MOLECULAR DEFECTS WITHIN THE SPECTRIN MEMBRANE SKELETON OF SICKLED CELLS

(改编自申请人的摘要)在本供资周期内， 调查人员已经证明了迟缓的一个主要原因 不可逆镰状细胞(ISC)膜骨架的解离，以及 导致ISC无法建模，这是一种单一的翻译后 ISCβ-肌动蛋白的修饰。在ISCβ-肌动蛋白中，二硫键是 在半胱氨酸284和半胱氨酸373之间形成，在 可逆性镰状细胞(RSC)或对照-β肌动蛋白。然而，幽灵蛋白是 也被证明对IS核心的缓慢解离有贡献 骨骼，尽管其结构缺陷仍有待确定。 此外，调查人员还证明，这些还原剂 二硫苏糖醇(DTT)和N-乙酰半胱氨酸均有抑制作用 在体外形成ISC。在拟议的授权期内，调查人员 将决定肌动蛋白的任何结构和功能改变 β-血影蛋白结合域(目标1)；利用兔自身抗体 它与对照阿尔法血影蛋白反应，但不与ISC阿尔法血影蛋白反应 定义阿尔法中存在的结构(和功能)修改 血影蛋白(目标2)；并开始人体试验，以确定N- 乙酰半胱氨酸可减少患者致密细胞数量和ISC 镰状细胞性贫血以及这种减少是否会导致 每年的镰状细胞危机(目标3)。

(Adapted from the Applicant's Abstract) During the current funding cycle, the investigators have demonstrated that a major cause of the slow dissociation of the irreversibly sickled cell (ISC) membrane skeleton, and resultant inability of the ISC to model, is a single post-translational modification in ISC beta-actin. In ISC beta-actin a disulfide bridge is formed between cysteine 284 and cysteine 373 which is not found in reversibly sickled cell (RSC) or control-beta actin. However, spectrin was also demonstrated to contribute to the slow dissociation of the IS core skeleton, although its structural defect remains to be determined. Furthermore, the investigators have demonstrated that the reducing agents dithiothreitol (DTT) and N-acetylcysteine both have the ability to inhibit ISC formation in vitro. In the proposed grant period the investigators will determine any structural and functional modification in the actin binding domain of beta spectrin (Aim 1); utilize a rabbit autoantibody which reacts with control alpha spectrin but not ISC alpha spectrin to define the structural (and functional) modifications that exist in alpha spectrin (Aim 2); and begin human trials to determine whether N- acetylcysteine can reduce the number of dense cells and ISC in patients with sickle cell anemia and whether this reduction will lead to less sickle cell crises per year (Aim 3).