MOLECULAR BASIS OF RENAL EPITHELIAL CYSTINE TRANSPORT

胱氨酸尿症是最常见的遗传性疾病之一，其特征在于： 尿中半胱氨酸和碱性氨基酸的过量排泄。 低 半胱氨酸的溶解性导致肾结石的形成， 最终导致肾衰竭。 这种疾病是由于高- 刷状缘膜半胱氨酸亲和转运系统 肾脏和肠上皮细胞。 的分子性质 参与上皮胱氨酸转运的蛋白质是不完全的， 明白 本实验室从大鼠肾脏中克隆的一种蛋白质（NBAT） 并显示其定位于肾和肠BBM， 与这次运输有关 虽然人类NBAT基因的突变 在一种形式的胱氨酸尿症中发现，有理由相信， 其他尚未鉴定的基因缺陷也可能涉及。 最近，该实验室表明，肾脏和肠道NBAT是 与另一种蛋白质（表示为P50）相关，并提出 异二聚体代表高亲和力胱氨酸的功能单元 在这些细胞膜中的转运蛋白。 这项建议的具体目标是 P50基因的克隆和鉴定及其在上皮细胞胱氨酸蛋白酶中的作用 运输 为实现这些目标而提出的方法包括 从大鼠肾脏中纯化并最终克隆P50。 位点特异 抗体将用于蛋白质的分子表征 以及其组织和细胞定位。 非洲爪蟾卵母细胞 表达系统将被用来研究P50的相对作用， 氨基酸转运中的NBAT。 如有必要，还可以使用其他表达系统（例如， C 0 S7和CHO细胞）。 拟议的研究是 预计将提高参与的分子机制的知识 上皮胱氨酸转运，并最终导致更完整的 了解导致癌症的分子和遗传因素 引起胱氨酸尿综合征。

Cystinuria, one of the most common genetic disorders, is characterized by excessive excretion of cysteine and basic amino acids in urine. The low solubility of cysteine results in formation of kidney stones which can eventually lead to renal failure. The disease is due to defects in a high- affinity transport system for cysteine in the brush border membranes (BBMs) of kidney and intestinal epithelial cells. The molecular properties of proteins involved in epithelial cystine transport are incompletely understood. A protein (NBAT), cloned in this laboratory from rat kidney and shown to be localized in the renal and intestinal BBMs, has been implicated in this transport. Although mutations in human NBAT gene have been found in one form of cystinuria, there are reasons to believe that defects in other, as yet uncharacterized, genes may also be involved. Recently, this laboratory showed that kidney and intestinal NBAT is associated with another protein (denoted P50) and proposed that the heterodimer represents the functional unit of the high-affinity cystine transporter in these membranes. The Specific Aims of this proposal are to clone and characterize P50 and elucidate its role in epithelial cystine transport. The methods proposed to achieve these objectives include purification and eventual cloning of P50 from rat kidney. Site-specific antibodies will be used for the molecular characterization of the protein and for its tissue and cellular localization. The Xenopus oocyte expression system will be used to investigate the relative role of P50 and NBAT in amino acid transport. If necessary, other expression systems (eg. COS7 and CHO cells) will also be employed. The proposed research is expected to enhance knowledge of the molecular mechanisms involved in epithelial cystine transport and, eventually, lead to a more complete understanding of the molecular and genetic factors responsible for producing the cystinuric syndrome.