173 results on '"Benedetti, Celso Eduardo"'
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2. Xanthomonas citri subsp. citri: host interaction and control strategies
- Author
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Martins, Paula Maria Moreira, de Oliveira Andrade, Maxuel, Benedetti, Celso Eduardo, and de Souza, Alessandra Alves
- Published
- 2020
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3. The expansin gene CsLIEXP1 is a direct target of CsLOB1 in citrus
- Author
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de Souza-Neto, Reinaldo Rodrigues, primary, N.C. Vasconcelos, Fernanda, additional, Teper, Doron, additional, Carvalho, Isis Gabriela Barbosa, additional, Takita, Marco Aurelio, additional, Benedetti, Celso Eduardo, additional, Wang, Nian, additional, and de Souza, Alessandra Alves, additional
- Published
- 2023
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4. Structure-function relationship of a citrus salicylate methylesterase and role of salicylic acid in citrus canker resistance
- Author
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Lima Silva, Caio Cesar de, Shimo, Hugo Massayoshi, de Felício, Rafael, Mercaldi, Gustavo Fernando, Rocco, Silvana Aparecida, and Benedetti, Celso Eduardo
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- 2019
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- View/download PDF
5. Suppression of citrus canker disease mediated by flagellin perception
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Andrade, Maxuel de Oliveira, primary, da Silva, Jaqueline Cristina, additional, Soprano, Adriana Santos, additional, Shimo, Hugo Massayoshi, additional, Leme, Adriana Franco Paes, additional, and Benedetti, Celso Eduardo, additional
- Published
- 2023
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- View/download PDF
6. Structural dynamics of SARS-CoV-2 nucleocapsid protein induced by RNA binding
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Ribeiro-Filho, Helder Veras, primary, Jara, Gabriel Ernesto, additional, Batista, Fernanda Aparecida Heleno, additional, Schleder, Gabriel Ravanhani, additional, Costa Tonoli, Celisa Caldana, additional, Soprano, Adriana Santos, additional, Guimarães, Samuel Leite, additional, Borges, Antonio Carlos, additional, Cassago, Alexandre, additional, Bajgelman, Marcio Chaim, additional, Marques, Rafael Elias, additional, Trivella, Daniela Barretto Barbosa, additional, Franchini, Kleber Gomes, additional, Figueira, Ana Carolina Migliorini, additional, Benedetti, Celso Eduardo, additional, and Lopes-de-Oliveira, Paulo Sergio, additional
- Published
- 2022
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7. An Extracytoplasmic Function Sigma Factor Required for Full Virulence in Xanthomonas citri pv. citri
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Lima, Lídia dos Passos, primary, Pereira, Juliana Biar, additional, Flores, Anthony Jhoao Fasabi, additional, Lorenzetti, Alan Péricles Rodrigues, additional, Boechat, Ana Laura, additional, Pereda, Maria Claudia, additional, Gualtieri, Sophia, additional, do Prado, Daniele Ferreira, additional, Rocha, Diego, additional, Ceseti, Lucas de Moraes, additional, Baldini, Regina Lúcia, additional, Farah, Chuck S., additional, Koide, Tie, additional, Benedetti, Celso Eduardo, additional, and Alvarez-Martinez, Cristina E., additional
- Published
- 2022
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8. Structural dynamics of SARS-CoV-2 nucleocapsid protein induced by RNA binding
- Author
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Filho, Helder Veras Ribeiro, primary, Jara, Gabriel Ernesto, additional, Batista, Fernanda Aparecida Heleno, additional, Schleder, Gabriel Ravanhani, additional, Tonoli, Celisa Caldana, additional, Soprano, Adriana Santos, additional, Guimarães, Samuel Leite, additional, Borges, Antonio Carlos, additional, Cassago, Alexandre, additional, Bajgelman, Marcio Chaim, additional, Marques, Rafael Elias, additional, Trivella, Daniela Barreto Barbosa, additional, Franchini, Kleber Gomes, additional, Figueira, Ana Carolina Migliorini, additional, Benedetti, Celso Eduardo, additional, and de Oliveira, Paulo Sergio Lopes, additional
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- 2021
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9. COI1 affects myrosinase activity and controls the expression of two flower-specific myrosinase-binding protein homologues in Arabidopsis
- Author
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Capella, Adriana Natalício, Menossi, Marcelo, Arruda, Paulo, and Benedetti, Celso Eduardo
- Published
- 2001
10. Molecular basis for diaryldiamine selectivity and competition with tRNA in a type 2 methionyl-tRNA synthetase from a Gram-negative bacterium
- Author
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Mercaldi, Gustavo Fernando, primary, Andrade, Maxuel de Oliveira, additional, Zanella, Jackeline de Lima, additional, Cordeiro, Artur Torres, additional, and Benedetti, Celso Eduardo, additional
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- 2021
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11. An Arabidopsis gene induced by wounding functionally homologous to flavoprotein oxidoreductases
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Costa, Cínthia Losano, Arruda, Paulo, and Benedetti, Celso Eduardo
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- 2000
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12. Altering the expression of the Chlorophyllase gene ATHCOR1 in transgenic Arabidopsis caused changes in the Chlorophyll-to-Chlorophyllide ratio (1)
- Author
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Benedetti, Celso Eduardo and Arruda, Paulo
- Subjects
Arabidopsis -- Genetic aspects ,Genetically modified plants -- Research ,Chlorophyll -- Genetic aspects ,Biological sciences ,Science and technology - Published
- 2002
13. The MAF1 Phosphoregulatory Region Controls MAF1 Interaction with the RNA Polymerase III C34 Subunit and Transcriptional Repression in Plants
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Oliveira Andrade, Maxuel, primary, Sforça, Mauricio Luis, additional, Batista, Fernanda Aparecida Heleno, additional, Figueira, Ana Carolina Migliorini, additional, and Benedetti, Celso Eduardo, additional
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- 2020
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14. Role of theCitrus sinensisRNA deadenylase CsCAF1 in citrus canker resistance
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Shimo, Hugo Massayoshi, primary, Terassi, Carolina, additional, Lima Silva, Caio Cesar, additional, Zanella, Jackeline de Lima, additional, Mercaldi, Gustavo Fernando, additional, Rocco, Silvana Aparecida, additional, and Benedetti, Celso Eduardo, additional
- Published
- 2019
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15. Síntese e caracterização de inibidores da deadenilase CAF1 (CNOT7) humana
- Author
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Terassi, Carolina, primary, Benedetti, Celso Eduardo, primary, Shimo, Hugo M., primary, Nascimento, Thiago A. do, primary, and Rocco, Silvana A., primary
- Published
- 2018
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16. MAF1 Phosphoregulatory Region Controls MAF1 Interaction with the RNA Polymerase III C34 Subunit and Transcriptional Repression in Plants.
