Spectrin-Actin Junctions of the Erythrocyte Skeleton

红细胞骨架的血影蛋白-肌动蛋白连接

• 批准号: 7266948
• 负责人: SAMUEL E LUX
• 金额: $ 40.06万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7266948
• 关键词: Accounting; Actins; Affect; Affinity; Amino Acids; Area; Attention; Binding; Binding Proteins; Binding Sites; Biological Assay; CD47 gene; Cell membrane; Cells; Cellular biology; Complex; Crosslinker; Cytoskeleton; Defect; EF-Hand Domain; Electron Microscopy; Erythrocyte Ghost; Erythrocytes; F-Actin; Fostering; Freeze Drying; Freezing; Glutathione S-Transferase; Gold Colloid; Grant; In Situ; Lipid Binding; Localized; Maps; Measures; Membrane; Membrane Proteins; Methods; Microfilaments; Modeling; Molecular; Morphologic artifacts; Mus; Mutation; Organelles; Peptide Fragments; Peptides; Phenotype; Post-Translational Protein Processing; Proteins; Resolution; Role; Scientist; Sedimentation process; Series; Site; Skeletal system; Skeleton; Spectrin; Structure; Tail; Testing; Variant; Work; alpha Spectrin; base; cofactor; crosslink; interest; mutant; photolysis; protein 4.1; research study; shear stress; stoichiometry

DESCRIPTION (provided by applicant): The red blood cell (RBC) membrane skeleton is composed principally of short F-actin filaments cross-linked by a2B2 spectrin tetramers with the assistance of protein 4.1 R. Actin and 4.1 R bind to the actin binding domain (BSpABD) at the tail of the spectrin B chain. The adjacent end of a-spectrin, called the EF hand domain (aSpEF), has been considered inert; however, the sph(1J) mouse, which has very unstable RBC membranes, makes a mutant a-spectrin that lacks the last 13 amino acids (aSpEFAC13), showing that the domain has an important function in spectrin-actin binding. 1) We will determine what binds to aSpEF, using pull-down assays or photoactivatable cross-linking, and extracts containing all RBC membrane proteins. Initial studies show that aSpEF but not aSpEFAC13 binds to protein 4.2 in a Ca (2+)-dependent manner. We will characterize this and other aSpEF-protein interactions and investigate whether post-translational modifications or other factors affect the binding. 2) We will ascertain whether binding site fragments that interfere with aSpEF interactions decrease membrane stability measured in the ektacytometer. 3) We will prepare a series of mini-spectrins consisting of the BSpABD domain and variants of the aSpEF domain attached by the last 4 spectrin repeats in each chain. The binding of these mini-spectrins to F actin and the roles of 4.1 R, 4.2 and other aSpEF binding proteins will be assessed. 4) Finally, we will identify proteins adjacent to 4.1 R or 4.2 on the membrane, or adjacent to aSpEF in actin junctional complexes using photoactivatable crosslinkers, and we will visualize the structure of the normal unstretched membrane skeleton and the skeletal defects in 4.2(-) and sph (1J) mice using high resolution EM and immunoEM. These studies will provide new information about the molecular junctions that form the core of the membrane skeleton and will illuminate the functions of the previously little studied EF hand domain. Since plasma membranes of all cells and membranes of many organelles have spectrin skeletons, these studies will be of broad interest in cell biology.

描述（由申请人提供）：红细胞（RBC）膜骨架主要由蛋白4.1 R. acTIN R. art-R. art-R. actin R.肌动蛋白和4.1 r结合在Spectrin B链的尾部与肌动蛋白结合结构域（BSPABD）结合，由A2B2 Spectrin Tetramer交联短的F-肌动蛋白丝组成。 A-spectrin的相邻端被认为是惰性的。然而，SPH（1J）小鼠具有非常不稳定的RBC膜，它使缺乏最后13个氨基酸（Aspefac13）的突变体A-谱蛋白，表明该结构域在Spectrin-Actin结合中具有重要功能。 1）我们将使用下拉测定或可光活化的交联，并提取包含所有RBC膜蛋白的提取物。初步研究表明，Aspef而非ASPEFAC13以Ca（2+）依赖性方式与蛋白质4.2结合。我们将表征这种和其他Aspef蛋白相互作用，并研究翻译后修饰或其他因素是否影响结合。 2）我们将确定干扰ASPEF相互作用的结合位点片段是否会降低在Ektacytometer中测得的膜稳定性。 3）我们将准备一系列由BSPABD结构域和Aspef结构域的变体组成的一系列迷你光谱蛋白，该变体由每个链中的最后4个Spectrin重复序列附加。这些微光谱与F肌动蛋白的结合以及4.1 R，4.2和其他ASPEF结合蛋白的作用将得到评估。 4) Finally, we will identify proteins adjacent to 4.1 R or 4.2 on the membrane, or adjacent to aSpEF in actin junctional complexes using photoactivatable crosslinkers, and we will visualize the structure of the normal unstretched membrane skeleton and the skeletal defects in 4.2(-) and sph (1J) mice using high resolution EM and immunoEM.这些研究将提供有关构成膜骨骼核心的分子连接的新信息，并将阐明先前研究的EF手动域的功能。由于许多细胞器的所有细胞和膜的质膜都具有光谱骨骼，因此这些研究将对细胞生物学具有广泛的兴趣。