Production of Bioactive NO: Origin of Hemoglobin E Associated Pathophysiology

描述(申请人提供)：Hb(Hb)E(2E26K)是世界上最常见的自然发生的突变Hb，在1121界面发生突变。EE患者表现出轻微的慢性贫血，而HBe/2-地中海贫血患者表现出一系列的临床表现，包括高发病率和死亡，通常是由心脏功能障碍引起的。HBe在红细胞病理生理学和导致HBe病的分子机制中的重要作用仍是个谜。由于HBe已被证明具有正常的氧亲和力(Bunn等人，1972)，我们提出了一种可能的机制，即HBe可能降低了产生足够生物活性一氧化氮(NO)的能力(1)以保护来自2-地中海贫血的高水平膜损伤活性氧物种(ROS)，以及(2)作为内皮功能的次要NO来源。为了支持这一假设，我们从HBe获得了初步数据，显示与HBA相比，亚硝酸还原酶活性降低。本课题组最近获得了高分辨率的脱氧和连接的HBe结构(蛋白质数据库条目1YVQ，1YVT，3DUT)，并发现HBe的T和R四元结构中的三级构象相对于HBA发生了变化。拟议的项目建立在这两个发现的基础上，试图确定亚硝酸盐反应性改变的程度和分子来源。该项目将利用创新的溶胶-凝胶封装协议来捕获和表征HBe的T和R状态相对于一系列反应的反应性，这些反应被认为有助于产生生物活性NO。该项目试图确定HBe从亚硝酸盐中产生生物活性形式NO的反应性改变的来源是否源于变构、局部三级结构或T和R状态的氧化还原性质的变化。这些研究具有重要意义，因为它们可能为HBe衍生的病理生理学的起源提供一种新的机制，并可能对其他与乙肝相关的病理和与内皮功能障碍有关的疾病产生影响。有可能有一种新的范例来开发治疗HBe2-地中海贫血的方法。 公共卫生相关性：这项建议旨在了解引起血红蛋白E疾病的根本原因，其中一种可能导致身体和精神发育迟缓，并可能导致死亡，通常是由于心脏功能障碍。这项提议的目的是检验这一假设，即这种疾病起源于HBe的特性，这种特性降低了其从亚硝酸盐产生一氧化氮的能力。这些发现有可能改变医生对这种往往致命的HBe/2地中海贫血疾病的看法和治疗方式。

DESCRIPTION (provided by applicant): Hemoglobin (Hb) E (2E26K) is the most common worldwide naturally occurring mutant Hb with a mutation at the 1121 interface. EE individuals exhibit a mild, chronic anemia while HbE/2-thalassemia individuals show a range of clinical manifestations, including high morbidity, and death, often resulting from cardiac dysfunction. The significant role of HbE in the red blood cell pathophysiology and molecular mechanisms giving rise to the HbE diseases is enigmatic. Since, HbE has been shown to have normal oxygen affinity (Bunn et al., 1972), we have proposed a possible mechanism whereby HbE may have reduced capacity to generate sufficient bioactive nitric oxide (NO) (1) to confer protection against high levels of membrane damaging reactive oxygen species (ROS) arising from the 2-thalassemia and (2) as a secondary NO source for endothelial functioning. In support of this hypothesis we have obtained preliminary data from HbE showing decreased nitrite reductase activity compared to HbA. Our group recently obtained the high resolution deoxy and liganded HbE structures (Protein Data Bank entries 1YVQ, 1YVT, 3DUT) and found that the tertiary conformations within the T and R quaternary structures of HbE are altered relative to HbA. The proposed project which builds on these two findings, seeks to establish the extent and molecular origins of the altered nitrite reactivity. The project will utilize innovative sol-gel encapsulation protocols to trap and characterize the reactivity of the T and R state of HbE with respect to a series of reactions proposed to contribute to the production of bioactive NO. The project seeks to determine whether the source of HbE altered reactivity to generate bioactive forms of NO from nitrite arises from changes in allostery, in local tertiary structure or in the redox properties of the T and R states. These studies are of significance in that they are likely to provide a novel mechanism for the origin of HbE-derived pathophysiology with implications for other Hb related pathologies and diseases linked to endothelial dysfunction. There is the potential for a new paradigm in which to develop therapies for HbE 2-thalassemia. PUBLIC HEALTH RELEVANCE: This proposal aims to understand the fundamental causes that give rise to Hemoglobin E diseases, one of which can cause body and mental retardation and can lead to death, usually from heart malfunction. It is the aim of this proposal to test the hypothesis that this disease originates from properties of HbE that reduce its capacity to produce nitric oxide from nitrite. These findings have the potential to change the way doctors view and treat the often fatal HbE/2-thalassemia disease.