MOLECULAR BASIS OF RENAL EPITHELIAL CYSTINE TRANSPORT

胱氨酸尿症是最常见的遗传性疾病之一，其特点是 尿半胱氨酸和碱性氨基酸排泄过多。低谷 半胱氨酸的溶解性导致肾结石的形成，它可以 最终导致肾功能衰竭。这种疾病是由于一种高位的- 半胱氨酸在刷状边界膜中的亲和转运系统 肾脏和肠道上皮细胞。分子性质的研究 参与上皮胱氨酸转运的蛋白质不完全 明白了。本实验室从大鼠肾脏克隆的一种蛋白质(NBAT) 并被证明定位于肾脏和肠道的BBM，已经被 与这起运输事件有牵连。尽管人类NBAT基因突变具有 在一种形式的胱氨酸尿症中被发现，有理由相信 其他尚未定性的基因缺陷也可能与此有关。 最近，本实验室显示，肾和肠道NBAT是 与另一种蛋白质(表示为P50)相关联，并提出 杂二聚体是高亲和力胱氨酸的功能单位 这些膜上的转运蛋白。这项建议的具体目的是 P50基因的克隆、鉴定及其在上皮胱氨酸中的作用 运输。为实现这些目标而提出的方法包括 大鼠肾脏P50蛋白的纯化及最终克隆。特定于站点 抗体将用于蛋白质的分子表征 以及它的组织和细胞定位。非洲爪哇的卵母细胞 表达系统将用于研究P50和P50的相对作用 氨基酸转运中的NBAT。如有必要，其他表达系统(例如， COS7和CHO单元)也将被采用。拟议的研究是 有望加强对参与其中的分子机制的了解 上皮胱氨酸的运输，最终导致更完整的 对分子和遗传因素的理解 产生了胱氨酸尿症综合征。

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.