Abstract
BackgroundThe 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. MethodsThe stability of lysosomal alpha-galactosidase under different conditions was studied by urea-induced unfolding followed by limited proteolysis and Western blotting. ResultsWe measured the concentration of urea needed to obtain half-maximal unfolding because this parameter represents an objective indicator of protein stability. ConclusionsUrea-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 significanceScarce 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.
Highlights
The reduction in protein stability is the most common cause of monogenic diseases [1]
Pharmacological chaperones work because they stabilize mutant proteins and the method based on thermodynamic analysis is designed to assess this property straightforwardly
Free energies or enthalpies of unfolding, midpoint denaturant concentration, and m-values help us evaluate the stability of wild type and mutant forms of a protein under different environmental conditions, pH or ligands
Summary
The reduction in protein stability is the most common cause of monogenic diseases [1]. 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. 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. General significance: Scarce and dispersed populations pose a problem for the clinical trial of drugs for rare diseases This is true for pharmacological chaperones that must be tested on each mutation associated with a given disease. 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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