A thermodynamic assay to test pharmacological chaperones for Fabry disease

• Published in: Biochimica et biophysica acta Vol. 1840; no. 3; pp. 1214 - 1224
• Main Authors: Andreotti, Giuseppina, Citro, Valentina, Correra, Antonella, Cubellis, Maria Vittoria
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.03.2014; Elsevier Pub. Co
• Subjects: alpha-galactosidase; alpha-Galactosidase - chemistry; alpha-Galactosidase - genetics; Animals; Cell lysate; Chlorocebus aethiops; clinical trials; COS Cells; drugs; Fabry Disease - drug therapy; Fabry Disease - genetics; genes; HEK293 Cells; Humans; in vitro studies; Limited proteolysis; Lysosomal storage disorder; Molecular Chaperones - chemistry; mutants; Mutation; Pharmacological chaperone; Protein Folding; Protein Stability; proteins; proteolysis; Thermodynamics; urea; Urea - pharmacology; Urea-induced unfolding; CD; Urea-induced unfolding; PC; DGJ; Lysosomal storage disorder; Cell lysate; Limited proteolysis; Pharmacological chaperone; FD; AGAL; lysosomal alpha-galactosidase; Fabry disease; pharmacological chaperones; circular dichroism; 1-deoxy-galactonojirimycin
• ISSN: 0304-4165; 0006-3002; 1872-8006; 1878-2434
• DOI: 10.1016/j.bbagen.2013.12.018

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. The stability of lysosomal alpha-galactosidase under different conditions was studied by urea-induced unfolding followed by limited proteolysis and Western blotting. We measured the concentration of urea needed to obtain half-maximal unfolding because this parameter represents an objective indicator of protein stability. 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. 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. [Display omitted] •Pharmacological chaperones stabilize the folded state of proteins.•Only some Fabry mutations can be treated with pharmacological chaperones.•Urea-induced unfolding represents a novel assay to test the efficiency of drugs.•The test with urea can be applied to a tiny amount of mutants in raw extracts.•Responsiveness of Fabry mutations to drugs can be tested with urea-induced unfolding.