SYSTEM A AMINO ACID TRANSPORTER IN HUMAN FIBROBLASTS

人类成纤维细胞中的 A 系统氨基酸转运蛋白

• 批准号: 3233562
• 负责人: Shirley B. Russell
• 金额: $ 8.68万
• 依托单位: MEHARRY MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-02-01 至 1989-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3233562
• 关键词: acid base balance; aminoacid transport; antibody formation; antibody specificity; autoradiography; cell membrane; cortisol; densitometry; enzyme linked immunosorbent assay; fibroblasts; gel electrophoresis; gene mutation; glucocorticoids; hormone regulation /control mechanism; human tissue; keloid skin disorder; molecular biology; monoclonal antibody; radiotracer; tissue /cell culture; vesicle /vacuole

Hydrocortisone (HC) induces an increase in amino acid uptake by human fibroblasts that is 5-fold greater in keloid-derived fibroblasts than in cells from normal dermis. The increase involves primarily System A which preferentially transports proline and glycine. It requires RNA and protein synthesis and results from an elevated Vmax. These findings suggest that HC induces a protein specifically involved in System A amino acid transport. The goal of this research is to determine the molecular basis of the HC-induced increase in amino acid transport in normal and keloid-derived fibroblasts. 1) Membrane proteins from keloid and normal cells will be examined to determine whether there are alterations in amount of HC-induced proteins that correlate with HC-induced increases in System A amino acid transport. Several methods of labeling membrane proteins will be employed to maximize the chance of detecting differences in amount of System A component(s). Other modulators of System A activity that affect normal and keloid-derived cells equally will also be varied to aid in the identification of System A proteins. 2) Plasma membrane vesicles will be prepared to determine whether they retain HC-induced differences in System A activity and protein composition. Use of vesicles increases the chance of identifying System A proteins, due to the partial purification achieved and applicability of additional labeling procedures. 3) Monoclonal antibodies will be prepared to membrane proteins that are induced by HC to a greater extent in keloid-derived fibroblasts than in normal cells. These antibodies will be screened for their ability to differentially bind to the surface of normal and keloid-derived cells grown with and without HC, to bind to purified glucocorticoid-induced proteins, and to alter System A amino acid transport. Detection of amino acid transport proteins in mammalian cells has been hampered by a dearth of mutations that affect transport, and a lack of specific high affinity ligands. Keloids may represent the first example in dermal fibroblasts of a genetic mutation that directly or indirectly alters amino acid transport. In addition to providing that directly or indirectly alters amino acid transport. In addition to providing information important to our understanding of keloids, this research may provide a unique opportunity to study one of the small number of proteins included in the glucocorticoid domain and to isolate and characterize an amino acid transporter from human cells.

氢化可的松 (HC) 诱导人体氨基酸摄取增加 瘢痕疙瘩来源的成纤维细胞数量是瘢痕疙瘩来源的成纤维细胞的 5 倍 来自正常真皮的细胞。 此次增加主要涉及系统A，其中 优先运输脯氨酸和甘氨酸。 它需要RNA和蛋白质 合成和升高 Vmax 的结果。 这些发现表明 HC 诱导一种专门参与系统 A 氨基酸的蛋白质 运输。 这项研究的目标是确定分子基础 HC 诱导的正常和 瘢痕疙瘩衍生的成纤维细胞。 1) 疤痕疙瘩和正常细胞的膜蛋白 将检查细胞以确定数量是否发生变化 与 HC 诱导的系统 A 增加相关的 HC 诱导蛋白的数量 氨基酸运输。 标记膜蛋白的几种方法 用于最大程度地检测数量差异的机会 系统 A 组件。 影响系统 A 活动的其他调节剂 正常细胞和瘢痕疙瘩衍生细胞同样也会发生变化，以帮助 系统A蛋白的鉴定。 2）质膜囊泡将是 准备好确定它们是否保留 HC 引起的系统差异 活性和蛋白质组合物。 使用囊泡会增加机会 由于实现了部分纯化，因此可以鉴定系统 A 蛋白 以及附加标签程序的适用性。 3) 单克隆 将制备针对 HC 诱导的膜蛋白的抗体 在瘢痕疙瘩衍生的成纤维细胞中比在正常细胞中的程度更大。 这些 将筛选抗体差异结合的能力 在有或没有 HC 的情况下生长的正常细胞和瘢痕疙瘩衍生细胞的表面， 与纯化的糖皮质激素诱导的蛋白质结合，并改变系统 A 氨基酸运输。 哺乳动物细胞中氨基酸转运蛋白的检测 由于缺乏影响运输的突变以及缺乏 特异性高亲和力配体。 瘢痕疙瘩可能是第一个例子 真皮成纤维细胞的基因突变直接或间接改变 氨基酸运输。 除了直接或间接提供之外 改变氨基酸转运。 除了提供信息之外 这项研究对于我们了解疤痕疙瘩很重要，可能会提供一个 研究其中包含的少数蛋白质之一的独特机会 糖皮质激素结构域并分离和表征氨基酸 来自人体细胞的转运蛋白。