LYSOSOMAL PHYSIOLOGY & PROTEIN DEGRADATION IN CYSTINOSIS

这项拨款提案的长期重点是理解分子 胱氨酸及其相关物质溶酶体转运的生物学和生理学 流程。之前在这项拨款上的工作已经确定了蛋白质的作用 降解导致溶酶体半胱氨酸在肾病中蓄积 在这项建议中，我们将扩大这些研究，以包括 分析溶酶体在RNA降解中的潜在作用。 本申请附带的初步数据表明，RNA可以 在溶酶体中恢复。我们认为溶酶体在这一过程中起作用。 当刺激时通过降解信使RNA来调节基因表达 因为它的生产被终止了。我们还开发了一种 对膀胱癌成纤维细胞毒性较大的筛选系统 便于检测胱氨酸溶酶体运输基因。我们提出了一个 使用标准的人类转基因技术进行的一系列实验 基因组文库，以鉴定已经被 转染成正常表型。我们已经得到了一种转变 膀胱癌细胞株，以提高转基因细胞的回收率。我们有 还获得了从鼠/人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胱氨酸病 成纤维细胞使我们能够识别溶酶体所在的染色体 胱氨酸运输基因驻留。与我们早先对 溶酶体蛋白分解我们提出了有关解离的研究 二硫键连接的配体受体复合体通过 内吞系统。我们之前描述了一种运输系统，用于 半胱氨酸直接进入溶酶体。我们假设半胱氨酸是 一种允许二硫键连接的络合物解离的还原剂 受体可以被回收到质膜上。最后，我们提出了一种 确定半胱胺进入机制的一系列实验 变成半胱氨酸型溶酶体。这就完成了我们的一系列调查 定义孤儿药物半胱胺是如何用于治疗肾病的 胱氨酸病，完成其药理作用。成功 完成本赠款提案中所述的项目将 显著提高我们对溶酶体半胱氨酸的生理学知识 运输，并将进一步加强我们对肾病的了解 胱氨酸病。还将获得关于以下方面的重要新知识 溶酶体在RNA降解中的作用及其与半胱氨酸的关系 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.