Project 3/Struct. of the CDB3:ankyrin:protein 4.2 complex in erythrocytes by EPR

人类红细胞表现出不同寻常的双凹圆盘形状，同时具有显著的稳定性和 变形性，所有这些都是它们在循环系统中生存所必需的。它现在已经很成熟了 不寻常的细胞形状和膜的机械性能在很大程度上是由于一种 肌动蛋白广泛的膜骨架，主要由排列在内侧的血影蛋白和肌动蛋白组成 膜表面和膜骨架与本征之间的特定桥联作用 脂双层中的膜蛋白。幽灵蛋白-肌动蛋白骨架通过与膜双层结合。 两种类型的接触，一种涉及短肌动蛋白原丝和蛋白质4.1与细胞质相互作用 第二个涉及连接蛋白ankyrinR和蛋白4.2的相互作用 与阴离子交换蛋白(AE1)的细胞质结构域结合。这个项目试图刻画 在人类红细胞中形成这种第二类桥联作用的蛋白质复合体的结构。 这种关注是由以下知识决定的，即在第二类相互作用中的变化会导致球形 红细胞体积变小，脆性增加，临床上称为遗传性 球形细胞增多症(HS)。HS是一系列疾病，发生在目前2000到3000个家庭中的一个家庭 临床表现为不同程度的溶血性贫血，由球形红细胞溶血引起。 它们遍历微循环。最近的数据表明，15%-20%的HS病例可归因于AE1 约50%的HS病例是由锚蛋白缺陷引起的(4)。结合蛋白4.2的减少 膜也是常见的发现(5)。 过去三十年的研究导致了固有膜和外周膜的定义 聚集在一起以稳定红细胞膜的蛋白质(在(6，7)中回顾)。球队的核心球员 这些蛋白质的组装是AE1的细胞质结构域(也称为带3)。细胞质结构域 带3(Cdb3)被认为是结合ankyrinR(8)和蛋白质的组织中心 4.2(9)以及其他胞浆蛋白，包括GAPDH(10，11)，醛缩酶(12)，磷酸果糖激酶(13)， 血红蛋白(14)、半红细胞(15)和p72syk(16)。图1显示了一些键的示意图 已阐明的蛋白质-蛋白质相互作用。

Human erythrocytes exhibit an unusual biconcave disc shape together with remarkable stability and deformability, all of which are necessary for their survival in the circulatory system. It is now well established that the unusual cell shape and membrane mechanical properties are due in large part to the presence of an extensive membrane skeleton, composed primarily of the proteins spectrin and actin, that lines the inner membrane surface and to specific bridging interactions between this membrane skeleton and intrinsic membrane proteins in the lipid bilayer. The spectrin-actin skeleton associates with the membrane bilayer via two types of contacts, one involving short actin protofilaments and protein 4.1 interacting with the cytoplasmic domain of glycophorin C, and the second involving the bridging protein ankyrinR and protein 4.2 interacting with the cytoplasmic domain of the anion exchange protein (AE1). This project seeks to characterize the structure of the complex of proteins that forms this second class of bridging interactions in human erythrocytes. This focus is dictated by the knowledge that alterations in this second class of interactions results in spherical erythrocytes with decreased cell size and increased fragility, a condition known clinically as hereditary spherocytosis (HS). HS is a spectrum of diseases, occurring in one family out of 2,000 to 3,000, that present clinically as varying degrees of hemolytic anemia resulting from hemolysis of the spherical erythrocytes when they traverse the microcirculation. Recent data have indicated that 15-20% of HS cases are attributable to AE1 defects and ~50% of HS cases result from ankyrin defects (4). A reduction in protein 4.2 binding to the membrane is also a common finding (5). Studies over the past three decades have led to the definition of the intrinsic and peripheral membrane proteins that assemble to stabilize the erythrocyte membrane (reviewed in (6,7)). A central player in the assembly of these proteins is the cytoplasmic domain of AE1 (also known as band 3). The cytoplasmic domain of band 3 (cdb3) has been hypothesized to serve as an organizing center for binding ankyrinR (8) and protein 4.2 (9) as well as other cytosolic proteins including GAPDH (10,11), aldolase (12), phosphofructokinase (13), hemoglobin (14), hemichromes (15), and p72syk (16). Figure 1 shows a schematic diagram of some of the key protein-protein interactions that have been elucidated.