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(Referência obtida automaticamente do Web of Science, por meio da informação sobre o financiamento pela FAPESP e o número do processo correspondente, incluída na publicação pelos autores.)

Molecular determinants of improved cathepsin B inhibition by new cystatins obtained by DNA shuffling

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Autor(es):
Valadares, Napoleao F. [1] ; Dellamano, Marcia [2] ; Soares-Costa, Andrea [2] ; Henrique-Silva, Flavio [2] ; Garratt, Richard C. [1]
Número total de Autores: 5
Afiliação do(s) autor(es):
[1] Univ Sao Paulo, Phys Inst Sao Carlos, Dept Phys & Informat, Ctr Struct Mol Biotechnol, BR-13560970 Sao Carlos, SP - Brazil
[2] Univ Fed Sao Carlos, Dept Genet & Evolut, Mol Biol Lab, BR-13565905 Sao Carlos, SP - Brazil
Número total de Afiliações: 2
Tipo de documento: Artigo Científico
Fonte: BMC STRUCTURAL BIOLOGY; v. 10, SEP 30 2010.
Citações Web of Science: 3
Resumo

Background: Cystatins are inhibitors of cysteine proteases. The majority are only weak inhibitors of human cathepsin B, which has been associated with cancer, Alzheimer's disease and arthritis. Results: Starting from the sequences of oryzacystatin-1 and canecystatin-1, a shuffling library was designed and a hybrid clone obtained, which presented higher inhibitory activity towards cathepsin B. This clone presented two unanticipated point mutations as well as an N-terminal deletion. Reversing each point mutation independently or both simultaneously abolishes the inhibitory activity towards cathepsin B. Homology modeling together with experimental studies of the reverse mutants revealed the likely molecular determinants of the improved inhibitory activity to be related to decreased protein stability. Conclusion: A combination of experimental approaches including gene shuffling, enzyme assays and reverse mutation allied to molecular modeling has shed light upon the unexpected inhibitory properties of certain cystatin mutants against Cathepsin B. We conclude that mutations disrupting the hydrophobic core of phytocystatins increase the flexibility of the N-terminus, leading to an increase in inhibitory activity. Such mutations need not affect the inhibitory site directly but may be observed distant from it and manifest their effects via an uncoupling of its three components as a result of increased protein flexibility. (AU)

Processo FAPESP: 98/14138-2 - Center for Structural Molecular Biotechnology
Beneficiário:Glaucius Oliva
Linha de fomento: Auxílio à Pesquisa - Centros de Pesquisa, Inovação e Difusão - CEPIDs
Processo FAPESP: 08/58316-5 - Estudos cinéticos e funcionais das proteínas humanas da família septina
Beneficiário:Napoleão Fonseca Valadares
Linha de fomento: Bolsas no Brasil - Pós-Doutorado
Processo FAPESP: 05/59833-5 - Desenvolvimento de canas-de-açúcar transgênicas superexpressando cistatinas visando o aumento da resistência a insetos
Beneficiário:Andrea Soares da Costa Fuentes
Linha de fomento: Bolsas no Brasil - Pós-Doutorado