) to the mitochondria is dependent upon the kinase activity and the accumulation of PINK1 on damaged mitochondria. Mutations in either PINK1 or Parkin genes disrupt this protective pathway and lead to the accumulation of damaged mitochondria. From a clinical standpoint, mutations in the PARK2 gene have been associated with the progression and onset of autosomal recessive juvenile parkinsonism. Without the presence of a quality control system such as that of the PINK1/Parkin pathway, the accumulation of damaged mitochondria could lead to increased levels of oxidative stress, a decrease in ATP, and the progression towards cellular death. However, many of the details regarding the mechanism of Parkin-mediated ubiquitination and its involvement in mitophagy are not fully established. The intent of this thesis is to further explore the function of Parkin by utilizing the yeast-two hybrid system to identify novel Parkin interactors/substrates. A HeLa (cervical cell carcinoma) cDNA library was screened using Parkin124-465 as the "bait" protein. From this screening, six positive Parkin interactors were isolated and characterized. Using this approach it is possible to gain a better understanding of the function of Parkin in regulating cellular processes such as mitophagy.
25

Use of the yeast two-hybrid system to define the function of THAP5 protein

Popat, Paiyal V. 01 January 2009 (has links)
THAP5 is a protein which was recently isolated in the Zervos Lab as an interactor of a pro-apoptotic protein, Omi/HtrA2. THAP5 is unique because it shares no homology with mouse or rat and can only be found in humans. The only homology it shares with any other protein is its THAP domain. THAP proteins are zinc-dependent sequence specific DNA-binding factors belonging to the zinc-finger family of proteins (2). There are 12 identified members of TIIAP proteins in humans, THAP0-THAP 11. The roles of these THAP proteins include proliferation, apoptosis, cell cycle, chromosome segregation, chromosome modification, and transcriptional regulation (2). The function of THAP5 is still unclear and thus, a Yeast Two-Hybrid experiment will be done to further determine its function. The Yeast Two-Hybrid System is a common molecular biology technique used to identify interactors of a certain protein of interest. By identifying the protein interactors of THAP5 and their functions, it is possible to further determine the function of THAP5.
26

"Identificação do papilomavírus humano em gestantes adolescentes por meio da captura hibrida II:correlação com a colpocitologia oncótica convencional, em base líquida e colposcopia" / Human papillomavírus identification by hybrid capture II technique in pregnant teenagers: : comparison with conventional, liquid-based Pap test and colposcopic findings

Santos, Fernanda Erci dos 22 March 2006 (has links)
Estudo prospectivo para identificar a presença do papilomavírus humano em gestantes adolescentes por meio da captura híbrida II e correlacionar com colpocitologia oncótica convencional, em base líquida e colposcopia. O grupo constituído por 60 gestantes entre 12 a 18 anos e idade gestacional media de 23 semanas. A captura híbrida II foi positiva em 51,7%. A colpocitocologia oncótica convencional : normal em 90% e anormal em 10%. Os achados citológicos anormais: lesão intraepitelial escamosa de baixo grau em 8,3% e carcinoma invasor em 1,7%. A colpocitologia em base líquida: normal em 90% e anormal em 10%. Os achados citológicos anormais: lesão intraepitelial escamosa de baixo grau em 8,3% e lesão intraepitelial escamosa de alto grau em 1,7%. Os achados colposcópicos normais foram o epitélio escamoso normal em 20%, epitélio glandular em 18,3% e a zona de transformação normal em 40%. A zona de transformação anormal presente em 21,7% / Study delineated to identify human papillomavirus by hybrid capture II in pregnant teenagers and to correlate with conventional, liquid-based Pap test and colposcopic findings. The study group was constituted by 60 pregnant women aged between 12 and 18 years old; mean gestational age was 23 weeks. They were submitted to anamnese, Pap smear, hybrid capture technique and colposcopy. Hybrid capture II of human papillomavirus was positive in 51,7% of the cases. Conventional Pap test was normal in 90% and abnormal in 10% of the cases. Abnormal results: low-grade squamous intraepithelial lesion in 8,3% and Invasor carcinoma in 1,7%. Based-liquid Pap test was normal in 90% and abnormal in 10% of the cases. Abnormal results: 8,3% of low-grade squamous intraepithelial lesion and 1,7% of high-grade squamous intraepithelial lesion. Normal results: 20% of normal squamous epithelium, 18,3%of columnar epithelium and 40% of normal transformation zone. Abnormal transformation zone was seen in 21,7% of the cases
27

