Kurzfassung

The treatment of cystinotic patients by cysteamine is based on the presence, in the lysosomal membrane, of non-affected amino acid transporters which remain unknown at molecular level. After entering patients’ cells, cysteamine accumulates into lysosomes through cysteine/cysteamine transporters. In the lysosomal lumen, it reacts with cystine to form a cysteine-cysteamine conjugate and, because of its structural similarity to lysine, the mixed disulfide leaves lysosomes through another...The treatment of cystinotic patients by cysteamine is based on the presence, in the lysosomal membrane, of non-affected amino acid transporters which remain unknown at molecular level. After entering patients’ cells, cysteamine accumulates into lysosomes through cysteine/cysteamine transporters. In the lysosomal lumen, it reacts with cystine to form a cysteine-cysteamine conjugate and, because of its structural similarity to lysine, the mixed disulfide leaves lysosomes through another transporter selective for cationic amino acids. After reduction of the mixed disulfide to cysteamine and cysteine in the cytosol, cysteamine reenters lysosomes to remove more cystine. The lysosomal cationic amino acid transporter, known biochemically as lysosomal ‘system c’, thus represents a ‘salvage pathway’ which by-passes the need for a functional cystine transporter.
In preliminary experiments, we identified in a family of cationic amino acid transporters two members which localize to lysosomes, and thus represent candidate system c proteins. In this project, we will characterize their transport activity, in particular their ability to translocate the cysteamine-cysteine disulfide across membranes. For this purpose, we will develop whole cell transport assays based on the redirection of the proteins to the plasma membrane by mutagenesis of their intracellular sorting motifs. This approach, which facilitates the study of lysosomal transporters and has been previously applied to cystinosin and sialin, will be carried out using radiotracer flux or electrophysiological techniques. In parallel, we will alter the expression of the candidate system c transporters in cultured cells, using cDNA transfection or RNA interference techniques, and examine whether their expression level correlates with the velocity at which cysteamine depletes cystine from cystinotic cells.
This project should represent a first step to improve the pharmacological treatment of cystinosis. By providing a robust assay to screen the substrate selectivity of system c for mixed disulfides, it should help identifying sulfhydryl compounds acting on cystinotic cells at lower concentration than cysteamine, thus providing a rationale approach to reduce the side-effects and constraints of the cysteamine treatment (gastrointestinal distress, administration every 6 hours).
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