Aspects of lysosome biogenesis and gene expression with emphasis on the lysosomal cystine transporter gene, CTNS
Description
The CTNS gene encodes the lysosomal membrane transport protein cystinosin. Defects in CTNS result in the disorder cystinosis, characterized by the renal Fanconi syndrome, progressive renal failure, corneal crystals and retinopathy. A nonsense mutation (G753A) found in the CTNS gene encodes a premature termination codon (PTC; W138X), and results in severe nephropathic cystinosis. Aminoglycoside antibiotics induce faulty proofreading and allow translation of full-length protein from nonsense message. Treatment of fibroblasts containing the PTC mutation with the aminoglycoside antibiotic, gentamicin, results in cystine depletion. Transfection with PTC-GFP fusion constructs followed by treatment with gentamicin demonstrated that GFP expression was localized to lysosomes. Tissue expression of the mouse homolog of CTNS and the effect of lysosomal cystine on CTNS expression were examined To investigate the regulation of lysosome volume and number normal human fibroblasts were cultured in the presence of sucrose, which induces lysosomal swelling. Gene expression in three normal human fibroblast cell lines treated with 100 mM sucrose for 24 hours, as well as untreated time-matched controls, was analyzed by HGU95A microarray. Of the 12,500 genes thus examined, thirty-six were increased and eleven were decreased an average of 2-fold or greater, relative to matched controls. The majority of increased genes function in cholesterol biosynthesis and fatty acid metabolism or have lysosomal and intracellular vesicle related roles. The latter included lysosomal neuraminidase, CLN3, and Rab7L1 genes. Rab7L1 was examined by expression of a GFP-Rab7L1 fusion protein and found to localize primarily to the Golgi apparatus, and in some cells, to the membranes bounding vesicles Transcription factors activated in sucrose treated cells were analyzed using protein/DNA arrays. C/EBP activity was increased in nuclear extracts from cells exposed to sucrose for 6 and 12 hours, relative to matched controls, and the increased activity at 12 hours was confirmed by EMSAs Study of lysosome biogenesis and the regulation of gene expression using the sucrose-induced vacuolation model has yielded several genes of interest and a potentially relevant transcriptional regulator. A better understanding of the genes regulating lysosome function may improve our understanding of lysosomal storage disorders and yield new options for their treatment