Production of Bioactive NO: Origin of Hemoglobin E Associated Pathophysiology

项目概要和相关性 血红蛋白（Hb）E（² E26 K）是世界上最常见的天然突变血红蛋白，具有 在<$1 <$1界面处发生突变。EE个体表现出轻度慢性贫血，而HbE/<$-地中海贫血 个体表现出一系列临床表现，包括高发病率和死亡，通常导致 心脏功能障碍HbE在红细胞病理生理学中的重要作用， 引起HbE疾病的分子机制是谜。从那时起，HbE已被证明 具有正常的氧亲和力（邦恩等人，1972年），我们提出了一种可能的机制， HbE可能降低了产生足够的生物活性一氧化氮（NO）的能力（1）， 保护免受高水平的膜损伤活性氧（ROS）的产生， （2）作为内皮功能的次要NO来源。为支持这一 假设我们已经从HbE中获得了显示亚硝酸还原酶活性降低的初步数据 与HbA相比我们的团队最近获得了高分辨率的脱氧和配体HbE结构 （蛋白质数据库条目1 YVQ，1 YVT，3DUT），发现T HbE和R的四级结构相对于HbA改变。拟议的项目建立在 这两项发现旨在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反应性 的T和R状态的HbE相对于一系列的反应，提出了有助于 该项目旨在确定HbE的来源是否改变 从亚硝酸盐产生NO的生物活性形式的反应性由变构的变化引起，在局部 三级结构或T和R状态的氧化还原性质。这些研究对于 它们可能为HbE衍生的病理生理学的起源提供新的机制， 对与内皮功能障碍相关的其他Hb相关病理和疾病的影响。有 开发HbE-地中海贫血疗法的新范例的潜力。

PROJECT SUMMARY & RELEVANCE Hemoglobin (Hb) E (¿E26K) is the most common worldwide naturally occurring mutant Hb with a mutation at the ¿1¿1 interface. EE individuals exhibit a mild, chronic anemia while HbE/¿-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 ¿-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 ¿-thalassemia.