Functional diversification of myoglobin in the lungfish

肌红蛋白是一种呼吸蛋白，在许多生物领域中被用作模型系统。它的主要功能是在心脏和骨骼肌中输送和储存氧气。肌红蛋白也可以作为一种酶，在一氧化氮(NO)的动态平衡和活性氧(ROS)的解毒中发挥作用。几乎所有脊椎动物都只有一个肌红蛋白基因；只有一些鲤科鱼类有两个肌红蛋白基因。此外，肌红蛋白在肌肉外的表达也很少见。我们发现，西非肺鱼Protopterus anneens至少含有7个不同的肌红蛋白基因，这些基因在不同的组织中差异表达。肺鱼在了解四足动物进化和陆系化方面占有重要地位。出人意料的肌红蛋白多样性强烈暗示了肺鱼中该基因的亚功能化和/或新功能化。这项应用的目的是了解肺鱼肌红蛋白的功能多样性及其出现。我们将测量重组表达的肌红蛋白的氧结合动力学，分析它们的酶活性，并研究它们的组织和细胞分布。此外，还将在稳定转基因的脊椎动物细胞系中进行进一步的分析：测试不同肌红蛋白增强线粒体呼吸、介导对缺氧和ROS的耐受性、保护细胞免于凋亡的能力以及它们在一氧化氮生成中的作用。结合这些实验的结果，我们将能够识别每一种肺鱼肌红蛋白的功能，并有助于了解它们在进化过程中的功能多样化。

Myoglobin is a respiratory protein that has served as model system in a variety of biological fields. Its main function is to deliver and store O2 in the heart and skeletal) muscles. Myoglobin may also act as an enzyme and is instrumental in homeostasis of nitric oxide (NO) and detoxification of reactive oxygen species (ROS). Almost any vertebrate harbors only a single myoglobin gene; only some cyprinid fish have two myoglobin genes. In addition, expression of myoglobin outside the muscles has rarely been observed. We have found that the West African lungfish Protopterus annectens harbors at least seven distinct myoglobin genes that are differentially expressed in various tissues. Lungfishes occupy a key position for the understanding of tetrapod evolution and terrestrialization. The unexpected diversity of myoglobin strongly hints to subfunctionalization and/or neofunctionalization of this gene in the lungfish. The objective of this application is to understand the functional diversity of lungfish myoglobins and its emergence. We will measure the O2 binding kinetics of recombinantly expressed myoglobin, analyze their enzymatic activities, and study their tissue and cellular distributions. Additional analyses will be carried out in stably transfected vertebrate cell lines: the ability of different myoglobins to augment mitochondrial respiration, to mediate tolerance towards hypoxia and ROS, to protect from apoptosis and their roles in the generation of NO will be tested. The combination of the results from these experiments will allow us to identify the function of each single lungfish myoglobin and help to understand their functional diversification in evolution.