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Doctoral Thesis
DOI
10.11606/T.76.2010.tde-14062010-163659
Document
Author
Full name
Fernando Alessandro
Institute/School/College
Knowledge Area
Date of Defense
Published
São Carlos, 2010
Supervisor
Committee
Thiemann, Otavio Henrique (President)
Fragoso, Stenio Perdigão
Garratt, Richard Charles
Kobarg, Jorg
Malavazi, Iran
Title in Portuguese
Estudos estruturais e bioquímicos das septinas 7 e 9 humanas
Keywords in Portuguese
Atividade GTPásica
Caracterização estrutural
Clonagem
Expressão
Septina 7
Septina 9
Abstract in Portuguese
As proteínas pertencentes à família das septinas foram originalmente descobertas em 1971 em decorrencia de estudos genéticos em células mutantes. Essas proteínas encontradas em fungos e animais, mas não em plantas apresentam como principais características a presença de um domínio conservado de ligação aos nucleotídeos de guanina (GTP) e a formação de filamentos homo- e hetero-oligoméricos, que são estruturas altamente organizadas. Estudos filogenéticos e moleculares em humanos identificaram 14 septinas que são divididas em 4 grupos (I, II, III e IV). Estas moléculas associam-se com membranas celulares, actina, microtúbulos do citoesqueleto e estão envolvidas em inúmeros processos que ocorrem no córtex celular e requerem organização espacial, tais como: citocinese, ciclo celular, formação de barreiras de difusão, alinhamento de fuso. Alterações na expressão das septinas estão associadas a vários tipos de tumores e a doenças de Parkinson e Alzheimer. Neste trabalho, com o objetivo de obter informações estruturais e bioquímicas das septinas 7 e 9 humanas. Este projeto é parte de um esforço conjunto coordenado pelo Prof. Dr. Richard C. Garratt e conhecido informalmente como Septimoma. As construções recombinantes SEPT 7, SEPT 7G, e SEPT 9G foram expressas em Escherichia coli e as proteínas recombinantes obtidas. As análises em eletroforese SDS-Page e em gel nativo indicam que essas proteínas foram purificadas com sucesso. A atividade GTPase e o estado oligomérico na forma dimérica foram verificados. Estudos de dicroísmo circular e fluorescência determinaram que esses recombinantes são formados por uma mistura de estruturas secundárias &alfa; e β, e também que o C e o N terminais aumentam a estabilidade das proteínas. Foram obtidos cristais da SEPT 7G e, por meio da técnica de raios-X, foi determinado um modelo tridimensional da proteína com resolução de 3,4o.
Title in English
Structural and biochemical studies of human septins 7 e 9
Keywords in English
Cloning
Expression
Gtpase Activity
Septin 7
Septin 9
Structural Characterization
Abstract in English
Proteins belonging to the septin family were originally discovered in 1971 through genetic studies of mutant cells. These proteins found in fungi and animals, but not in plants present, as their main characteristics, a conserved guanine nucleotide-binding domain (GTP) and they also form homo and hetero-oligomeric filaments that are highly organized structures. Phylogenetic and molecular studies in humans have identified 14 septins which are divided into 4 subfamilies (groups I, II, III and IV). These molecules associate with cell membranes, actin, cytoskeleton microtubules and they are related to a number of processes that take place in the cell cortex and that require spatial organization, such as cytokinesis, cell cycle, diffusion barrier formation and spindle alignment. Alterations in the expression of septins are associated with several types of tumors and with Parkinsons and Alzheimers diseases. In this work, with the goal of obtaining structural and biochemical information of human septins 7 and 9, the recombinants SEPT 7, SEPT 7G and SEPT 9G were expressed in E. coli. Analyses both in SDS-Page electroforesis and in native gel suggest that these proteins were purified successfully for they are soluble and homogeneous. GTpase activity has been verified in all of these recombinants, which shows that these proteins are present in native form and that additional molecules are not needed for this activity. It was possible to determine through different techniques such as molecular exclusion chromatography and SAXS that all the molecules in solution are grouped as dimeric form. Circular dichroism and fluorescence spectroscopic studies have determined both that such recombinants are formed by means of a mixture of &alfa; and β secondary structures and that the C and N-terminals increase the stability of proteins. Protein stability studies under different pH and temperature conditions show that the raise of the latter produces a greater molecular aggregation. Measurements of fluorescence emissions have indicated that the SEPT 7, SEPT 7G and SEPT 9G form structures of amyloid-like filaments found in many septins. Crystal structures of SEPT 7G have been obtained and, by means of the X-ray technique, a 3-D model of the protein has been determined with a resolution of 3.4o. It has been possible to predict, with molecular modeling studies, regions formed by loops that showed low electronic density in the GTPase crystallographic model. Therefore, it has been possible to add more structural information to this domain and to form the complete polypeptide without cuts.
 
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Publishing Date
2010-06-16
 
WARNING: The material described below relates to works resulting from this thesis or dissertation. The contents of these works are the author's responsibility.
  • Serrão, Vitor Hugo Balasco, et al. Promiscuous interactions of human septins: The GTP binding domain of SEPT7 forms filaments within the crystal [doi:10.1016/j.febslet.2011.10.043]. FEBS Letters [online], 2011, vol. 585, p. 3868-3873.
  • ALESSANDRO, F, et al. Structure-function analysis of human septin 7 and 9. GTPase domain. In XXXVIII Annual Meeting of the Brazilian Biochemistry and Molecular Biology Society, Águas de Lindóia, 2009. Program and Index.São Paulo : Sociedade Brasileira de Bioquímica e Biologia Molecular, 2009. Resumo.
  • ALESSANDRO, F, and THIEMANN, O H. Structural and biochemical studies of human Septin 7. In 53º Congresso Brasileiro de Genética, Águas de Lindóia, 2007. Resumos. : SBG, 2007. Resumo.
  • ALESSANDRO, F, e THIEMANN, O H. Acesso à diversidade microbiana na busca de novos compostos anti-leishmania por analise metagenômica. In IX Workshop da Pós-Graduação no Instituto de Física de São Carlos - USP, São Carlos, 2005. Caderno de Resumos., 2005. Resumo.
  • ALESSANDRO, F, e THIEMANN, O H. Construção de bibliotecas metagênomicas em Escherichi coli e sondagem de moléculas heterólogas que apresenta atividade antileishmania. In X Workshop da Pós-Graduação do IFSC, São Carlos, 2006. Caderno de Resumos.São Carlos : Instituto de Física de São Carlos, 2006. Resumo.
  • ALESSANDRO, F, e THIEMANN, O H. Estudos estruturais e bioquímicos de septina humana 7. In XI Workshop da Pós-Graduação do IFSC, São Carlos, 2007. Livro de Resumos., 2007. Resumo.
  • CALDAS, V., ALESSANDRO, F, and THIEMANN, O H. Study of microbial diversity of brazilian savanna conserved regions and a eucalyptus culture. In XXXVII Reunião Anual da Sociedade Brasileira de Bioquímica e Biologia Molecular - SBBq, Águas de Lindóia, 2008. Program and Index., 2008. Resumo.
  • SERRAO, V. H., et al. Structural and biochemical studies of human septin 7. In XXXVII Reunião Anual da Sociedade Brasileira de Bioquímica e Biologia Molecular - SBBq, Águas de Lindóia, 2008. Program and Index., 2008. Resumo.
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