α-Galactosidase Aggregation Is a Determinant of Pharmacological Chaperone Efficacy on Fabry Disease Mutants

Abstract

Background: Deficiency in ␣-galactosidase activity leads to Fabry disease, for which treatment employing pharmacological chaperones is being developed. Results: Aggregating ␣-galactosidase mutants are not responsive to the treatment with the pharmacological chaperone, 1-deoxygalactonojirimycin (DGJ). Conclusion: Aggregation of ␣-galactosidase is a decisive factor for DGJ efficiency. Significance: Combining pharmacological chaperones treatment with suppression of aggregation might be beneficial for future therapeutic strategy against Fabry disease.

Misfolding caused by the pathogenic mutation G47R on the minor allele of alanine:glyoxylate aminotransferase and chaperoning activity of pyridoxine

“…The whole cell extract was separated by centrifugation (29,200 g, 10 min, 4°C) to obtain the soluble fraction. Pellets were then resuspended in an equal volume of denaturing gel loading buffer to obtain the insoluble fraction.…”

“…SEC analyses of lysates of CHO-GO cells were performed according to the method of Siekierska A. et al [29]. Four 10 cm diameter dishes of confluent CHO-GO-G47R-Mi cells were grown in the absence or in the presence of PN (3 μM), harvested and lysed in 500 μl of PBS as previously described.…”

Fabry_CEP: a tool to identify Fabry mutations responsive to pharmacological chaperones

“…Upon selection, the site affected by the mutation is shown on a space-filling model and colored in yellow or in blue, if it occurs at disulphide bridge sites or in the active site, in magenta otherwise. Recently, it was proposed that mutations promoting protein aggregation do not respond to PC [7]. We ran the program TANGO [8] and we found that a small minority, twelve out of 130 mutations experimentally tested Fabry, is predicted to promote aggregation.…”

Three Essential Resources to Improve Differential Scanning Fluorimetry (DSF) Experiments

“…High-throughput DSF screens are used to identify candidate binders to proteins of interest independent of enzymatic activity 7,12,21 . DSF has also been used to search for small-molecule correctors for genetically destabilized proteins [22][23][24][25] which underlie many misfolding diseases 13,26,27 such as cystic fibrosis 28,29 , Gaucher's disease 30 and Fabry's disease 31 . For example, our lab conducted a primary chemical screen by DSF to identify ligands for cataract-associated forms of alpha B-crystallin 32 .…”