Mechanisms of Hemoglobin Adaptation to Hypoxia in High-altitude Rodents

描述(申请人提供)：拟议的研究项目的目的是阐明生理适应高原低氧的分子基础，高原低氧是呼吸组织细胞氧气供应减少所导致的一种状况。具体地说，拟议的研究将涉及对血红蛋白变异的结构和功能分析，这与高原鹿小鼠(Permyscus Manulatus)血液生化和有氧代谢的适应性变异有关。深入了解允许高海拔动物在慢性缺氧条件下生存和发挥功能的分子机制，有助于我们理解和管理危害氧气运输系统的人类疾病过程。通过确定耐缺氧的分子基础，有可能用基于药物的新疗法、基因疗法和基于血红蛋白的血液替代品来复制这一机制。这项高度跨学科的研究将整合分子种群遗传学、分子进化、结构生物学和蛋白质生物化学的工具和理论。这项研究项目的具体目的是(1)确定导致血红蛋白对低氧适应的特定氨基酸突变；(2)评估血红蛋白结构的改变是否也与循环红细胞中不同血红蛋白亚型的组成化学计量学的调节调整有关；(3)评估观察到的结构和调节变化的功能后果。在首先对DNA序列变异进行群体水平调查以确定高原鹿小鼠珠蛋白基因自然发生的突变后，这项研究将包括群体遗传学分析以推断哪些观察到的氨基酸变化可能归因于达尔文式的积极选择，对mRNA和蛋白质水平的调控变异进行分析，对预测对血红蛋白-氧亲和力的影响的计算机分析，以及评估已发现的结构和调控变化如何影响内在氧亲和力以及对温度、质子(玻尔效应)、变构效应以及活性氧物种和一氧化氮代谢的敏感性的体外实验分析。通过识别在高海拔啮齿动物自然种群中进化的血红蛋白适应机制，拟议的研究项目应该为耐缺氧的分子基础提供新的见解。与公共健康相关：拟议研究项目的目标是确定在高海拔环境中进化的小鼠的血红蛋白功能的具体变化。通过识别使高海拔动物能够在低氧条件下生存和发挥功能的特定分子机制，有可能将这种机制复制到危害氧气运输系统的人类疾病的治疗方法中。

DESCRIPTION (provided by applicant): The purpose of the proposed research project is to elucidate the molecular basis of physiological adaptation to high-altitude hypoxia, a condition resulting from a reduced supply of oxygen to the cells of respiring tissues. Specifically, the proposed research will involve a structural and functional analysis of hemoglobin variation that is associated with adaptive variation in the blood biochemistry and aerobic metabolism of high-altitude deer mice (Peromyscus maniculatus). Insights into the molecular mechanisms that allow high-altitude animals to survive and function under conditions of chronic hypoxia can aid our understanding and management of disease processes in humans that compromise the oxygen transport system. By identifying the molecular underpinnings of hypoxia tolerance, it may be possible to replicate the mechanism with novel drug-based therapy, gene therapy, and hemoglobin-based blood substitutes. This highly interdisciplinary study will integrate the tools and theory of molecular population genetics, molecular evolution, structural biology, and protein biochemistry. The specific aims of this research project are (1) To identify the specific amino acid mutations that are responsible for hemoglobin adaptation to hypoxia; (2) To assess whether modifications of hemoglobin structure are also associated with regulatory adjustments in the composition stoichiometry of different hemoglobin isoforms in circulating red blood cells; and (3) To assess the functional consequences of the observed structural and regulatory changes. After first conducting a population-level survey of DNA sequence variation to identify naturally occurring mutations in the globin genes of high-altitude deer mice, this study will involve a population-genetic analysis to infer which of the observed amino-acid changes may be attributable to positive Darwinian selection, an analysis of regulatory variation at the mRNA and protein levels, an 'in silico' computational analysis to predict effects on hemoglobin-oxygen affinity, and an 'in vitro' experimental analysis to assess how the identified structural and regulatory changes influence intrinsic oxygen affinity, as well as sensitivities to temperature, protons (Bohr effect), allosteric effectors, and metabolism of reactive oxygen species and nitric oxide. By identifying mechanisms of hemoglobin adaptation that have evolved in natural populations of high-altitude rodents, the proposed research project should provide novel insights into the molecular basis of hypoxia tolerance. PUBLIC HEALTH RELEVANCE: The goal of the proposed research project is to identify the specific changes in hemoglobin function that have evolved in mice that are native to high-altitude environments. By identifying the specific molecular mechanisms that have enabled high-altitude animals to survive and function under low oxygen conditions, it may be possible to replicate the mechanism in therapeutic treatments of human diseases that compromise the oxygen transport system.