LYSOSOMAL PHYSIOLOGY & PROTEIN DEGRADATION IN CYSTINOSIS

这项拨款提案的长期重点是了解分子 胱氨酸和相关的溶酶体转运的生物学和生理学 流程. 这项资助的前期工作已经确定了蛋白质的作用 降解导致肾病患者溶酶体胱氨酸蓄积 胱氨酸病，在本提案中，我们将扩展这些研究，包括 分析溶酶体在RNA降解中的潜在作用。 本申请所附的初步数据表明，RNA可以被 在溶酶体内回收。 我们认为，溶酶体发挥了作用，在 当刺激物被释放时，通过降解信使RNA调节基因表达 因为它的生产被终止了。 我们还开发了一个 选择系统对胱氨酸成纤维细胞有毒， 有助于检测胱氨酸溶酶体转运基因。 我们提出了一个 使用人的标准转染技术的一系列实验 基因组文库，以允许鉴定已被 转染成正常表型。 我们得到了一个 胱氨酸化细胞系以增强转染细胞的恢复。 我们有 还获得了从小鼠/人类杂交中产生微细胞的能力， 使我们能够将正常的人类染色体显微注射到 使我们能够识别染色体上的溶酶体 胱氨酸转运基因存在。 与我们先前的研究一致， 溶酶体蛋白水解，我们提出的研究有关的解离 二硫键连接的配体受体复合物，因为它们通过 内吞系统 我们先前已经描述了用于以下的传输系统： 半胱氨酸直接进入溶酶体。 我们假设半胱氨酸是 还原剂，其允许二硫键连接的复合物解离，使得 受体可以再循环到质膜。 最后，我们提出了一个 确定半胱胺进入机制的一系列实验 进入胱氨酸化溶酶体。 我们的一系列调查到此结束 定义孤儿药半胱胺如何用于治疗肾病 胱氨酸病，完成其药理活性。 成功 完成本赠款建议中描述的项目将 显著提高我们对溶酶体胱氨酸生理学的认识 运输，并将进一步加强我们对肾病的理解 胱氨酸病 此外，还将获得关于以下方面作用的重要新知识： 溶酶体在RNA降解中的作用以及半胱氨酸在 二硫键连接的受体-配体相互作用。

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.