"Identificação do papilomavírus humano em gestantes adolescentes por meio da captura hibrida II:correlação com a colpocitologia oncótica convencional, em base líquida e colposcopia" / Human papillomavírus identification by hybrid capture II technique in pregnant teenagers: : comparison with conventional, liquid-based Pap test and colposcopic findings

Fernanda Erci dos Santos 22 March 2006 (has links)
Estudo prospectivo para identificar a presença do papilomavírus humano em gestantes adolescentes por meio da captura híbrida II e correlacionar com colpocitologia oncótica convencional, em base líquida e colposcopia. O grupo constituído por 60 gestantes entre 12 a 18 anos e idade gestacional media de 23 semanas. A captura híbrida II foi positiva em 51,7%. A colpocitocologia oncótica convencional : normal em 90% e anormal em 10%. Os achados citológicos anormais: lesão intraepitelial escamosa de baixo grau em 8,3% e carcinoma invasor em 1,7%. A colpocitologia em base líquida: normal em 90% e anormal em 10%. Os achados citológicos anormais: lesão intraepitelial escamosa de baixo grau em 8,3% e lesão intraepitelial escamosa de alto grau em 1,7%. Os achados colposcópicos normais foram o epitélio escamoso normal em 20%, epitélio glandular em 18,3% e a zona de transformação normal em 40%. A zona de transformação anormal presente em 21,7% / Study delineated to identify human papillomavirus by hybrid capture II in pregnant teenagers and to correlate with conventional, liquid-based Pap test and colposcopic findings. The study group was constituted by 60 pregnant women aged between 12 and 18 years old; mean gestational age was 23 weeks. They were submitted to anamnese, Pap smear, hybrid capture technique and colposcopy. Hybrid capture II of human papillomavirus was positive in 51,7% of the cases. Conventional Pap test was normal in 90% and abnormal in 10% of the cases. Abnormal results: low-grade squamous intraepithelial lesion in 8,3% and Invasor carcinoma in 1,7%. Based-liquid Pap test was normal in 90% and abnormal in 10% of the cases. Abnormal results: 8,3% of low-grade squamous intraepithelial lesion and 1,7% of high-grade squamous intraepithelial lesion. Normal results: 20% of normal squamous epithelium, 18,3%of columnar epithelium and 40% of normal transformation zone. Abnormal transformation zone was seen in 21,7% of the cases
28

Identification of TEF cofactor(s) in skeletal muscles utilizing yeast two hybrid system

Zhang, Aijing. January 2004 (has links)
Thesis (M.S.)--University of Missouri--Columbia, 2004. / Typescript. Vita. Includes bibliographical references (leaves 70-75). Also issued on the Internet.
29

Caractérisation biochimique et moléculaire du complexe SCF (SKP1-CULLIN-FBOX) chez le blé tendre / Biochemical and molecular characterization of the SCF complex (SKP1-CULLIN-FBOX) in soft wheat

