MOLECULAR DEFECTS WITHIN THE SPECTRIN MEMBRANE SKELETON OF SICKLED CELLS

镰状细胞血影蛋白膜骨架内的分子缺陷

• 批准号: 6241966
• 负责人: STEVEN Richard GOODMAN
• 金额: $ 12.93万
• 依托单位: UNIVERSITY OF SOUTH ALABAMA
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-04-01 至 1998-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6241966
• 关键词: actins; chemical aggregate; cytoskeleton; electron microscopy; erythrocyte membrane; high performance liquid chromatography; human subject; human tissue; mass spectrometry; molecular pathology; polymerization; posttranslational modifications; protein 4.1; protein sequence; protein structure; sickle cell anemia; spectrin; temperature

The long term objective of this project is to identify the molecular defect(s) within the spectrin membrane skeleton of irreversibly sickled cells which cause these cells to lock into the sickled shape. In preliminary studies we have demonstrated that core skeletons which contain only spectrin, protein 4.1, and actin remain sickled when isolated from high density sickled cells. Furthermore, we have demonstrated that spectrin membrane skeletons. from normal red cells dissociate when prepared at 37 degrees C, while the skeletons from high density sickle cells remain associated- The fact that irreversibly sickled core skeletons remain associated at 37 degrees C is not due to covalent linkages; because these skeletons dissociate completely to spectrin, protein 4.1, and actin in the presence of SDS. Our hypothesis is that posttranslational modification(s) of spectrin, protein 4.1, or actin lead to an irreversibly sickled skeleton which resists dissociation at 37 degrees C. We will test this hypothesis with the following studies. The morphology of normal and sickle cell core skeletons prepared at 0 degrees C and 37 degrees C will he studied by negative staining and electronmicroscopy. The protein which is functionally defective will he identified by [1] studying the spectrin-4.1-f-actin interaction and spectrin dimer-dimer interaction when spectrin is isolated from control versus sickled cells. [2] Studying the spectrin-4.1-f-actin interaction when protein 4.1 is isolated from control versus sickled cells. [3] Determining the rate of polymerization of sickle cell versus control actin, and the ability of sickle cell versus normal actin protofilaments to serve as seeds for actin polymerization. Once the abnormal protein(s) have been identified the posttranslational modification(s) causing the problem will he identified by reverse phase HPLC separation of proteolytic digests of the sickle cell versus control skeletal protein, followed by protein sequencing of altered peptides by MS/MS mass spectrometry.

该项目的长期目标是确定分子 不可逆镰状血影蛋白膜骨架内的缺陷 导致这些细胞锁定成镰状形状的细胞。在 初步研究我们已经证明核心骨架 只含有血影蛋白、蛋白质 4.1 和肌动蛋白，当 从高密度镰状细胞中分离出来。此外，我们还有 证明了血影蛋白膜骨架。来自正常红细胞 在 37°C 下制备时会解离，而来自高温的骨架 密度镰状细胞仍然相关 - 不可逆转的事实 镰状核心骨架在 37 摄氏度下仍然保持联系并不是因为 共价键；因为这些骨骼完全解离 SDS 存在下的血影蛋白、蛋白质 4.1 和肌动蛋白。我们的假设 是血影蛋白、蛋白质 4.1 或 肌动蛋白导致形成不可逆的镰状骨架，抵抗解离 37 摄氏度。我们将用以下方法检验这个假设 研究。正常和镰状细胞核心骨架制备的形态 在 0 摄氏度和 37 摄氏度下，他将通过负染色进行研究 电子显微镜。功能缺陷的蛋白质将被 通过研究血影蛋白 4.1-f-肌动蛋白相互作用 [1] 确定 当血影蛋白与对照分离时，血影蛋白二聚体-二聚体相互作用 与镰状细胞相比。 [2] 研究血影蛋白-4.1-f-肌动蛋白相互作用 当从对照细胞和镰状细胞中分离出蛋白质 4.1 时。 [3] 确定镰状细胞与对照的聚合速率 肌动蛋白，以及镰状细胞与正常肌动蛋白原丝的能力 作为肌动蛋白聚合的种子。一旦出现异常蛋白质 已确定导致翻译后修饰 问题将通过反相 HPLC 分离来确定 镰状细胞与对照骨骼蛋白的蛋白水解消化， 然后通过 MS/MS 质量对改变的肽进行蛋白质测序 光谱测定法。