THE RED CELL CYTOSKELETON AND SICKLE CELL DISEASE

镰状细胞性贫血的特征是多种临床表现和 细胞异常，由于存在的结果， 突变基因产生血红蛋白S（HbS）。 虽然很可能很多人 伴随的细胞缺陷的获得，机制， 它们的出现仍然是未知的。 本提案的长期目标 是确定负责膜的发展的机制， SS细胞异常。 为了实现这一目标，我们将测试 假设氧化损伤在发展中起作用， SS细胞膜和细胞骨架蛋白功能异常。 我们将重点关注氧化对蛋白质关键成分的影响， 红细胞膜和细胞骨架，包括血影蛋白、锚蛋白和 波段3。 蛋白质氧化损伤将通过与 巯基特异性化学探针和通过直接分析蛋白质 个氨基酸 进一步的研究将检查蛋白质的功能， 确定功能是否因氧化修饰而改变。 血影蛋白和钙调蛋白之间的相互作用将被研究，以确定 如果这些蛋白质之间的异常相互作用在 产生不可逆的镰状细胞。 功能研究将 确定是否酶参与维持蛋白质巯基 稳态，如巯基转移酶，在SS细胞中被破坏。 的作用 镰状化事件本身在蛋白质氧化 将在氧合-脱氧循环研究中检查病变。 的 将测量确定的SS细胞群的抗氧化能力， 确定氧化损伤是否是由于某些细胞的能力降低而导致的， SS细胞修复氧化损伤。 蛋白质氧化在 将通过以下方法研究形态学异常的发展： 使用巯基特异性化学物质的荧光和电子显微术 probes. 蛋白质氧化在发育中的作用 细胞体积调节的异常将使用定义的 K：C1共转运活性高或低的细胞。 HbS的作用 结合到膜和铁去分解在 细胞形态和蛋白质功能异常的发展将 在具有确定量的HbS的转基因HbS小鼠中进行测试。 在一起， 这些研究可能有助于阐明 SS细胞膜异常的发展，以及我们更好的 了解这种疾病和其他疾病的病理生理学 以不稳定的血红蛋白为特征的血红蛋白病。

Sickle cell anemia is characterized by a multitude of clinical and cellular abnormalities that arise as a consequence of the presence of the mutant gene produce hemoglobin S (HbS). Although it is likely that many of the attendant cellular defects are acquired, the mechanisms by which they arise are still unknown. The long-term objective of this proposal is to define the mechanisms responsible for the development of membrane abnormalities in SS cells. To achieve this objective we will test the hypothesis that oxidative damage plays a role in the development of membrane and cytoskeletal protein functional abnormalities in SS cells. We will focus on the effects of oxidation on key protein components of the red cell membrane and cytoskeleton, including spectrin, ankyrin and band 3. Protein oxidative damage will be assessed by binding to sulfhydryl-specific chemical probes and by direct analysis of protein amino acids. Additional studies will examine protein function to determine if function has been altered by oxidative modifications. Interactions between spectrin and calmodulin will be studied to determine if abnormal interactions between these proteins plays a role in the generation of irreversibly-sickled cells. Functional studies will determine if enzymes involved in maintaining protein sulfhydryl homeostasis, such as thioltransferase, are damaged in SS cells. The role of the sickling event per se in the development of protein oxidative lesions will be examined in oxygenated-deoxygenated cycling studies. The antioxidant capacity of defined SS cell populations will be measured to determine if oxidative damage results from a decreased capacity of some SS cells to repair oxidative lesions. The role of protein oxidation in the development of morphologic abnormalities will be studied by fluorescent and electron microscopy using sulfhydryl-specific chemical probes. The role of protein oxidation in the development of abnormalities in cell volume regulation will be studied using defined cells with high of low K:C1 cotransport activity. The role of HbS binding to the membrane and iron decompartmentalization in the development of cell morphologic and protein functional abnormalities will be tested in transgenic HbS mice with defined amounts of HbS. Together, these studies may help to elucidate the mechanisms responsible for the development of membrane abnormalities in SS cells and better our understanding of the pathophysiology of this disease and of other hemoglobinopathies characterized by unstable hemoglobins.