Incidence of Infection in Young Children with Sickle Cell Disease in Africa

本项目的总体目标是进一步阐明脱氧血红蛋白S的聚合机理。 探讨HB-S聚合物中蛋白质相互作用部位的氨基酸作用及构型开发 抑制Hb S聚合和改善镰状细胞的抗病药物，包括多肽 疾病(SCD)。最近我们发现Hb C-Harlem(c_：13，.6Gi，-_-_w7，.3)的均相成核过程 A，_A，O)比HbS慢约10倍。此外，我们的微分干涉对比度(DIC) Hb C-Harlem的电子显微镜研究显示有晶体形成，但没有形成纤维或其他 聚合产物具有HB S特征的聚合产物，如大纤维或大束这些发现表明ITAE 1373对Hb-S聚合物性能的影响，并由此提出了氨基酸在Hb-Hb-Hb上的作用假说 血红蛋白聚合物的轴向和侧向接触。我们假设(1)Hb S分子的运动 在聚合物形成之前的核内是由协同分布的相互作用能控制的 侧向和轴向接触，例如136、t322和1373位氨基酸通过疏水、静电和氢气 (2)~(134)Thr对B73Asp氢键作用的增强 I-T5 S聚合物促进14股纤维的形成，而减弱这种相互作用会导致双股纤维的形成 晶体的形成。为了测试这些亚型，我们将使用突变策略来设计HB S的变种 关键相互作用部位的突变。我们将评估动力学和热力学性质以及聚合物 由此产生的血红蛋白变体的结构。在特定目标2中，基于蛋白质的基本信息 HB S聚合物的相互作用我们将识别和开发基于结构的试剂，包括多肽， 它们与HB S聚合物的轴向和侧向接触位置相互作用。我们将首先进行反聚合 含有134Thr的A-螺旋结构域上的肽图测试，预计将强烈 结合含有1373Asp或1373His的E-螺旋结构域。利用基于计算机的结构分析，我们将 寻找与E-螺旋上的1373Asp和/或Ahelix上的134Thr相互作用的基于结构的化学物质或肽 抑制Hb S的成核和聚合。我们还将使用噬菌体展示技术来生产 与E-螺旋结构域结合的多肽，并筛选这些多肽以找到与其具有更好结合性能的多肽 1373Asp，而不是HbS的天然A-螺旋结构域，然后对鉴定的多肽进行检测 用于使用体外测试的反聚合性能。我们认为通过病毒同时传入 这些多肽的载体(阻断关键的蛋白质-蛋白质相互作用)和γ-珠蛋白链(形成Hb F)在 SCD枯萎病的基因治疗为治疗这种疾病提供了一种新的治疗方法。

The overall goals of this project are to further elucidate the mechanism of polymerization of deoxy Hb S by exploring the role of amino acids at protein-protein interaction sites of Hb S polymers and to develop structurebased anti-sickling agents, including peptides, that will inhibit Hb S polymerization and ameliorate sickle cell disease (SCD). We recently found that the homogeneous nucleation process of Hb C-Harlem (c_:13,.6GI,-_-_w7,.3 A,_A,o) is about 10:?-fold slower than that of Hb S. In addition, our differential interference contrast (DIC and electron microscopic studies of Hb C-Harlem showed crystal formation but no formation of fiber or othe polymerization products characteristic of Hb S, such as macrofibers or bundles. These findings indicate itae influence of 1373on properties of Hb S polymers and led to our hypotheses regarding the role of amino acids at the axial and lateral contacts of hemoglobin polymers. We hypothesize that (1) movement of Hb S molecules within nuclei prior to polymer formation is controlled by synergistically distributed interaction energies of lateral and axial contacts, e.g., amino acids at 136,t322 and 1373by hydrophobic, electrostatic and hydrogen bond interactions, respectively, and (2) enhancement of the B73 Asp hydrogen bond interaction with 134Thr in I-t5 S polymers promotes 14-stranded fiber formation, while weakening this interaction leads to two-stranded crystal formation. To test these hypo;heses, we will use mutagenesis strategies to engineer Hb S variants with mutations at critical interaction sites. We will evaluate kinetic and thermodynamic properties as well as polymer structure of the resultant hemoglobin variants. In specific aim 2, based on the basic information of the proteinprotein interactions of Hb S polymers we will identify and develop structure-based agents, including peptides, which interact with axial and lateral contact sites of Hb S polymers. We will first perform an antipolymerization test of peptide mapping in the A-helix domains containing 134Thr, which is expected to strongly bind the E-helix domain containing 1373Asp or 1373His. Using computer-based structural analysis, we will seek structure-based chemicals or peptides that interact with 1373Asp on the E-helix and/or 134Thr on the Ahelix to inhibit Hb S nucleation and polymerization. We will also use phage display technologies to produce peptides that bind to the E-helix domain and screen these peptides to find those with better binding properties to the 1373Asp in the E-helix than the native A-helix domains of Hb S. The identified peptides then will be tested for anti-polymerization properties using in vitro assays. We believe that simultaneous introduction by viral vectors of these peptides (blocking critical protein-protein interactions) and of y-globin chains (to form Hb F) in gene therapy of SCD wilt result in the generation of a novel therapeutic way for treating this disorder.