Studies of Erythrocyte Membrane Structure

红细胞膜结构的研究

• 批准号: 8056620
• 负责人: PHILIP Stewart LOW
• 金额: $ 44.26万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-07-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8056620
• 关键词: Actins; Address; Affinity; Anions; Ankyrins; Antigens; Binding; Binding Sites; Blood; Blood Clot; Blood coagulation; Blood flow; CD47 gene; Carbon Dioxide; Cell Volumes; Cell membrane; Cells; Clinical; Complex; Crystallization; Cytoplasmic Protein; Cytoplasmic Tail; Cytoskeleton; Defect; Diffusion; Erythrocyte Anion Exchange Protein 1; Erythrocyte Membrane; Erythrocytes; Excision; Family member; Glucose Transporter; Glycophorin A; Health; Human; Image; Inherited; Intervention; Ion Transport; Ions; Knock-in Mouse; Lead; Link; Lipid Bilayers; Lipids; Lung; Malaria; Maps; Mechanics; Mediating; Membrane; Membrane Proteins; Membrane Structure and Function; Metabolism; Methods; Modeling; Molecular; Monitor; Monoclonal Antibodies; Mus; Mutate; Mutation; Nature; Nitric Oxide; Normal Cell; Parasites; Pathology; Phase; Population; Process; Property; Proteins; Reaction; Regulation; Reporting; Research; Resolution; Rupture; Site; Skeleton; Spectrin; Structure; Testing; Tissues; Videotape; Wound Healing; adducin; base; deoxyhemoglobin; imaging modality; in vivo; member; mutant; novel strategies; oxygen transport; prevent; protein 4.1; receptor; senescence; solute; tool; water channel

DESCRIPTION (provided by applicant): The human erythrocyte membrane (RBCM) is the most thoroughly studied plasma membrane, not only because it serves as an accessible model of other human membranes, but also because inherited and acquired defects in its components lead to serious pathologies. Despite this scrutiny, fundamental aspects of the structure and function of the RBCM remain poorly understood. In Aim 1, we will characterize the structure and function of a newly discovered bridge (band 3 to 2-adducin to spectrin) that links the lipid bilayer to the spectrin-actin skeleton. We have recently demonstrated that ~1/3 of the band 3 population is anchored to the spectrin/actin junctional complex via this bridge and that rupture of the bridge leads to membrane fragmentation. To evaluate the significance of the bridge in vivo, we will map the binding site of adducin on band 3, identify mutations in band 3 that prevent adducin binding, generate a mouse containing the mutant band 3, and evaluate the morphological and mechanical properties of the mutant erythrocytes. Included as a major component of this aim is the characterization of a method to image the diffusion of single band 3 molecules in intact erythrocytes as a tool to sensitively monitor perturbations of RBCM structure. The membrane-spanning domain of band 3 (msdb3) not only mediates anion transport across the membrane, but also organizes a complex of membrane-spanning proteins, including glycophorin A, CD47, Rh proteins, aquaporin, and several transporters. In order to understand the function of msdb3 at a molecular level, we will solve its crystal structure at high resolution (This will be the first structure of any member of solute carrier class IV). We currently have crystals that diffract to <6E, and although we could solve a 6E structure now, we are confident that we can generate much higher resolution crystals using the novel strategies outlined in Aim 2. Strong evidence suggests that 1) red cell metabolism, 2) membrane structural properties, and 3) ion transport are regulated by O2. Because deoxyhemoglobin (but not oxyHb) binds with high affinity to band 3, and since band 3 associates with proteins responsible for each of the above properties, we hypothesize that the reversible association of band 3 with deoxyHb constitutes the "molecular switch" through which red cell oxygenation regulates membrane properties. In Aim 3, we will test this hypothesis using recently discovered band 3 mutations that either: i) eliminate all affinity for deoxyHb, or ii) enhance the affinity of deoxyHb for band 3 so significantly that deoxyHb can neither release its O2 nor dissociate from band 3, even at saturating O2. We propose to generate knock-in mice that express these two mutant band 3s, and use the mice to determine if the above O2- regulated properties are permanently "switched on" or permanently "switched off", as predicted. PUBLIC HEALTH RELEVANCE: The red blood cell performs a variety of functions critical to our survival, including transport of oxygen from the lungs to the tissues and carbon dioxide from the tissues to the lungs, delivery of nitric oxide to facilitate blood flow, participation in blood clotting to facilitate wound healing, and regulation of a number of other reactions that occur in the blood. Our research seeks to understand the molecular basis of each of these important functions, and where possible, to define clinical interventions that might enable treatment of conditions where the above processes malfunction.

描述（由申请人提供）：人类红细胞膜（RBCM）是最彻底研究的质膜，这不仅是因为它是其他人类膜的可访问模型，而且还因为其成分中的遗传和获得的缺陷导致了严重的病理学。尽管进行了审查，但RBCM的结构和功能的基本方面仍然知之甚少。在AIM 1中，我们将表征将脂质双层连接到Spectrin-actin骨骼的新发现的桥（带3至2-杜非蛋白与光谱）的结构和功能。我们最近证明，3个频带3个种群的〜1/3通过这座桥锚定在谱蛋白/肌动蛋白连接络合物上，并且桥的破裂导致膜碎片。为了评估体内桥的显着性，我们将绘制Adducin在频段3上的结合位点，鉴定在3个带3中的突变，以防止Adducin结合，产生含有突变体带3的小鼠，并评估突变型红细胞的形态和机械性能。作为此目的的主要组成部分，包括一种方法来描述单个条带3分子在完整的红细胞中的扩散，以敏感地监视RBCM结构的扰动的工具。带3（MSDB3）的膜跨膜结构域不仅介导整个膜的阴离子转运，而且还组织了一系列跨膜蛋白质，包括糖蛋白A，CD47，RH蛋白，水普通道蛋白和几个转运蛋白。为了了解MSDB3在分子水平上的功能，我们将在高分辨率下解决其晶体结构（这将是溶质载体IV类的任何成员的第一个结构）。我们目前的晶体衍射为<6e，尽管我们现在可以解决6E结构，但我们相信我们可以使用AIM 2中概述的新型策略产生更高的分辨率晶体。有力的证据表明1）1）红细胞代谢，2）膜结构和3）O2调节离子运输。由于脱氧血红蛋白（而非oxyHb）与频带3具有高亲和力结合，并且由于频带3与负责上述每个特性的蛋白质相关联，因此我们假设频带3与脱氧hb的可逆关联构成了红细胞氧化的“分子开关”，从而调节了红细胞的氧化物。在AIM 3中，我们将使用最近发现的频带3突变检验这一假设，即要么：i）消除对脱氧hb的所有亲和力，或ii）增强了对频段3的亲和力，因此即使在饱和的O2饱和的O2时，DeoxyHb也无法释放其O2也不能与3频段分离。我们建议生成表达这两个突变带3的敲击小鼠，并使用小鼠确定上述O2调节特性是永久性地“打开”或永久性地“关闭”的。公共卫生相关性：红细胞发挥了各种对我们生存至关重要的功能，包括从肺部到组织的氧气和二氧化碳从组织到肺的运输，一氧化氮的传递，以促进血液流动，促进血液凝结的血液凝结以促进伤口愈合，并调节其他发生在血液中的反应。我们的研究试图了解这些重要功能中每一个的分子基础，并在可能的情况下定义临床干预措施，以便可以治疗上述过程故障的情况。