- Author
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Andrade, Maxuel Oliveira, Sforça, Mauricio Luis, Batista, Fernanda Aparecida Heleno, Figueira, Ana Carolina Migliorini, and Benedetti, Celso Eduardo
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- 2020
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17. Regulation of tRNA biogenesis in plants and its link to plant growth and response to pathogens
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Soprano, Adriana Santos, primary, Smetana, Juliana Helena Costa, additional, and Benedetti, Celso Eduardo, additional
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- 2018
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18. BigR is a sulfide sensor that regulates a sulfur transferase/dioxygenase required for aerobic respiration of plant bacteria under sulfide stress
- Author
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de Lira, Nayara Patricia Vieira, primary, Pauletti, Bianca Alves, additional, Marques, Ana Carolina, additional, Perez, Carlos Alberto, additional, Caserta, Raquel, additional, de Souza, Alessandra Alves, additional, Vercesi, Aníbal Eugênio, additional, Paes Leme, Adriana Franco, additional, and Benedetti, Celso Eduardo, additional
- Published
- 2018
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19. Crystal structure and regulation of the citrus pol III repressor MAF1 by auxin and phosphorylation
- Author
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Sao Paulo Research Foundation, Conselho Nacional de Desenvolvimento Científico e Tecnológico (Brasil), Fundação de Amparo à Pesquisa do Estado de São Paulo, Santos Soprano, Adriana, Oliveira de Giuseppe, Priscila, Massayoshi Shimo, Hugo, Brenelli Lima, Tatiani, Heleno Batista, Fernanda Aparecida, Lima Righetto, Germanna, Carvalho Pereira, José Geraldo de, Campos Granato, Daniela, Ziem Nascimento, Andrey Fabricio, Cesar Gozzo, Fabio, Lopesde Oliveira, Paulo Sérgio, Migliorini Figueira, Ana Carolina, Costa Smetana, Juliana Helena, Franco Paes Leme, Adriana, Murakami, Mario Tyago, Benedetti, Celso Eduardo, Sao Paulo Research Foundation, Conselho Nacional de Desenvolvimento Científico e Tecnológico (Brasil), Fundação de Amparo à Pesquisa do Estado de São Paulo, Santos Soprano, Adriana, Oliveira de Giuseppe, Priscila, Massayoshi Shimo, Hugo, Brenelli Lima, Tatiani, Heleno Batista, Fernanda Aparecida, Lima Righetto, Germanna, Carvalho Pereira, José Geraldo de, Campos Granato, Daniela, Ziem Nascimento, Andrey Fabricio, Cesar Gozzo, Fabio, Lopesde Oliveira, Paulo Sérgio, Migliorini Figueira, Ana Carolina, Costa Smetana, Juliana Helena, Franco Paes Leme, Adriana, Murakami, Mario Tyago, and Benedetti, Celso Eduardo
- Abstract
MAF1 is the main RNA polymerase (Pol) III repressor that controls cell growth in eukaryotes. The Citrus ortholog, CsMAF1, was shown to restrict cell growth in citrus canker disease but its role in plant development and disease is still unclear. We solved the crystal structure of the globular core of CsMAF1, which reveals additional structural elements compared with the previously available structure of hMAF1, and explored the dynamics of its flexible regions not present in the structure. CsMAF1 accumulated in the nucleolus upon leaf excision, and this translocation was inhibited by auxin and by mutation of the PKA phosphorylation site, S45, to aspartate. Additionally, mTOR phosphorylated recombinant CsMAF1 and the mTOR inhibitor AZD8055 blocked canker formation in normal but not CsMAF1-silenced plants. These results indicate that the role of TOR on cell growth induced by Xanthomonas citri depends on CsMAF1 and that auxin controls CsMAF1 interaction with Pol III in citrus
- Published
- 2017
20. Additive roles of PthAs in bacterial growth and pathogenicity associated with nucleotide polymorphisms in effector‐binding elements of citrus canker susceptibility genes
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Abe, Valeria Yukari and Benedetti, Celso Eduardo
- Subjects
Citrus ,Polymorphism, Genetic ,Xanthomonas ,Base Sequence ,Nucleotides ,Ficusin ,food and beverages ,Original Articles ,Genes, Plant ,Bacterial Proteins ,Host-Pathogen Interactions ,Disease Susceptibility ,Promoter Regions, Genetic ,Gene Deletion ,Plant Diseases ,Plant Proteins ,Protein Binding - Abstract
Citrus canker, caused by Xanthomonas citri, affects most commercial citrus varieties. All X. citri strains possess at least one transcription activator-like effector of the PthA family that activates host disease susceptibility (S) genes. The X. citri strain 306 encodes four PthA effectors; nevertheless, only PthA4 is known to elicit cankers on citrus. As none of the PthAs act as avirulence factors on citrus, we hypothesized that PthAs 1-3 might also contribute to pathogenicity on certain hosts. Here, we show that, although PthA4 is indispensable for canker formation in six Brazilian citrus varieties, PthAs 1 and 3 contribute to canker development in 'Pera' sweet orange, but not in 'Tahiti' lemon. Deletions in two or more pthA genes reduce bacterial growth in planta more pronouncedly than single deletions, suggesting an additive role of PthAs in pathogenicity and bacterial fitness. The contribution of PthAs 1 and 3 in canker formation in 'Pera' plants does not correlate with the activation of the canker S gene, LOB1 (LATERAL ORGAN BOUNDARIES 1), but with the induction of other PthA targets, including LOB2 and citrus dioxygenase (DIOX). LOB1, LOB2 and DIOX show differential PthA-dependent expression between 'Pera' and 'Tahiti' plants that appears to be associated with nucleotide polymorphisms found at or near PthA-binding sites. We also present evidence that LOB1 activation alone is not sufficient to elicit cankers on citrus, and that DIOX acts as a canker S gene in 'Pera', but not 'Tahiti', plants. Our results suggest that the activation of multiple S genes, such as LOB1 and DIOX, is necessary for full canker development.
- Published
- 2016
21. Crystal Structure and Regulation of the Citrus Pol III Repressor MAF1 by Auxin and Phosphorylation
- Author
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Soprano, Adriana Santos, primary, Giuseppe, Priscila Oliveira de, additional, Shimo, Hugo Massayoshi, additional, Lima, Tatiani Brenelli, additional, Batista, Fernanda Aparecida Heleno, additional, Righetto, Germanna Lima, additional, Pereira, José Geraldo de Carvalho, additional, Granato, Daniela Campos, additional, Nascimento, Andrey Fabricio Ziem, additional, Gozzo, Fabio Cesar, additional, de Oliveira, Paulo Sérgio Lopes, additional, Figueira, Ana Carolina Migliorini, additional, Smetana, Juliana Helena Costa, additional, Paes Leme, Adriana Franco, additional, Murakami, Mario Tyago, additional, and Benedetti, Celso Eduardo, additional
- Published
- 2017
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22. Role of the Citrus sinensis RNA deadenylase CsCAF1 in citrus canker resistance.
- Author
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Shimo, Hugo Massayoshi, Terassi, Carolina, Lima Silva, Caio Cesar, Zanella, Jackeline de Lima, Mercaldi, Gustavo Fernando, Rocco, Silvana Aparecida, and Benedetti, Celso Eduardo
- Subjects
CITRUS canker ,CANKER (Plant disease) ,ORANGES ,XANTHOMONAS campestris ,RNA ,MESSENGER RNA ,GENE expression - Abstract
Summary: Poly(A) tail shortening is a critical step in messenger RNA (mRNA) decay and control of gene expression. The carbon catabolite repressor 4 (CCR4)‐associated factor 1 (CAF1) component of the CCR4‐NOT deadenylase complex plays an essential role in mRNA deadenylation in most eukaryotes. However, while CAF1 has been extensively investigated in yeast and animals, its role in plants remains largely unknown. Here, we show that the Citrus sinensis CAF1 (CsCAF1) is a magnesium‐dependent deadenylase implicated in resistance against the citrus canker bacteria Xanthomonas citri. CsCAF1 interacted with proteins of the CCR4‐NOT complex, including CsVIP2, a NOT2 homologue, translin‐associated factor X (CsTRAX) and the poly(A)‐binding proteins CsPABPN and CsPABPC. CsCAF1 also interacted with PthA4, the main X. citri effector required for citrus canker elicitation. We also present evidence suggesting that PthA4 inhibits CsCAF1 deadenylase activity in vitro and stabilizes the mRNA encoded by the citrus canker susceptibility gene CsLOB1, which is transcriptionally activated by PthA4 during canker formation. Moreover, we show that an inhibitor of CsCAF1 deadenylase activity significantly enhanced canker development, despite causing a reduction in PthA4‐dependent CsLOB1 transcription. These results thus link CsCAF1 with canker development and PthA4‐dependent transcription in citrus plants. [ABSTRACT FROM AUTHOR]
- Published
- 2019
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23. Additive roles of PthAs in bacterial growth and pathogenicity associated with nucleotide polymorphisms in effector-binding elements of citrus canker susceptibility genes
- Author
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Abe, Valeria Yukari, primary and Benedetti, Celso Eduardo, additional
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- 2016
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24. Citrus MAF1, a Repressor of RNA Polymerase III, Binds the Xanthomonas citri Canker Elicitor PthA4 and Suppresses Citrus Canker Development1
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Soprano, Adriana Santos, Abe, Valeria Yukari, Smetana, Juliana Helena Costa, and Benedetti, Celso Eduardo
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Citrus ,Xanthomonas ,Molecular Sequence Data ,food and beverages ,RNA Polymerase III ,Saccharomyces cerevisiae ,Genes, Development, and Evolution ,Repressor Proteins ,Humans ,Amino Acid Sequence ,Sequence Alignment ,Conserved Sequence ,Phylogeny ,Plant Diseases ,Plant Proteins - Abstract
Transcription activator-like (TAL) effectors from Xanthomonas species pathogens act as transcription factors in plant cells; however, how TAL effectors activate host transcription is unknown. We found previously that TAL effectors of the citrus canker pathogen Xanthomonas citri, known as PthAs, bind the carboxyl-terminal domain of the sweet orange (Citrus sinensis) RNA polymerase II (Pol II) and inhibit the activity of CsCYP, a cyclophilin associated with the carboxyl-terminal domain of the citrus RNA Pol II that functions as a negative regulator of cell growth. Here, we show that PthA4 specifically interacted with the sweet orange MAF1 (CsMAF1) protein, an RNA polymerase III (Pol III) repressor that controls ribosome biogenesis and cell growth in yeast (Saccharomyces cerevisiae) and human. CsMAF1 bound the human RNA Pol III and rescued the yeast maf1 mutant by repressing tRNA(His) transcription. The expression of PthA4 in the maf1 mutant slightly restored tRNA(His) synthesis, indicating that PthA4 counteracts CsMAF1 activity. In addition, we show that sweet orange RNA interference plants with reduced CsMAF1 levels displayed a dramatic increase in tRNA transcription and a marked phenotype of cell proliferation during canker formation. Conversely, CsMAF1 overexpression was detrimental to seedling growth, inhibited tRNA synthesis, and attenuated canker development. Furthermore, we found that PthA4 is required to elicit cankers in sweet orange leaves and that depletion of CsMAF1 in X. citri-infected tissues correlates with the development of hyperplastic lesions and the presence of PthA4. Considering that CsMAF1 and CsCYP function as canker suppressors in sweet orange, our data indicate that TAL effectors from X. citri target negative regulators of RNA Pol II and Pol III to coordinately increase the transcription of host genes involved in ribosome biogenesis and cell proliferation.
