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A thermodynamic assay to test pharmacological chaperones for Fabry disease.
Andreotti, Giuseppina; Citro, Valentina; Correra, Antonella; Cubellis, Maria Vittoria.
Afiliação
  • Andreotti G; Istituto di Chimica Biomolecolare, CNR, Pozzuoli, Italy. Electronic address: giuseppina.andreotti@icb.cnr.it.
  • Citro V; Istituto di Genetica e Biofisica 'A. Buzzati Traverso,' CNR, Napoli, Italy. Electronic address: v_citro@libero.it.
  • Correra A; Istituto di Genetica e Biofisica 'A. Buzzati Traverso,' CNR, Napoli, Italy; Dipartimento di Biologia, Università Federico II, Napoli, Italy. Electronic address: correraantonella@gmail.com.
  • Cubellis MV; Dipartimento di Biologia, Università Federico II, Napoli, Italy; Istituto di Biostrutture e Bioimmagini, CNR, Napoli, Italy. Electronic address: cubellis@unina.it.
Biochim Biophys Acta ; 1840(3): 1214-24, 2014 Mar.
Article em En | MEDLINE | ID: mdl-24361605
BACKGROUND: The majority of the disease-causing mutations affect protein stability, but not functional sites and are amenable, in principle, to be treated with pharmacological chaperones. These drugs enhance the thermodynamic stability of their targets. Fabry disease, a disorder caused by mutations in the gene encoding lysosomal alpha-galactosidase, represents an excellent model system to develop experimental protocols to test the efficiency of such drugs. METHODS: The stability of lysosomal alpha-galactosidase under different conditions was studied by urea-induced unfolding followed by limited proteolysis and Western blotting. RESULTS: We measured the concentration of urea needed to obtain half-maximal unfolding because this parameter represents an objective indicator of protein stability. CONCLUSIONS: Urea-induced unfolding is a versatile technique that can be adapted to cell extracts containing tiny amounts of wild-type or mutant proteins. It allows testing of protein stability as a function of pH, in the presence or in the absence of drugs. Results are not influenced by the method used to express the protein in transfected cells. GENERAL SIGNIFICANCE: Scarce and dispersed populations pose a problem for the clinical trial of drugs for rare diseases. This is particularly true for pharmacological chaperones that must be tested on each mutation associated with a given disease. Diverse in vitro tests are needed. We used a method based on chemically induced unfolding as a tool to assess whether a particular Fabry mutation is responsive to pharmacological chaperones, but, by no means is our protocol limited to this disease.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Termodinâmica / Doença de Fabry / Dobramento de Proteína / Alfa-Galactosidase / Chaperonas Moleculares Tipo de estudo: Guideline Limite: Animals / Humans Idioma: En Revista: Biochim Biophys Acta Ano de publicação: 2014 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Termodinâmica / Doença de Fabry / Dobramento de Proteína / Alfa-Galactosidase / Chaperonas Moleculares Tipo de estudo: Guideline Limite: Animals / Humans Idioma: En Revista: Biochim Biophys Acta Ano de publicação: 2014 Tipo de documento: Article