Dysregulation of the Sickle Cell Membrane Skeleton

项目负责人实验室的主要目标是了解纯合镰状细胞（SS）红细胞（RBC）成为不可逆镰状细胞（ISCs）和致密的机制;朝着设计阻止ISCs形成和细胞脱水的治疗方法迈出了一步。我们已经证明，ISC膜骨架在37摄氏度下分解得比可逆镰状细胞（RSC）和对照膜骨架慢得多。这表明，ISCs不能改变形状的原因是因为它们的膜骨架不能动态地分解和重新组装。此外，我们证明了这种缓慢的分解是血影蛋白和肌动蛋白修饰的线索。ISC β-肌动蛋白中的修饰已被证明是C284-C373二硫桥，其导致肌动蛋白丝缓慢分解。我们还不知道ISC血影蛋白的修饰会导致 血影蛋白-4.1-肌动蛋白三元复合物的较慢解离。血影蛋白是一种E2泛素结合酶，也是泛素的靶点。E2硫酯位点和一个靶位点位于血影蛋白重复序列α 20/21内。有趣的是，SS血影蛋白在其DTT敏感的E2位点和DTT不敏感的靶位点的泛素化降低了80 - 90%，可能是由于E2半胱氨酸的谷胱甘肽化。由于α 20/21重复序列是异二聚体成核位点，并与β-血影蛋白上的蛋白4.1和内收蛋白位点相关，我们假设SS血影蛋白的泛素化缺乏将导致异二聚体形成的更快速率和更高亲和力的血影蛋白-4.1-肌动蛋白和血影蛋白-内收蛋白-肌动蛋白三元复合物的形成。这将提供一个答案，为什么ISC骨骼解离远慢于RSC或控制骨骼。在这个建议中，我们将确定确切的半胱氨酸和赖氨酸参与E2 (and可能是E3）和α 20/21的靶位点;利用同位素编码的亲和标签和LC-MS/MS（Aim 1）的蛋白质组学技术。然后我们将确定幽灵蛋白的作用 泛素化在调节异源二聚体形成（Aim 2）和血影蛋白-4.1-肌动蛋白和血影蛋白-内收蛋白-肌动蛋白形成和分解（Aim 3）中的作用。

The major goal of the Project Head's laboratory is to understand the mechanisms by which homozygous sickle cell (SS) red blood cells (RBCs) become irreversible sickled cells (ISCs) and dense; a step towards devising therapeutics that block formationof ISCs and cellular dehydration. We have demonstrated that ISC membrane skeletons disassemble far more slowly at 37 degrees C than reversible sickled cells (RSCs) and control membrane skeletons. This suggested that the reason that ISCs cannot change shape is because their membrane skeleton cannot dynamically disassemble and reassemble. Furthermore, we demonstrated that this slow disassembly was clue to modifications in spectrin and actin. The modification in ISC beta-actin has been demonstrated to be a C284-C373 disulfide bridge which leads to actin filaments that slowly disassemble. We do not yet know the modification of ISC spectrin that leads to a slower disassociation of the spectrin-4.1-actin ternary complex. Spectrin is an E2 ubiquitin conjugating enzyme as well as a target for ubiquitin.The E2 thioester site and one target site are within spectrin repeats alpha 20/21. Interestingly, ubiquitination of SS spectrin in its DTT sensitive E2 site and DTT insensitive target sites is reduced by 80 to 90% probably due to glutathiolation of the E2 cysteine. Since the alpha 20/21 repeat is the heterodimer nucleation site and associated with the protein 4.1 and adducin sites on beta-spectrin, we hypothesize that lack of ubiquitination of SS spectrin would lead to faster rates of heterodimer formation and higher affinity spectrin-4.1-actin and spectrin-adducin-actin ternary complex formation. This would supply an answer to why the ISC skeleton dissociates far more slowly than the RSC or control skeleton. In this proposal we will identify the precise cysteines and lysines involved in E2 (and possibly E3) and target sites of alpha 20/21 respectively; utilizing the proteomics technology of isotope coded affinity tags and LC-MS/MS (Aim 1). We will then determine the role of spectrin ubiquitination in regulating heterodimer formation (Aim 2) and spectrin-4.1-actin and spectrin-adducin-actin formation and disassembly (Aim 3).