- Published
- 2013
25. Correction: The TAL Effector PthA4 Interacts with Nuclear Factors Involved in RNA-Dependent Processes Including a HMG Protein That Selectively Binds Poly(U) RNA
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de Souza, Tiago Antonio, primary, Soprano, Adriana Santos, additional, de Lira, Nayara Patricia Vieira, additional, Quaresma, Alexandre José Christino, additional, Pauletti, Bianca Alves, additional, Leme, Adriana Franco Paes, additional, and Benedetti, Celso Eduardo, additional
- Published
- 2015
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26. Structure and Mechanism of Dimer–Monomer Transition of a Plant Poly(A)-Binding Protein upon RNA Interaction: Insights into Its Poly(A) Tail Assembly
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Domingues, Mariane Noronha, primary, Sforça, Mauricio Luis, additional, Soprano, Adriana Santos, additional, Lee, Jack, additional, de Souza, Tatiana de Arruda Campos Brasil, additional, Cassago, Alexandre, additional, Portugal, Rodrigo Villares, additional, de Mattos Zeri, Ana Carolina, additional, Murakami, Mario Tyago, additional, Sadanandom, Ari, additional, de Oliveira, Paulo Sergio Lopes, additional, and Benedetti, Celso Eduardo, additional
- Published
- 2015
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27. The Xanthomonas citri effector protein PthA interacts with citrus proteins involved in nuclear transport, protein folding and ubiquitination associated with DNA repair
- Author
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Benedetti, Celso Eduardo
- Subjects
CANCRO (DOENÇA DE PLANTA) - Published
- 2010
28. Crystallization and preliminary X-ray analysis of BigR, a transcription repressor from Xylella fastidiosa involved in biofilm formation
- Author
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Barbosa, Rosicler Lazaro, 1978, Rinaldi, Fabio Cupri, 1980, Guimarães, Beatriz Gomes, Benedetti, Celso Eduardo, 1964, and UNIVERSIDADE ESTADUAL DE CAMPINAS
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Xylella fastidiosa ,Biofilms ,Biofilm growth-associated repressor ,Artigo original ,Transcription repressors ,Biofilmes - Abstract
Agradecimentos: This work was supported by FAPESP grants (03/08316-5, Smolbnet 00/10266-8 and Cepid 98/14138-2). RLB and FCR received PhD fellowships from FAPESP (02/12329-2 and 03/12875-0). CEB received a fellowship from CnPq Abstract: BigR (biofilm growth-associated repressor) is a novel repressor protein that regulates the transcription of an operon implicated in biofilm growth in both Xylella fastidiosa and Agrobacterium tumefaciens. This protein binds to a palindromic TA-rich element located in the promoter of the BigR operon and strongly represses transcription of the operon. BigR contains a helix-turn-helix (HTH) domain that is found in some members of the ArsR/SmtB family of metal sensors, which control metal resistance in bacteria. Although functional studies have suggested that BigR does not act as a metal sensor, the presence of two cysteines and a methionine in its primary structure raised the possibility of BigR being a metal-ligand protein. In order to gain new insights into the protein structure and its possible interaction with a metal ion or effector ligand, BigR from X. fastidiosa was crystallized in native and selenomethionine ( SeMet) labelled forms using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected from native and SeMet crystals to resolutions of 1.95 and 2.2 angstrom, respectively. Both crystals belong to space group P321 and contain one molecule per asymmetric unit FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP Fechado
- Published
- 2007
29. Increased Resistance Against Citrus Canker Mediated by a Citrus Mitogen-Activated Protein Kinase
- Author
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de Oliveira, Maria Luiza Peixoto, primary, de Lima Silva, Caio Cesar, additional, Abe, Valéria Yukari, additional, Costa, Marcio Gilberto Cardoso, additional, Cernadas, Raúl Andrés, additional, and Benedetti, Celso Eduardo, additional
- Published
- 2013
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30. Citrus MAF1, a Repressor of RNA Polymerase III, Binds the Xanthomonas citri Canker Elicitor PthA4 and Suppresses Citrus Canker Development
- Author
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Soprano, Adriana Santos, primary, Abe, Valeria Yukari, additional, Smetana, Juliana Helena Costa, additional, and Benedetti, Celso Eduardo, additional
- Published
- 2013
- Full Text
- View/download PDF
31. Correction: TAL Effectors Target the C-Terminal Domain of RNA Polymerase II (CTD) by Inhibiting the Prolyl-Isomerase Activity of a CTD-Associated Cyclophilin
- Author
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Domingues, Mariane Noronha, primary, de Campos, Bruna Medeia, additional, de Oliveira, Maria Luiza Peixoto, additional, de Mello, Uli Quirino, additional, and Benedetti, Celso Eduardo, additional
- Published
- 2013
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32. A Redox 2-Cys Mechanism Regulates the Catalytic Activity of Divergent Cyclophilins
- Author
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Campos, Bruna Medéia, primary, Sforça, Mauricio Luis, additional, Ambrosio, Andre Luis Berteli, additional, Domingues, Mariane Noronha, additional, Brasil de Souza, Tatiana de Arruda Campos, additional, Barbosa, João Alexandre Ribeiro Gonçalvez, additional, Leme, Adriana Franco Paes, additional, Perez, Carlos Alberto, additional, Whittaker, Sara Britt-Marie, additional, Murakami, Mario Tyago, additional, Zeri, Ana Carolina de Matos, additional, and Benedetti, Celso Eduardo, additional
- Published
- 2013
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33. TAL Effectors Target the C-Terminal Domain of RNA Polymerase II (CTD) by Inhibiting the Prolyl-Isomerase Activity of a CTD-Associated Cyclophilin
- Author
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Domingues, Mariane Noronha, primary, de Campos, Bruna Medeia, additional, de Oliveira, Maria Luiza Peixoto, additional, de Mello, Uli Quirino, additional, and Benedetti, Celso Eduardo, additional
- Published
- 2012
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34. The TAL Effector PthA4 Interacts with Nuclear Factors Involved in RNA-Dependent Processes Including a HMG Protein That Selectively Binds Poly(U) RNA
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de Souza, Tiago Antonio, primary, Soprano, Adriana Santos, additional, Lira, Nayara Patricia Vieira de, additional, Quaresma, Alexandre José Christino, additional, Pauletti, Bianca Alves, additional, Leme, Adriana Franco Paes, additional, and Benedetti, Celso Eduardo, additional
- Published
- 2012
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35. The repeat domain of the type III effector protein PthA shows a TPR‐like structure and undergoes conformational changes upon DNA interaction
- Author
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Murakami, Mário Tyago, primary, Sforça, Mauricio Luis, additional, Neves, Jorge Luiz, additional, Paiva, Joice Helena, additional, Domingues, Mariane Noronha, additional, Pereira, André Luiz Araujo, additional, de Mattos Zeri, Ana Carolina, additional, and Benedetti, Celso Eduardo, additional
- Published
- 2010
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36. The Xanthomonas citri effector protein PthA interacts with citrus proteins involved in nuclear transport, protein folding and ubiquitination associated with DNA repair
- Author
