LYSOSOMAL PHYSIOLOGY & PROTEIN DEGRADATION IN CYSTINOSIS

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The long range focus of this grant proposal is to understand the molecular biology and physiology of lysosomal transport of cystine and related processes. Prior work on this grant has defined the role of protein degradation in leading to lysosomal cystine accumulation in nephropathic cystinosis, and in this proposal we will extend these studies to include an analysis of the potential role of lysosomes in the degradation of RNA. Preliminary data enclosed with this application demonstrate that RNA can be recovered within lysosomes. We suggest that lysosomes play a role in the modulation of gene expression by degrading messenger RNA when the stimulus for its production is terminated. We additionally have developed a selection system toxic for cystinotic fibroblasts which should greatly facilitate detection of the cystine lysosomal transport gene. We propose a series of experiments using standard transfection techniques of a human genomic library to allow identification of cystinotic cells which have been transfected to the normal phenotype. We have obtained a transformed cystinotic cell line to enhance recovery of transfected cells. We have also acquired the ability to produce microcells from mouse/human hybrids to enable us to microinject normal human chromosomes into cystinotic fibroblasts to permit us to identify the chromosome on which the lysosomal cystine transport gene resides. In line with our earlier studies on lysosomal proteolysis we propose studies relating to the dissociation of disulfide-linked ligand receptor complexes as they pass through the endocytic system. We have previously described a transport system for cysteine directed into lysosomes. We hypothesize that cysteine is the reductant which permits disulfide-linked complexes to dissociate such that receptors can be recycled to the plasma membrane. Finally, we propose a series of experiments to determine the mechanism of entry of cysteamine into cystinotic lysosomes. This completes our series of investigations defining how the orphan drug, cysteamine, used to treat nephropathic cystinosis, accomplishes its pharmacologic activities. Successful completion of the projects described in this grant proposal will significantly enhance our knowledge of the physiology of lysosomal cystine transport, and will additionally enhance our understanding of nephropathic cystinosis. Significant new knowledge will also be gained on the role of lysosomes in RNA degradation and on the role of cysteine with regard to disulfide-linked receptor-ligand interaction.