title: A thermodynamic assay to test pharmacological chaperones for Fabry disease.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Istituto di Chimica Biomolecolare, CNR, Pozzuoli, Italy. Electronic address: giuseppina.andreotti@icb.cnr.it.
authors.forename: Giuseppina
authors.initials: G
authors.lastname: Andreotti
authors.affiliation: Istituto di Genetica e Biofisica 'A. Buzzati Traverso,' CNR, Napoli, Italy. Electronic address: v_citro@libero.it.
authors.forename: Valentina
authors.initials: V
authors.lastname: Citro
authors.affiliation: Istituto di Genetica e Biofisica 'A. Buzzati Traverso,' CNR, Napoli, Italy; Dipartimento di Biologia, Università Federico II, Napoli, Italy. Electronic address: correraantonella@gmail.com.
authors.forename: Antonella
authors.initials: A
authors.lastname: Correra
authors.affiliation: Dipartimento di Biologia, Università Federico II, Napoli, Italy; Istituto di Biostrutture e Bioimmagini, CNR, Napoli, Italy. Electronic address: cubellis@unina.it.
authors.forename: Maria Vittoria
authors.initials: MV
authors.lastname: Cubellis
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D000795:Fabry Disease / Q000188:drug therapy / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D018832:Molecular Chaperones / Q000737:chemistry*; D009154:Mutation; D017510:Protein Folding; D055550:Protein Stability; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology; D000519:alpha-Galactosidase / Q000737:chemistry* / Q000235:genetics
chemical_list: D018832:Molecular Chaperones; D014508:Urea; D000519:alpha-Galactosidase
keywords: 1-deoxy-galactonojirimycin; AGAL; CD; Cell lysate; DGJ; FD; Fabry disease; Limited proteolysis; Lysosomal storage disorder; PC; Pharmacological chaperone; Urea-induced unfolding; circular dichroism; lysosomal alpha-galactosidase; pharmacological chaperones