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DOMINGUES, MARIANE NORONHA, primary, DE SOUZA, TIAGO ANTONIO, additional, CERNADAS, RAÚL ANDRÉS, additional, DE OLIVEIRA, MARIA LUIZA PEIXOTO, additional, DOCENA, CÁSSIA, additional, FARAH, CHUCK SHAKER, additional, and BENEDETTI, CELSO EDUARDO, additional
- Published
- 2010
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37. Role of auxin and gibberellin in citrus canker development and in the transcriptional control of cell-wall remodeling genes modulated by Xanthomonas axonopodis pv. citri
- Author
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Cernadas, Raúl Andrés, primary and Benedetti, Celso Eduardo, additional
- Published
- 2009
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38. Transcriptional analysis of the sweet orange interaction with the citrus canker pathogensXanthomonas axonopodispv.citriandXanthomonas axonopodispv.aurantifolii
- Author
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CERNADAS, RAÚL ANDRÉS, primary, CAMILLO, LUCIANA RODRIGUES, additional, and BENEDETTI, CELSO EDUARDO, additional
- Published
- 2008
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39. The solution structure of the outer membrane lipoprotein OmlA from Xanthomonas axonopodis pv. citri reveals a protein fold implicated in protein-protein interaction
- Author
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Vanini, Marina Marques Teixeira, primary, Spisni, Alberto, additional, Sforça, Maurício Luis, additional, Pertinhez, Thelma Aguiar, additional, and Benedetti, Celso Eduardo, additional
- Published
- 2008
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- View/download PDF
40. Crystallization and preliminary X-ray analysis of BigR, a transcription repressor fromXylella fastidiosainvolved in biofilm formation
- Author
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Barbosa, Rosicler Lázaro, primary, Rinaldi, Fábio Cupri, additional, Guimarães, Beatriz Gomes, additional, and Benedetti, Celso Eduardo, additional
- Published
- 2007
- Full Text
- View/download PDF
41. Plasmodium falciparum histidine-rich protein II binds to actin, phosphatidylinositol 4,5-bisphosphate and erythrocyte ghosts in a pH-dependent manner and undergoes coil-to-helix transitions in anionic micelles
- Author
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Benedetti, Celso Eduardo, primary, Kobarg, Jörg, additional, Pertinhez, Thelma Aguiar, additional, Gatti, Reynaldo Mascagni, additional, Souza, Osmar Norberto de, additional, Spisni, Alberto, additional, and Meneghini, Rogério, additional
- Published
- 2003
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42. Altering the Expression of the Chlorophyllase GeneATHCOR1 in Transgenic Arabidopsis Caused Changes in the Chlorophyll-to-Chlorophyllide Ratio
- Author
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Benedetti, Celso Eduardo, primary and Arruda, Paulo, additional
- Published
- 2002
- Full Text
- View/download PDF
43. A Redox 2-Cys Mechanism Regulates the Catalytic Activity of Divergent Cyclophilins.
- Author
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Medéia Campos, Bruna, Sforça, Mauricio Luis, Berteli Ambrosio, Andre Luis, Domingues, Mariane Noronha, de Arruda Campos Brasil de Souza, Tatiana, Gonçalvez Barbosa, João Alexandre Ribeiro, Paes Leme, Adriana Franco, Perez, Carlos Alberto, Whittaker, Sara Britt-Marie, Murakami, Mario Tyago, de Matos Zeri, Ana Carolina, and Benedetti, Celso Eduardo
- Subjects
ORANGES ,CYCLOPHILINS ,XANTHOMONAS campestris ,GENETIC research ,PLANT genetics ,IMMUNOPHILINS - Abstract
The citrus (Citrus sinensis) cyclophilin CsCyp is a target of the Xanthomonas citri transcription activator-like effector PthA, required to elicit cankers on citrus. CsCyp binds the citrus thioredoxin CsTdx and the carboxyl-terminal domain of RNA polymerase II and is a divergent cyclophilin that carries the additional loop KSGKPLH, invariable cysteine (Cys) residues Cys-40 and Cys-168, and the conserved glutamate (Glu) Glu-83. Despite the suggested roles in ATP and metal binding, the functions of these unique structural elements remain unknown. Here, we show that the conserved Cys residues form a disulfide bond that inactivates the enzyme, whereas Glu-83, which belongs to the catalytic loop and is also critical for enzyme activity, is anchored to the divergent loop to maintain the active site open. In addition, we demonstrate that Cys-40 and Cys-168 are required for the interaction with CsTdx and that CsCyp binds the citrus carboxyl-terminal domain of RNA polymerase II YSPSAP repeat. Our data support a model where formation of the Cys-40-Cys-168 disulfide bond induces a colfformational change that disrupts the interaction of the divergent and catalytic loops, via Glu-83, causing the active site to close. This suggests a new type of allosteric regulation in divergent cyclophilins, involving disulfide bond formation and a loop-displacement mechanism. [ABSTRACT FROM AUTHOR]
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- 2013
- Full Text
- View/download PDF
44. Transcriptional analysis of the sweet orange interaction with the citrus canker pathogens Xanthomonas axonopodis pv. citri and Xanthomonas axonopodis pv. aurantifolii.
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CERNADAS, RAÚL ANDRÉS, CAMILLO, LUCIANA RODRIGUES, and BENEDETTI, CELSO EDUARDO
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XANTHOMONAS ,PLANT physiology ,PLANT diseases ,GENE expression ,PLANT cell walls ,PLANT metabolism ,CELL division ,DEVELOPMENTAL biology ,NITROGEN - Abstract
Xanthomonas axonopodis pv. citri (Xac) and Xanthomonas axonopodis pv. aurantifolii pathotype C (Xaa) are responsible for citrus canker disease; however, while Xac causes canker on all citrus varieties, Xaa is restricted to Mexican lime, and in sweet oranges it triggers a defence response. To gain insights into the differential pathogenicity exhibited by Xac and Xaa and to survey the early molecular events leading to canker development, a detailed transcriptional analysis of sweet orange plants infected with the pathogens was performed. Using differential display, suppressed subtractive hybridization and microarrays, we identified changes in transcript levels in approximately 2.0% of the ~32 000 citrus genes examined. Genes with altered expression in response to Xac/Xaa surveyed at 6 and 48 h post-infection (hpi) were associated with cell-wall modifications, cell division and expansion, vesicle trafficking, disease resistance, carbon and nitrogen metabolism, and responses to hormones auxin, gibberellin and ethylene. Most of the genes that were commonly modulated by Xac and Xaa were associated with basal defences triggered by pathogen-associated molecular patterns, including those involved in reactive oxygen species production and lignification. Significantly, we detected clear changes in the transcriptional profiles of defence, cell-wall, vesicle trafficking and cell growth-related genes in Xac-infected leaves between 6 and 48 hpi. This is consistent with the notion that Xac suppresses host defences early during infection and simultaneously changes the physiological status of the host cells, reprogramming them for division and growth. Notably, brefeldin A, an inhibitor of vesicle trafficking, retarded canker development. In contrast, Xaa triggered a mitogen-activated protein kinase signalling pathway involving WRKY and ethylene-responsive transcriptional factors known to activate downstream defence genes. [ABSTRACT FROM AUTHOR]
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- 2008
- Full Text
- View/download PDF
45. Crystallization and preliminary X-ray analysis of BigR, a transcription repressor from Xylella fastidiosa involved in biofilm formation.