El Beji, Imen 18 July 2011 (has links)
Les modifications post-traductionnelles des protéines constituent un niveau crucial de régulation de l’expression des gènes. Parmi elles, la conjugaison peptidique impliquant l’ubiquitine intervient entre autre dans la régulation de la stabilité protéique. La fixation de ce peptide de 76 acides aminés, extrêmement conservé, sous forme de chaîne de polyubiquitine, nécessite l’intervention de trois enzymes (E1, E2 et E3) et constitue un signal de dégradation de la protéine ainsi modifiée. Cette voie de régulation intervient dans de très nombreux processus biologiques. Les complexes SCF sont impliqués dans la voie de protéolyse ciblée. Ils représentent l' une des classes les plus fréquentes d'ubiquitine ligase E3 et ils sont composés de quatre sous-unités (Rbx, Cullin, SKP1, et F-box). La structure et la fonction des complexes SCF, ont été étudiées chez la levure, l’Homme et la plante modèle A. thaliana. Cependant, peu de travaux ont été réalisés chez des plantes cultivées, en particulier les céréales, telles que le blé. Cinq gènes codant pour la sous-unité Skp1 (TSK1, TSK3, TSK6, TSK11 et TSK16), cinq gènes codant pour la sous-unité F-box (ZTL, ATFBL5, EBF, TIR1 et ABA-T), un gène codant pour la sous-unité Cullin1 et un gène codant pour la protéine RBX du complexe SCF du blé, ont été isolés et clonés. Les différents tests d’interaction entre les quatre sous-unités du complexe SCF ont été réalisés par la méthode du double-hybride dans la levure en utilisant la technologie Gateway. Ces études ont montré que les deux protéines, TSK1 et TSK3, fixent spécifiquement différentes sous-unités F-box. Parallèlement, nous avons montré que la protéine TSK11 représente une structure particulière. Des études d’insertion/délétion sur la protéine TSK11 ont permis d’identifier un nouveau domaine indispensable à l’interaction. Les analyses par PCR semi-quantitative des différents gènes codant pour la sous-unité Skp1, dans trois tissus différents (feuille tige et racine), ont mis en évidence une expression constitutive des gènes TSK3, TSK6 et TSK11. Tandis que les gènes TSK1 et TSK16 sont exprimés préférentiellement dans les racines. Les analyses par PCR semi-quantitative sur des plantules de blé à différents stades de développement, ont mis en évidence une surexpression du gène TSK11 au moment de la floraison. Ce qui suggère que TSK11 est probablement un équivalent fonctionnel d’ASK1 chez Arabidopsis thaliana. / The selective degradation of proteins is an important means of regulating gene expression and plays crucial roles in the control of various cellular processes. The Ubiquitin (Ub)–Proteasome System (UPS) is the principal non-lysosomal proteolytic pathway in eukaryotic cells and is required for the degradation of key regulatory proteins. Ubiquitin is a 76-residue protein that can be attached covalently to target proteins through an enzymatic conjugation cascade involving three enzymes denoted, E1, E2 and E3.The SCF complex is a type of ubiquitin-protein ligase (E3) that acts as the specific factor responsible for substrate recognition and ubiquitination. Some polyubiquitinated proteins are then targeted to the 26S proteasome for degradation. The SCF complex consists of four components including SKP1, Cullin1, Rbx1 and a large gene family of F-box proteins. Twenty one SKP1-related genes have been described in the Arabidopsis genome and some of these genes have been analyzed genetically. By contrast, little is known about the function and structure of SKP1 homologues in wheat. Some of the Triticum SKP1-related protein (TSKs) have been characterized in this study. Five complete sequences of SKP1 (TSK1, TSK3, TSK6, TSK11 and TSK16), five F-box (ZTL, ATFBL5, EBF, TIR1 and ABA-T), one Cullin1 and one Rbx, were successfully cloned and biochemically characterized. Yeast two-hybrid analysis showed that TSK1 and TSK3 are capable of interacting with different F-box proteins. Furthermore, TSK11 contains an additional domain that changed its interaction capabilities. In vitro analysis using a chimeric protein showed that this additional domain could modify the interaction between a SKP-like protein and two F-box proteins. Expression analyses revealed that TSK1 and TSK16 were expressed predominantly in roots. While, TSK3, TSK6 and TSK11 were expressed in several wheat organs. In addition, the TSK11 was up-regulated in the leaves at the flowering stage.
30

Analise das proteinas Ki-1/57 e PRMT1 : identificação, mapeamento e caracterização funcional da interação com outras proteinas / Analysis of the proteins Ki-1/57 and PRMT1: identification, mapping and characterization of the interaction with other proteins

