A thermodynamic assay to test pharmacological chaperones for Fabry disease

Biochimica Et Biophysica Acta
Giuseppina AndreottiMaria Vittoria Cubellis

Abstract

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 pharmacol...Continue Reading

References

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Apr 24, 2009·Protein Science : a Publication of the Protein Society·Moon-Soo KimChiwook Park
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Nov 26, 2010·Orphanet Journal of Rare Diseases·Dominique P Germain
Dec 9, 2010·Orphanet Journal of Rare Diseases·Giuseppina AndreottiMaria Vittoria Cubellis
Apr 5, 2011·Expert Opinion on Therapeutic Patents·Juan M BenitoCarmen Ortiz Mellet
Oct 19, 2011·Orphanet Journal of Rare Diseases·Giuseppina AndreottiMaria Vittoria Cubellis
Nov 1, 2011·Journal of Inherited Metabolic Disease·Patrick B Deegan
Jan 14, 2012·Proceedings of the Japan Academy. Series B, Physical and Biological Sciences·Satoshi Ishii
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Jul 17, 2013·BMC Bioinformatics·Marco CammisaMaria Vittoria Cubellis
Jul 26, 2013·Orphanet Journal of Rare Diseases·Marco CammisaMaria Vittoria Cubellis

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Citations

Apr 8, 2015·Drug Testing and Analysis·Giuseppina AndreottiMaria Vittoria Cubellis
Apr 7, 2015·Biochimie·Maria Vittoria CubellisGiuseppina Andreotti
Apr 22, 2014·Pharmacological Research : the Official Journal of the Italian Pharmacological Society·Ya-Juan WangTing-Wei Mu
Sep 7, 2016·Protein Science : a Publication of the Protein Society·Aurangazeb KabirKazuo Kuwata
Sep 28, 2017·International Journal of Molecular Sciences·Chiara CimmarutaValentina Citro
Jan 17, 2020·International Journal of Molecular Sciences·Ludovica LiguoriGiuseppina Andreotti

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Methods Mentioned

BETA
xenografts
transfections
transfection
circular dichroism
glycosylation
thermal shift
electrophoresis
Assay

Software Mentioned

Synergy
KaleidaGraph

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