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Barbosa, Rosicler Lázaro, Rinaldi, Fábio Cupri, Guimarães, Beatriz Gomes, and Benedetti, Celso Eduardo
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CRYSTALLIZATION ,PROTEINS ,OPERONS ,GENETIC regulation ,GENETIC repressors - Abstract
BigR ( biofilm growth-associated repressor) is a novel repressor protein that regulates the transcription of an operon implicated in biofilm growth in both Xylella fastidiosa and Agrobacterium tumefaciens. This protein binds to a palindromic TA-rich element located in the promoter of the BigR operon and strongly represses transcription of the operon. BigR contains a helix–turn–helix (HTH) domain that is found in some members of the ArsR/SmtB family of metal sensors, which control metal resistance in bacteria. Although functional studies have suggested that BigR does not act as a metal sensor, the presence of two cysteines and a methionine in its primary structure raised the possibility of BigR being a metal-ligand protein. In order to gain new insights into the protein structure and its possible interaction with a metal ion or effector ligand, BigR from X. fastidiosa was crystallized in native and selenomethionine (SeMet) labelled forms using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected from native and SeMet crystals to resolutions of 1.95 and 2.2 Å, respectively. Both crystals belong to space group P321 and contain one molecule per asymmetric unit. [ABSTRACT FROM AUTHOR]
- Published
- 2007
- Full Text
- View/download PDF
46. Expressão diferencial de genes de laranja doce em resposta a infecção por Xanthomonas axonopodis pv. citri e axonopodis pv. aurantifolii
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Cernadas, Raul Andres, Benedetti, Celso Eduardo, 1964, Vincentz, Michel Georges Albert, Machado, Marcos Antonio, Fontes, Elizabeth Pacheco Batista, Maia, Ivan de Godoy, Universidade Estadual de Campinas. Instituto de Biologia, Programa de Pós-Graduação em Genética e Biologia Molecular, and UNIVERSIDADE ESTADUAL DE CAMPINAS
- Subjects
Citrus canker ,Xanthomonas axonopodis pv. citri ,Cancro citrico ,Gene expression ,Expressão gênica - Abstract
Orientador: Celso Eduardo Benedetti Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: A forma mais agressiva de cancro cítrico, cancrose A, é causada pela bactéria Xanthomonas axonopodis pv citri (Xac), que ataca qualquer variedade ou espécie de citros. Além de Xac, Xanthomonas axonopodis pv aurantifolii (Xaa) causa um tipo mais leve da doença, cancrose C, restrita ao limão Galego (Citrus aurantifolii). Na laranja Pêra (Citrus sinensis), Xaa provoca uma resposta de defesa que impede o desenvolvimento dos sintomas do cancro incluindo a ruptura da epiderme que é fundamental à disseminação da bactéria. Neste trabalho, abordamos a patogenicidade diferencial entre Xac e Xaa na laranja Pêra para estudar a expressão gênica na planta associada ao desenvolvimento dos sintomas (hipertrofia e hiperplasia celular) e à resposta de defesa. O projeto apresenta a identificação e caracterização de genes expressos diferencialmente em resposta à infecção por Xac e Xaa responsáveis pelo cancro cítrico. A partir de três estratégias independentes (Display diferencial de PCR, Hibridação subtrativa suprimida e micro arranjos de DNA) detalhou-se o perfil transcricional de folhas de laranja infiltradas com Xaa, Xac ou água como controle. Mais de 120 genes candidatos foram validados através de PCR quantitativo em tempo real ou hibridação Northern. Os genes diferencialmente expressos a 6 ou 48 hs após da infecção (hai) aparecem agrupados em categorias funcionais como: remodelamento de parede celular, divisão e expansão celular, tráfego de vesículas, resposta de defesa, metabolismo de nitrogênio e carbono e sinalização de hormônios como auxina, giberelina e etileno. Inicialmente, tanto Xaa quanto Xac induzem respostas de defesa associadas com ataque de patógenos incluindo a produção de espécies reativas de oxigênio e lignificação da parede celular. Notavelmente, a mudança no perfil transcricional mostra que Xac suprime as defesas da planta entre 6 e 48 hai, ao mesmo tempo em que induz genes relacionados com o metabolismo da parede celular, divisão celular e trafego de vesículas, entre outros. A conseqüência dessa manipulação transcricional no hospedeiro por parte de Xac dá origem à um estado fisiológico típico de expansão celular (hipertrofia). O inibidor de trafego de vesículas, Brefeldina A, atrasou o desenvolvimento dos sintomas do cancro, indicando que essa atividade está mais relacionada com hipertrofia celular do que com respostas de defesa. No perfil transcricional de folhas infectadas com Xaa destaca-se a ativação de uma via de sinalização por proteína quinase ativada por patógeno, envolvendo também fatores de transcrição do tipo WRKY, assim como elementos de resposta a etileno. Tanto Xac quanto Xaa parecem modular a transcrição de genes relacionados com sinalização e transporte de auxinas e biosíntese de giberelina. O tratamento de folhas de laranja com, ácido naftalenoacético (NAA) e ácido giberélico (GA3) demonstrou que esses hormônios induzem a expressão de genes de remodelamento de parede celular, biosíntese de giberelina e sinalização de auxina. Além disso, o inibidor de biosíntese de giberelina (Chlorocoline Chloride) diminuiu significativamente a expressão de genes induzidos por NAA sugerindo que exista uma regulação cruzada entre auxina e giberelina que controla a expressão de genes relacionados com divisão e expansão celular em citros. Finalmente, duas regiões promotoras de genes que codificam proteínas relacionadas com patogenicidade (PR-1 e PR-5) foram isoladas e caracterizadas. Esses genes são fortemente induzidos por Xaa e Xac em 6 e 48 hai. Substancialmente, existem elementos similares com o "upa box" nas regiões promotoras de PR-1 e PR-5. Mediante experimentos de Electrophoretic Mobility Shift Assays (EMSA) demonstrou-se que a proteína PthA4 de Xanthomonas é capaz de ligar-se no "upa box" presente nos promotores de PR-1 e PR-5. Tanto o promotor de PR-1 quanto o de PR-5 dirigem a expressão transitória do gene repórter uidA em N. benthamiana mediante Agro infiltração. Abstract: The most aggressive form of the citrus canker disease is caused by the bacteria Xanthomonas axonopodis pv. citri (Xac), which can infect all commercial varieties or species of citrus. In addition to Xac, Xanthomonas axonopodis pv. aurantifolii (Xaa) causes a weaker form of the disease, known as cancrose C, which is restricted to Mexican Lime (Citrus aurantifolii). In sweet orange (Citrus sinensis), Xaa triggers a defense response that halts the canker-symptoms development including the epidermal rupture, which is essential for bacterial dissemination. In this work, we approached the differential pathogenicity existing between Xac and Xaa in order to survey the host gene expression associated with symptoms development (cellular hypertrophy and hyperplasia) and defense response in sweet orange. The project presents the identification and initial characterization of differentially expressed genes in response to the infection with the citrus canker pathogens, Xac and Xaa. Three independent strategies were conducted in order to have a detailed transcriptional profiling of orange leaves infected with Xaa, Xac or water as mock control (Differential Display PCR, Suppression Subtractive Hybridization and GeneChip Microarrays). More than 120 candidate genes were validated through quantitative real time PCR or Northern blot. The differentially expressed genes at 6 or 48 hours after infection (hai) were grouped into functional categories: cell-wall remodeling, cell division and expansion, vesicle trafficking, carbon and nitrogen metabolism or hormone signaling, auxin, gibberellin and ethylene. Initially, both Xaa and Xac elicit a defense response associated with pathogen attack that includes the production of reactive oxygen species (ROS) and cell-wall lignification. Notably, the changes in the transcriptional profiles show that Xac suppresses the plant defenses between 6 and 48 hai and at the same time it induces genes related to the cell-wall metabolism, cell division and vesicle trafficking among others. The consequence of this host manipulation by Xac originates a physiological state very similar to that of cell expansion (hypertrophy). The vesicle trafficking inhibitor, Brefeldin A, delayed the development of canker symptoms indicating that this activity is more related to the cellular enlargement than to the defense responses. The transcriptional profile of Xaa-infiltrated leaves highlights the activation of amitogen-activated protein kinase (MAPK) signaling pathway related to pathogen attack with the involvement of other components of defense responses like WRKY-transcriptional factors and ethylene-response elements. Both Xac and Xaa appear to modulate the transcription of genes related to auxin signaling and transport and gibberellin biosynthesis. Treatments of orange leaves with 1- naphthylphthalamic acid (NAA) . Resumo: A forma mais agressiva de cancro cítrico, cancrose A, é causada pela bactéria Xanthomonas axonopodis pv citri (Xac), que ataca qualquer variedade ou espécie de citros. Além de Xac, Xanthomonas axonopodis pv aurantifolii (Xaa) causa um tipo mais leve da doença, cancrose C, restrita ao limão Galego (Citrus aurantifolii). Na laranja Pêra (Citrus sinensis), Xaa provoca uma resposta de defesa que impede o