Dysregulation of the Sickle Cell Membrane Skeleton

镰状细胞膜骨架失调

• 批准号: 7409091
• 负责人: STEVEN Richard GOODMAN
• 金额: $ 24.19万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7409091
• 关键词: Actins; Active Sites; Affinity; Alabama; Amelia; Amino Acids; Applications Grants; Award; Binding; Binding Proteins; C-terminal; Cell membrane; Cells; Cellular biology; Cleaved cell; Clinical; Complex; Computer Analysis; Computers; Cysteine; Dehydration; Dissociation; Disulfides; Elasticity; Enzymes; Erythrocytes; Erythropoiesis; Event; F-Actin; Funding; Glycophorin C; Goals; Grant; Head; Institution; Integral Membrane Protein; Isotopically-Coded Affinity Tagging; Laboratories; Lead; Lysine; Maps; Mechanics; Medical center; Membrane; Microfilaments; Modification; Molecular; Morbidity - disease rate; N-terminal; Object Attachment; Personal Satisfaction; Pliability; Principal Investigator; Progress Reports; Property; Proteins; Proteomics; Rate; Regulation; Research; Role; Scientist; Shapes; Sickle Cell; Sickle Cell Anemia; Site; Skeletal system; Skeleton; Spectrin; Structure; Surface; System; Tail; Techniques; Technology; Temperature; Testing; Therapeutic; Ts-72; Ubiquitin; Ubiquitin-Conjugating Enzymes; Ubiquitination; United States National Institutes of Health; Universities; adducin; adduct; base; beta Actin; beta Spectrin; depolymerization; falls; interest; monomer; mortality; multicatalytic endopeptidase complex; professor; programs; protein 4.1; protein degradation; protein protein interaction; recombinant peptide; thioester

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).

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) 变成不可逆镰状细胞 (ISC) 和致密的机制；朝着设计阻止 ISC 形成和细胞脱水的疗法迈出了一步。我们已经证明，ISC 膜骨架在 37 摄氏度下的分解速度比可逆镰状细胞 (RSC) 和对照膜骨架慢得多。这表明ISC不能改变形状的原因是它们的膜骨架不能动态拆卸和重组。此外，我们证明这种缓慢的分解是血影蛋白和肌动蛋白修饰的线索。 ISC β-肌动蛋白的修饰已被证明是 C284-C373 二硫键，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解离速度较慢。血影蛋白是一种 E2 泛素结合酶，也是泛素的靶标。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 E2 位点和 DTT 不敏感靶位点中的泛素化减少了 80% 至 90%，可能是由于 E2 半胱氨酸的谷胱甘肽化。由于 α 20/21 重复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，因此我们假设 SS 血影蛋白缺乏泛素化将导致更快的异二聚体形成速率和更高的亲和力血影蛋白-4.1-肌动蛋白和血影蛋白-内收蛋白-肌动蛋白三元复合物形成。这可以解释为什么 ISC 骨架的解离速度比 RSC 或控制骨架慢得多。在此提案中，我们将确定 E2 中涉及的精确半胱氨酸和赖氨酸 （可能还有 E3）和 alpha 20/21 的靶位点；利用同位素编码亲和标签和 LC-MS/MS 的蛋白质组学技术（目标 1）。然后我们将确定血影蛋白的作用 泛素化调节异二聚体形成（目标 2）以及血影蛋白 4.1 肌动蛋白和血影蛋白内收蛋白肌动蛋白形成和分解（目标 3）。