MOLECULAR DEFECTS WITHIN THE SPECTRIN MEMBRANE SKELETON OF SICKLED CELLS

（改编自申请人摘要）在本供资周期内， 调查人员已经证明， 不可逆镰状细胞（ISC）膜骨架的解离，和 结果ISC无法建模，是一个单一的翻译后 ISC β-肌动蛋白的修饰。在ISC β-肌动蛋白中， 在半胱氨酸284和半胱氨酸373之间形成，其在 可逆镰状细胞（RSC）或对照β肌动蛋白。然而，血影蛋白是 也被证明有助于IS核心的缓慢解离 骨骼，虽然其结构缺陷仍有待确定。 此外，研究人员已经证明， 二硫苏糖醇（DTT）和N-乙酰半胱氨酸都具有抑制 体外ISC形成。在研究期间，研究人员 将决定肌动蛋白的任何结构和功能修饰 β血影蛋白结合域（Aim 1）;利用兔自身抗体 其与对照α血影蛋白反应，但不与ISC α血影蛋白反应， 定义alpha中存在的结构（和功能）修饰 血影蛋白（目标2）;并开始人体试验，以确定是否N- 乙酰半胱氨酸可以减少患者的致密细胞和ISC数量 镰状细胞性贫血，以及这种减少是否会导致 镰状细胞病危机（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).