desenvolvimento dos sintomas do cancro incluindo a ruptura da epiderme que é fundamental à disseminação da bactéria. Neste trabalho, abordamos a patogenicidade diferencial entre Xac e Xaa na laranja Pêra para estudar a expressão gênica na planta associada ao desenvolvimento dos sintomas (hipertrofia e hiperplasia celular) e à resposta de defesa. O projeto apresenta a identificação e caracterização de genes expressos diferencialmente em resposta à infecção por Xac e Xaa responsáveis pelo cancro cítrico. A partir de três estratégias independentes (Display diferencial de PCR, Hibridação subtrativa suprimida e micro arranjos de DNA) detalhou-se o perfil transcricional de folhas de laranja infiltradas com Xaa, Xac ou água como controle. Mais de 120 genes candidatos foram validados através de PCR quantitativo em tempo real ou hibridação Northern. Os genes diferencialmente expressos a 6 ou 48 hs após da infecção (hai) aparecem agrupados em categorias funcionais como: remodelamento de parede celular, divisão e expansão celular, tráfego de vesículas, resposta de defesa, metabolismo de nitrogênio e carbono e sinalização de hormônios como auxina, giberelina e etileno. Inicialmente, tanto Xaa quanto Xac induzem respostas de defesa associadas com ataque de patógenos incluindo a produção de espécies reativas de oxigênio e lignificação da parede celular. Notavelmente, a mudança no perfil transcricional mostra que Xac suprime as defesas da planta entre 6 e 48 hai, ao mesmo tempo em que induz genes relacionados com o metabolismo da parede celular, divisão celular e trafego de vesículas, entre outros. A conseqüência dessa manipulação transcricional no hospedeiro por parte de Xac dá origem à um estado fisiológico típico de^expansão celular (hipertrofia). O inibidor de trafego de vesículas, Brefeldina A, atrasou o desenvolvimento dos sintomas do cancro, indicando que essa atividade está mais relacionada com hipertrofia celular do que com respostas de defesa.No perfil transcricional de folhas infectadas com Xaa destaca-se a ativação de uma via de sinalização por proteína quinase ativada por patógeno, envolvendo também fatores de transcrição do tipo WRKY, assim como elementos de resposta a etileno. Tanto Xac quanto Xaa parecem modular a transcrição de genes relacionados com sinalização e transporte de auxinas e biosíntese de giberelina. O tratamento de folhas de laranja com, ácido naftalenoacético (NAA) e ácido giberélico (GA3) demonstrou que esses hormônios induzem a expressão de genes de remodelamento de parede celular, biosíntese de giberelina e sinalização de auxína. Além disso, o inibidor de biosíntese de giberelina (Chlorocoline Chloridè) diminuiu significativamente a expressão de genes induzidos por NAA sugerindo que exista uma regulação cruzada entre auxina e giberelina que controla a expressão de genes relacionados com divisão e expansão celular em citros. Finalmente, duas regiões promotoras de genes que codificam proteínas relacionadas com patogenicidade (PR-1 e PR-5) foram isoladas e caracterizadas. Esses genes são fortemente induzidos por Xaa e Xac em 6 e 48 hai. Substancialmente, existem elementos similares com o "upa box" nas regiões promotoras de PR-1 e PR-5. Mediante experimentos de Electrophoretic Mobility Shift Assays (EMSA) demonstrou-se que a proteína PthA4 de Xanthomonas é capaz de ligar-se no "upa box" presente nos promotores de PR-1 e PR-5. Tanto o promotor de PR-1 quanto o de PR-5 dirigem a expressão transitória do gene repórter uidA em N. benthamiana mediante Agro infiltração Abstract: The most aggressive form of the citrus canker disease is caused by the bacteria Xanthomonas axonopodis pv. citri (Xac), which can infect all commercial varieties or species of citrus. In addition to Xac,Xanlhomonas axonopodis pv. aurantifolii (Xaa) causes a weaker form of the disease, known as cancrose C, which is restricted to Mexican Lime {Citrus aurantifolii). In sweet orange {Citrus sinensis), Xaa triggers a defense response that halts the canker-symptoms development including the epidermal rupture, which is essential for bacterial dissemination. In this work, we approached the differential pathogenicity existing between Xac and Xaa in order to survey the host gene expression associated with symptoms development (cellular hypertrophy and hyperplasia) and defense response in sweet orange. The project presents the identification and initial characterization of differentially expressed genes in response to the infection with the citrus canker pathogens, Xac and Xaa. Three independent strategies were conducted in order to have a detailed transcriptional profiling of orange leaves infected with Xaa, Xac or water as mock control (Differential Display PCR, Suppression Subtractive Hybridization and GeneChip Microarrays). More than 120 candidate genes were validated through quantitative real time PCR or Northern blot. The differentially expressed genes at 6 or 48 hours after infection (hai) were grouped into functional categories: cell-wall remodeling, cell division and expansion, vesicle trafficking, carbon and nitrogen metabolism or hormone signaling, auxin, gibberellin and ethylene. Initially, both Xaa and Xac elicit a defense response associated with pathogen attack that includes the production of reactive oxygen species (ROS) and cell-wall lignification. T Notably, the changes in the transcriptional profiles show that Xac suppresses the plant defenses between 6 and 48 hai and at the same time it induces genes related to the cell-wall metabolism, cell division and vesicle trafficking among others. The consequence of this host manipulation by Xac originates a physiological state very similar to that of cell expansion (hypertrophy). The vesicle trafficking inhibitor, Brefeldin A, delayed the development of canker symptoms indicating that this activity is more related to the cellular enlargement than to the defense responses. The transcriptional profile of Xaa-infiltrated leaves highlights the activation of a mitogen-activated protein kinase (MAPK) signaling pathway related to pathogen attack with the involvement of other components of defense responses like WRKY-transcriptional factors and ethylene-response elements. Both Xac and Xaa appear to modulate the transcription of genes related to auxin signaling and transport and gibberellin biosynthesis. Treatments of orange leaves with 1-naphthylphihalamic acid (NAA) e Gihherellic Acid (GKi) demonstrated that these hormones induce the expression of genes related to cell-wall remodeling, gibberellin biosynthesis and auxin signaling. Moreover, the gibberellin-biosynthesis inhibitor (Chlorocoline Chloride) decreased significantly the expression of auxin-induced genes suggesting a cross-talk regulation between auxin and gibberellin controlling the expression of citrus genes associated with cell division and expansion. Finally, two promoter regions of the pathogenesis related (PR) genes, PR-I and PR-5.2, were isolated and characterized. These genes are strongly up-regulated by Xaa and Xac at 6 or 48 hai. Substantially, there are "upd" box-like motifs in the promoter regions of both PR-I and PR-5.2. By means of Electrophoretic Mobility Shift Assays (EMSA) we have demonstrated that PthA4 from Xac is capable of binding the "upd" box-like regions found in PR-1 e PR-5 promoters. Furthermore, both of the isolated promoters (PR-1 and PR-5) address the transient expression of the reporter gene uidA in JV. bethamiana leaves through agro infiltration Doutorado Genética Vegetal e Melhoramento Doutor em Genética e Biologia Molecular
- Published
- 2021
47. Isolamento e caracterização de uma flavoproteina oxido-redutase de Arabidopsis thaliana induzida por injuria mecanica e metil jasmonato
- Author
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Costa, Cinthia Losano, Benedetti, Celso Eduardo, 1964, Arruda, Paulo, 1952, Universidade Estadual de Campinas. Instituto de Biologia, and UNIVERSIDADE ESTADUAL DE CAMPINAS
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Plantas - Efeito dos genes ,Plantas - Desenvolvimento ,Cinetica de enzimas - Abstract
Orientadores: Celso Eduardo Benedetti, Paulo Arruda Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia Mestrado
- Published
- 2021
48. Analise funcional e estrutural da proteina BigR de Xylella fastidiosa envolvida na regulação do operon Xf0768-0764
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Barbosa, Rosicler Lazaro, 1978, Benedetti, Celso Eduardo, 1964, Marques, Marilis do Valle, Nunes, Luiz Roberto, Souza, Anete Pereira de, Souza, Alessandra Alves de, Universidade Estadual de Campinas. Instituto de Biologia, Programa de Pós-Graduação em Genética e Biologia Molecular, and UNIVERSIDADE ESTADUAL DE CAMPINAS
- Subjects
Xylella fastidiosa ,Regulação da expressão gênica ,Gene expression regulation ,BigR protein ,Fatores de transcrição ,Biofilm ,Transcriptional factor ,Proteina bigR ,Biofilmes - Abstract
