Role of hemoglobin b93cys residue in nitric oxide bioactivity

描述(申请人提供)：血红蛋白293cys残基是保守的，最近受到广泛关注，因为它是红细胞如何影响血管一氧化氮(NO)新陈代谢和功能的潜在调节剂。然而，涉及的确切功能和机制仍不清楚。了解这种残基如何控制血管一氧化氮功能是至关重要的，因为这些机制的功能障碍可能导致许多血管病理状态。在这项建议中，我们建立在新的小鼠模型产生的初步数据基础上，这些模型只表达野生型人类血红蛋白或人类血红蛋白，其中293cys残基已被红细胞中的ALA取代。具体地说，我们提供的数据表明，在生理条件下，红细胞和血红蛋白的脱氧激活了亚硝酸还原酶的活性，导致阴离子亚硝酸盐被单电子还原为NO。这一过程是通过控制血红蛋白的氧亲和力来变构调节的，有趣的是，它还受到293cys残基的调节。在此背景下，我们认为293cys残基在连接血红蛋白氧感应和亚硝酸盐来源的NO生物活性方面起着关键作用。相反，在急性炎症性疾病脓毒症期间，我们认为293cys是亚硝化应激形成S亚硝基血红蛋白(SNOHb)的靶点，而SNOHb又促进了与该疾病相关的血管和肺功能障碍。后者的数据表明，含有SNO的红细胞可以刺激中性粒细胞与肺内皮细胞的黏附，并以一种不依赖变构调节的方式引起血管扩张。肺部炎症和肺损伤都是脓毒症的特征。在这项提案中，这些新的概念将通过以下假设进行研究：在急性炎症过程中，293cys残基作为血管NO信号调节器的作用从依赖亚硝酸盐还原酶的机制转变为SNOHb依赖的机制，这将通过以下特定目标进行测试：1)确定293cys调节RBC依赖的NO血管细胞信号的机制；2)确定293cys残基在体内控制亚硝酸盐还原和NO依赖的血管细胞信号转导中的作用；3)确定293cys残基在脓毒症诱导的低血压和肺部炎症过程中影响RBC效应的作用。这些目标的实现将带来对机制的洞察，新的治疗靶点专注于红细胞如何调节NO代谢。 公共卫生相关性：红细胞在控制血管内稳态机制中起着重要作用。我们在此提出，通过调节红细胞控制一氧化氮的功能，血红蛋白的一个特定氨基酸残基(293cys残基)在这方面是至关重要的。在这项建议中，我们旨在阐明293cys残基在正常生理条件下和与脓毒症相关的炎症期间控制一氧化氮功能的具体机制，并希望通过这样做来确定新的治疗靶点和策略。

DESCRIPTION (provided by applicant): The hemoglobin 293cys residue is conserved and received much attention recently as a potential modulator of how red cells affect vascular nitric oxide (NO) metabolism and function. However, the precise function and mechanisms involved remain unclear. Understanding how this residue controls vascular nitric oxide function is critical since dysfunction in these mechanisms may contribute to a number of vascular pathological states. In this proposal we build upon preliminary data generated from novel mouse models that express exclusively either wild-type human hemoglobin or human hemoglobin in which the 293cys residue has been replaced with an Ala in their red cells. Specifically, we present data indicating that under physiological conditions, deoxygenation of red cells and hemoglobin activates a nitrite reductase activity that results in the one-electron reduction of the anion nitrite to NO. This process is regulated allosterically by controlling hemoglobin oxygen affinity and interestingly modulated by the 293cys residue. In this context we propose that the 293cys residue is critical in coupling hemoglobin oxygen sensing with nitrite derived NO-bioactivity. In contrast, during the acute inflammatory disease Sepsis, we propose that the 293cys is a target for nitrosative stress forming S-nitrosohemoglobin (SNOHb), which in turn contributes to the vascular and pulmonary dysfunction associated with this disease. The latter is indicated by data showing SNO-containing red cells can stimulate neutrophil adhesion to pulmonary endothelial cells and elicit vasodilation in a manner that is independent on allosteric regulation. Both pulmonary inflammation and lung injury are features of sepsis. These novel concepts will be investigated in this proposal by pursuit of the hypothesis that during acute inflammation, the role of the 293cys residue as a modulator of vascular NO-signaling changes from a nitrite-reductase dependent to SNOHb dependent mechanism which will be tested via the following specific aims 1) Determine the mechanism by which 293cys regulates RBC dependent NO vascular cell signaling., 2) Determine the role of 293cys residue in controlling nitrite reduction and NO- dependent vascular cell signaling in vivo, 3) Determine the role of the 293cys residue in affecting RBC effects during Sepsis induced hypotension and pulmonary inflammation. Accomplishment of these aims will yield insights into the mechanisms novel therapeutic targets focusing on how RBCs modulate NO-metabolism. PUBLIC HEALTH RELEVANCE: Red blood cells play important roles in controlling vascular homeostasis mechanisms. We propose herein that a specific amino acid residue of the hemoglobin protein (the 293cys residue) is critical in this regard by modulating how red cells control nitric oxide function. In this proposal we aim to elucidate the specific mechanisms by which the 293cys residue controls nitric oxide function both during normal physiological conditions and during inflammation associated with the disease Sepsis and in doing so, hope to identify novel therapeutic targets and strategies.