Mechanisms of Hemoglobin Adaptation to Hypoxia in High-altitude Rodents

描述（由申请人提供）：拟建研究项目的目的是阐明高原缺氧生理适应的分子基础，高原缺氧是由呼吸组织细胞氧气供应减少引起的。具体来说，拟议的研究将涉及与高海拔鹿小鼠（Peromyscus maniculatus）血液生化和有氧代谢适应性变化相关的血红蛋白变化的结构和功能分析。深入了解高海拔动物在慢性缺氧条件下生存和运作的分子机制，有助于我们理解和管理损害氧气运输系统的人类疾病过程。通过确定低氧耐受性的分子基础，有可能用新的药物治疗、基因治疗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群体遗传学、分子进化、结构生物学和蛋白质生物化学的工具和理论。本研究项目的具体目标是：(1)确定负责血红蛋白适应缺氧的特定氨基酸突变；(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同种异构体组成化学计量的调节调节有关；(3)评估观察到的结构和监管变化对功能的影响。在首先进行种群水平的DNA序列变异调查，以确定高海拔鹿小鼠的珠蛋白基因自然发生的突变之后，本研究将涉及种群遗传分析，以推断哪些观察到的氨基酸变化可能归因于积极的达尔文选择，mRNA和蛋白质水平的调控变异分析，以及“计算机”计算分析，以预测对血红蛋白-氧亲和力的影响。以及“体外”实验分析，以评估已确定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）、变构效应和活性氧和一氧化氮代谢的敏感性。通过确定高海拔啮齿类动物自然种群中进化出的血红蛋白适应机制，该研究项目将为低氧耐受性的分子基础提供新的见解。公共卫生相关性：拟议研究项目的目标是确定在高海拔环境中进化的小鼠血红蛋白功能的特定变化。通过确定使高海拔动物在低氧条件下生存和发挥功能的特定分子机制，可能有可能在损害氧气运输系统的人类疾病的治疗中复制该机制。

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.