Orientador: Celso Eduardo Benedetti Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: O gene XF0767 de Xylella fastidiosa está localizado num operon composto por mais quatro genes classificados como proteínas hipotéticas (XF0768-XF0767-XF0766-XF0765-XF0764). Este operon está conservado em uma série de bactérias associadas a plantas, incluindo Agrobacterium tumefaciens, Mezorhizobium loti e Sinorhizobium meliloti. A região upstream ao códon de iniciação deste operon possui seqüências canônicas correspondentes a sítios -35 e ¿10 de regiões promotoras reguladas por fatores sigma70. O objetivo deste trabalho foi caracterizar o sistema de regulação do operon pela proteína XF0767, nomeada BigR (biofilm growth-associated repressor), visando à compreensão deste sistema e sua importância para a bactéria. A proteína BigR se liga na seqüência repetida invertida (9-4-9) localizada na região ¿10 do promotor do operon XF0768-0764, denominada BigRbox. BigR inibe a atividade do operon reprimindo a expressão do gene repórter GFP em Escherichia coli, Agrobacterium tumefaciens e Xylella fastidiosa. Mutações no BigRbox dos promotores de Xylella e Agrobacterium afetam a ligação do repressor e abole a transcrição do gene repórter. Esses dados indicam, portanto, que BigR compete com a RNA polimerase pelo mesmo sítio de ligação ao DNA, revelando assim o mecanismo de regulação do operon. BigR apresenta similaridade com fatores transcricionais de bactérias contendo domínio HTH (helix-turn-helix) de ligação ao DNA. Apesar de BigR ter sido inicialmente classificada como uma proteína da família SmtB/ArsR, as quais regulam operons relacionados à resistência a metais, nossos dados indicam que BigR não atua como sensor de metal. A adição de cádmio, ferro e cobre na mistura de ligação causam uma aparente dissociação do complexo DNA/BigR, entretanto, estudos in vivo na presença de metais não evidenciaram nenhuma alteração na expressão do gene repórter. Mutantes deficientes em BigR também não apresentam diferença de crescimento na presença de metais. O gene XF0768 codifica uma proteína nomeada BLH (beta-lactamase-like hydrolase) por pertencer à superfamília das metalo-beta-lactamases. Dada a presença de BigR e BLH no operon de Xylella e Agrobacterium, a atividade do operon foi analisada em diferentes condições como crescimento das células repórter in planta, co-cultivo com bactérias endofíticas e deficiência nutricional. Entretanto, uma maior atividade do operon em Xylella e Agrobacterium foi detectada apenas na condição de biofilme. Células de Agrobacterium aderidas em raiz de tabaco também apresentaram maior expressão do gene repórter quando comparadas às células em suspensão. Mutantes de Agrobacterium deficientes em BigR (bigR-) apresentam maior expressão do gene repórter, confirmando que BigR age como o repressor transcricional do operon. A quantificação da formação de biofilme das células mutante e selvagem revelou uma maior densidade de biofilme no mutante bigR- em superfície de vidro e também maior quantidade de células aderidas em raiz de tabaco, indicando que o operon pode estar envolvido com o processo de adesão ou desenvolvimento do biofilme bacteriano. Do ponto de vista estrutural, foi mostrado que a proteína BigR truncada no N-terminal (?BigR) encontra-se estruturada na forma de trímero, diferindo do observado para as proteínas com domínio HTH que em geral formam dímeros e monômeros. BigR é estável termicamente, começando a perder estrutura à 74oC, mas retendo um sinal residual de a-hélice até 90oC. Tratamentos com altas concentrações de (NH4) SO 2 4 interferiram na ligação da proteína ao DNA alvo e no conteúdo de estrutura secundária sem alterar, contudo, sua estabilidade térmica. A purificação e cristalização da proteína ?BigR resultou na coleta de alguns conjuntos de dados da proteína nativa e derivada, através de soaking ou marcada com SeMet. Apesar dos dados obtidos serem de boa qualidade, até o momento, não foi possível a resolução da estrutura cristalográfica desta proteína Abstract: The XF0767 gene from Xylella fastidiosa is located in an operon composed by a set of genes (XF0768-XF0767-XF0766-XF0765-XF0764) of unknown function. This operon is conserved in a number of plant-associated bacteria including Agrobacterium tumefaciens, Mezorhizobium loti e Sinorhizobium meliloti. The DNA region upstream of the operon has canonical sequences corresponding to ¿35 and ¿10 elements found in sigma70-regulated promoters. The aim of this work was to elucidate the biological function of the protein encoded by XF0767, named BigR (biofilm growth-associated repressor), as a transcriptional regulator and its importance to the bacteria. BigR binds to an inverted repeat sequence (9-4-9) located in the ¿10 region of the XF0768-0764 operon promoter. This sequence was named BigRbox. BigR repressed transcription of its own operon upon binding to the BigRbox in Xylella fastidiosa and Agrobacterium tumefaciens. Mutations in the BigRbox significantly affected the repressor binding and abolished transcription of the reporter gene in both bacteria, indicating that BigR compete with the RNA polymerase for the same promoter site. BigR is similar to HTH transcriptional factors of the SmtB/ArsR family, which control tolerance and detoxification of heavy metals in prokaryotes. Despite the similarities, BigR does not appear to function as a metal sensor, as initially predicted. Although binding of BigR to its target DNA was diminished in the presence of cadmium, copper and iron, operon regulation in response to metals was not demonstrated in vivo. In addition, Agrobacterium mutants deficient in BigR did not show changes in growth rates in the presence of metals. BLH is an unusual beta-lactamase-like hydrolase coded by the XF0768 gene. To gain insights into the possible function of the operon, the activity of reporter cells was observed in the presence of different compounds and conditions including in planta growth, effect of endophytic competition and nutrient deficiency. Significantly, an increased operon activity was observed in Xylella and Agrobacterium biofilms. Agrobacterium cells attached to tobacco roots also showed high levels of reporter gene expression in comparison to cells in suspension. A. tumefaciens mutants deficient in the BigR showed constitutive expression of the operon, confirming that BigR acts as a transcriptional repressor. Biofilm quantification showed increased biofilm formation in glass surfaces as well as in tobacco roots, indicating that the operon may play a role in cell adherence or biofilm development. Structurally, the truncated BigR protein (?BigR) is a trimmer in solution, as opposed to most HTH regulators known, which are usually found as dimmers or monomers. BigR is stable at high temperatures (74oC); however, treatments with high concentrations of ammonium sulfate interfere with the protein secondary structure and its DNA binding capacity, without affecting its thermal stability. Good quality X-ray diffraction data were collected for the native and derivative ?bigR protein; however, structure resolution was not possible probably due to problems with the crystals Doutorado Genética de Microorganismos Doutor em Genética e Biologia Molecular
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- 2021
49. Expressão e caracterização da proteina tirosina fosfatase de soja e analise do perfil kinomico de raizes em germinação
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Medeiros, Luciana de Campos Leite, Aoyama, Hiroshi, 1943, Benedetti, Celso Eduardo, 1964, Kobarg, Jörg, Granjeiro, Jose Mauro, Ciancaglini, Pietro, Prazeres, Janaina Nicanuzia dos, Universidade Estadual de Campinas. Instituto de Biologia, Programa de Pós-Graduação em Biologia Funcional e Molecular, and UNIVERSIDADE ESTADUAL DE CAMPINAS
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Tissue distribution ,Soja ,Proteína tirosina fosfatase ,Germinação ,Germination ,Raízes (Botânica) ,Soybean ,Roots ,Protein tyrosine phosphatase - Abstract
Orientadores: Hiroshi Aoyama, Celso Eduardo Benedetti Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: Em nosso laboratório foram purificadas quatro isoformas de fosfatases ácidas, a partir de sementes de soja quiescentes, tendo sido também estudadas suas propriedades cinéticas e físico-químicas. Estudos físicos e estruturais destas enzimas requerem uma grande quantidade da proteína pura, o que exigiria várias purificações convencionais, que, em geral, são bastante trabalhosas. Devido a este fato e a existência de poucas de tais proteínas clonadas e expressas, nos propusemos a clonar e expressar uma proteína tirosina fosfatase (PTP) de soja. O estudo da cinética da PTP recombinante revelou que esta enzima possui características típicas de uma fosfatase e maior especificidade para tirosina-fosfato em relação a outros substratos analisados. Foi feito o estudo de desnaturação térmica da enzima, através de dicroísmo circular, que mostrou que a enzima recombinante apresenta baixa estabilidade térmica. Identificamos por western blot a presença da GmPTP nos diferentes tecidos de soja, germinados tanto no claro como no escuro. Estes resultados, confrontados com os obtidos no PCR quantitativo, mostram uma expressão aumentada do tecido raiz, quando comparada aos outros tecidos avaliados, que está em concordância com o encontrado na literatura, referente à importância fisiológica da enzima neste tecido. Níveis adequados de fósforo são necessários para o crescimento e desenvolvimento de todos os organismos para o bom funcionamento de funções como estrutura molecular, geração de energia e regulação metabólica. A demanda por fósforo e sua conseqüente absorção do solo pelas raízes, aumenta dramaticamente durante o período de rápido crescimento e divisão, por exemplo, na germinação de sementes. Sendo assim, como supridores importantes de fósforo para o metabolismo da planta, podemos citar as fitases e fosfatases, que, além de contribuírem para prover nutrientes, as fosfatases também desempenham papel