Passos, Dario Oliveira dos 31 August 2006 (has links)
Orientador: Jorg Kobarg / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-07T08:03:42Z (GMT). No. of bitstreams: 1 Passos_DarioOliveirados_D.pdf: 4831709 bytes, checksum: 0aa3e031d4e82dc447636416b68401e8 (MD5) Previous issue date: 2006 / Resumo: A proteína Ki-1/57 que é encontrada tanto no núcleo quanto no citoplasma está associada com atividade de proteína quinase serina/treonina e é fosforilada nestes resíduos após ativação celular. Neste trabalho verificamos que Ki-1/57 interage com a proteína Chromatin-Helicase-DNA-binding domain 3 (CHD3) e com a proteína adaptadora/sinalizadora RACK1 no núcleo. Pelo sistema do duplo híbrido de levedura (SDHL) a proteína arginina metiltransferase 1 (PRMT1) foi selecionada como outra proteína de interação. A PRMT1 integra uma família representada por nove enzimas humanas que catalisam reações de metilação em resíduos de arginina. Em seguida, usando agora a PRMT1 como isca - no SDHL - identificamos as proteínas Ki-1/57 e hnRNPQ, juntamente com outras 13. A maioria delas contêm motivos ¿RGG-box¿ em suas seqüências de aminoácidos, que são conhecidos alvos para metilação. Posteriormente verificamos que Ki-1/57 e seu provável parálogo CGI-55 conservam dois motivos ¿RGG/RXR-box¿ e que são substratos in vitro para a metilação de argininas pela PRMT1. Estudos de mapeamento mostraram que todos os fragmentos contendo o motivo ¿RGG/RXR-box¿ interagem com a PRMT1 e são alvos à metilação in vitro. Ki-1/57 endógena, imunoprecipitada de células L540, mostrou ser metilada in vivo, além de ser um alvo a metilação pela PRMT1 in vitro, somente quando as células são previamente tratadas com o inibidor da metilação Adox. Tratamento das células Hela com o inibidor da metilação (Adox) causa desaparecimento da imuno-marcação citoplasmática de Ki-1/57 e relativa redistribuição do parálogo CGI-55 para o citosol. Assim, pode ser especulado que a metilação destas proteínas deve ser um evento importante para suas localizações subcelulares e conseqüentemente para suas funções. Em resumo, nossos dados sugerem que o SDHL é um método efetivo na identificação de novos substratos celulares para a PRMT1 e poderia ser estendido para a identificação e caracterização de novos substratos para os outros integrantes da família das PRMTs humanas / Abstract: The protein Ki-1/57 that is found both in the cytoplasm and nucleus is associated with serine/threonine protein kinase activity and gets phosphorylated on serine and threonine residues upon cellular activation. We demonstrated that Ki-1/57 interacts with the Chromatin-Helicase-DNA-binding domain protein 3 (CHD3) and with the adaptor/signaling protein RACK1 in the nucleus. By utilizing the yeast two-hybrid system (YTHS), we were further able to find the protein arginine-methylatranseferase-1 (PRMT1) as another interacting protein. PRMT1 is a member of the family constituted by 9 human enzymes that catalyze methylation reactions on arginine residues. Afterwards, by using PRMT1 as bait in the YTHS we identified both Ki-1/57 and NSAP1 as interacting proteins, along with 13 other proteins. The majority of them present RGG-box clusters in their amino acid sequences, which are known to be targets for arginine methylation. We further found that Ki-1/57 and its putative paralogue CGI-55 have two RGG/RXR-box clusters conserved between them and that they are substrates for arginine-methylation by PRMT1 in vitro. In mapping studies, we observed that all Ki-1/57 protein fragments containing the RGG/RXRbox clusters interact with PRMT1 and are targets for methylation in vitro. Endogenous cellular Ki-1/57 seems to be methylated in vivo and is a target for methylation by PRMT1 in vitro, only when cells have been previously treated with the methylation inhibitor Adox. Treatment of Hela cells with the inhibitor of methylation (Adox) causes the disappearance of the immuno-staining of Ki-1/57 in the cytoplasm and a relative redistribution of the paralogue CGI-55 to the cytosol. It can therefore be speculated that the methylation of these proteins is important for their sub-cellular localization and in consequence for their function. In summary our data suggest that the YTHS is an effective method for the identification of novel cellular PRMT substrates and could be extended for the identification and characterization of novel substrates to the other components of the human PRMT1 family / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

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