importante no controle de diferentes funções da planta, particularmente na regulação das cascatas de transdução de sinal. Utilizamos microarranjos de quinases como ferramenta para avaliação do quinoma da raiz nas plântulas de soja germinadas na presença e ausência de luz. Assim, concluímos que fatores ambientais, como a presença de luz e disponibilidade de nutrientes fazem com que diferentes vias estejam ativas e que diferentes fitormônios estejam agindo predominantemente. Relacionando as condições de germinação das plântulas de soja com esses fatores e às quinases mais ativadas no chip, propomos um modelo de sinalização onde o hormônio ácido abscísico responde ao estresse através da ativação de vias como as de MAPKs e de cálcio, causando modulação da atividade de fosfatases e quinases, resultando em respostas como proliferação e rearranjo do citoesqueleto das raízes. Neste trabalho, nós descrevemos a clonagem, expressão de uma de soja, sua caracterização cinética, localização tecidual e investigação da possível relação com a sinalização disparada pela presença ou ausência de luz. A investigação e caracterização de PTPs em plantas podem fornecer informações relevantes no campo de pesquisa do metabolismo vegetal. Sendo assim, nós disponibilizamos dados bioquímicos relevantes os quais sugerem que as plântulas de soja contêm PTP típicas que são principalmente expressas em raízes e aparentemente desempenham papel importante durante a germinação da semente. Abstract: Our group previously described the purification and characterization of four acid phosphatase isoforms obtained from mature soybean seeds, and determined the kinetic and physico-chemical properties of these enzymes. Structural and physical studies demand high amounts at purity levels of these enzymes, requiring efforts in laborious conventional purifications. Due to this fact and to the existence of few plant proteins that already has been cloned and expressed, we proposed to clone and express a protein tyrosine phosphatase (PTP) from soybean. The kinetic studies of the recombinant PTP revealed an enzyme with phosphatase typical features and higher specificity toward Tyr-phosphate when compared to other analysed substrates. The analysis of thermal inactivation of the enzyme was carried out by circular dichroism, which showed that GmPTP had low thermal stability. The immunolocalization of GmPTP, by western blot, identified the enzyme in different soybean tissues, which were germinated in the presence and absence of light. These results are in agreement with those obtained from the quantitative PCR results, that showed a higher expression of GmPTP in roots when compared with other evaluated tissues. The physiological importance of this tissue to the plant metabolism corroborates our results, because adequate levels of phosphorus are required for the good operation of metabolic functions, as growth and development. The phosphorus demand and the consequent soil absorption through the roots dramatically increase in periods of high growth rate, for instance, during germination. Therefore, phytases and phosphatases are important suppliers for plant metabolism and besides the contribution to provide nutrients, phosphatases could play an important role in regulation of signal transduction cascades. Kinase microarrays were employed as a strong tool to analyse the root kinome in soybean plantlets germinated in light and dark conditions. We concluded that abiotic factors as light presence and nutrients availability were responsible for the activation of different pathways and different phytormones prevalence. Relating the germination conditions of soybean plantlets to these factors and to the analysis of the most activated kinases on the chip, we propose a signaling model to explain that, when the hormone abscisic acid is the main response of stress stimuli, it triggers the activation of MAPK and calcium signaling, and the resulting modulation of kinases and phosphatases activities led to proliferation and cytoskeleton rearrangement in roots. In this work, we described the cloning and expression of a soybean PTP, its kinetic characterization, tissue distribution and investigation of a putative relationship with the signaling pathway triggered by light and dark. The investigation and characterization of PTPs from plants can add relevant information in the plant metabolism research field. Therefore, we provided a wealth biochemical data which suggest that soybean plantlets contain typical PTP, which is mainly expressed in roots and apparently plays a critical role during germination. Doutorado Bioquímica Doutor em Biologia Funcional e Molecular
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- 2021
50. Mutagenese da proteina HBx do virus da hepatite B e estudo da interação com RNA e proteinas humanas
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Rui, Edmilson, Kobarg, Jörg, 1965, Costa, Fabio Trindade Maranhão, Benedetti, Celso Eduardo, Hyslo, Stephen, Marangoni, Sergio, Universidade Estadual de Campinas. Instituto de Biologia, and UNIVERSIDADE ESTADUAL DE CAMPINAS
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Oncogeneses ,Ativação viral ,Fígado - Câncer ,Liver- Cancer ,Virus activation - Abstract
Orientador: Jorg Kobarg Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: A hepatite B constitui um grave problema de saúde pública. Dados epidemiológicos estimam que aproximadamente 350 milhões de pessoas são portadores do vírus da hepatite B (HBV). Admite-se que a infecção evolui para a cura em média 95% dos casos, entretanto nos portadores crônicos a infecção pode evoluir para cirrose e carcinoma hepatocelular (HCC). Há um crescente acúmulo de evidências que relaciona a proteína HBx do HBV ao desenvolvimento do HCC. A maioria dos estudos sobre a função da proteína HBx sugere que ela é uma proteína reguladora de funções pleiotrópicas com a capacidade de induzir o crescimento tumoral. Isso é possivelmente devido à sua capacidade de interagir com uma vasta gama de proteínas celulares. No presente estudo investigamos os aspectos moleculares da estrutura e função da proteína HBx e os resultados foram contextualizados no processo de transformação celular. Observamos pela primeira vez a capacidade da proteína HBx em se ligar ao RNA contendo seqüências ricas em adenina e uracila (AU), que são presentes em alguns proto-oncogenes como c-myc e c-fos. A geração de proteínas truncadas permitiu mapear a região de interação entre HBx e RNA. Realizamos ensaios de mutação sítio-dirigida em HBx e substituímos todas as cisteínas por serinas. Nossos resultados sugerem que as cisteínas na proteína HBx são de menor importância para a sua interação com o RNA e com as proteínas humanas p53 e RXR. Descobrimos ainda uma nova proteína humana que interage com HBx: E4F. Este fator de transcrição humano está relacionado com o controle do ciclo celular, com a segregação cromossômica e com a embriogênese. Ensaios em leveduras e in vitro mostraram que HBx selvagem, bem como as suas formas mutadas, foram capazes de interagir e regular a função de E4F, indicando um possível novo mecanismo para a transformação celular e a regulação da transcrição viral, uma vez que E4F exibiu uma capacidade de interagir com a região ¿enhancer II¿ do genoma do HBV Abstract: Viral hepatitis is an important global public health problem. Epidemiological data show that worldwide 350 million people are chronically infected with the hepatitis B virus. About 95% of the infections cure spontaneously, but the chronically infected patients may develop liver cirrhosis or even hepatocellular carcinoma (HCC). A large body of evidence points to the viral onco-protein HBx as the principal cause for the cellular transformation. The majority of studies on HBx function suggest that it is a regulatory protein with pleiotropic functions and its capacity to induce tumor growth may be due to its ability to interact with a diverse array of cellular proteins. In the present study we investigated molecular and structural aspects of the function of the HBx protein in order to be able to shed light on the process of cellular transformation. We were able to demonstrate that HBx protein has the ability to bind to an AU-rich RNA sequences present in the mRNAs of certain proto-oncogenes such as c-myc and c-fos. The generation of truncated proteins allowed to map the region of interaction of HBx with the RNA. Furthermore, we performed site directed mutagenesis studies of HBx protein and substituted all of its cysteine residues with serines. Our data suggest that the cysteine residues in the HBx protein are of minor importance for its interaction with RNA, p53 and RXR proteins. Finally, we discovered in E4F a new human interacting protein partner for the HBx protein. The transcription factor E4F has been functionally implicated in the control of the cell cycle, the chromosomal segregation and embriogenesis. In studies using the yeast we further showed that wild-type HBx, as well as its mutated forms, can physically interact with the E4F protein and regulate its function. This indicates a possible new mechanism of cellular transformation and the regulation of the viral transcription, since the human protein E4F demonstrated the capacity to bind to the enhancer II region of the HBV genome Doutorado Bioquímica Doutor em Biologia Funcional e Molecular
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- 2021
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