Functional diversification of myoglobin in the lungfish

肌红蛋白是一种呼吸系统蛋白质，在许多生物学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O2。肌红蛋白也可以作为一种酶，并有助于一氧化氮（NO）的体内平衡和活性氧（ROS）的解毒。几乎所有的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，很少观察到肌红蛋白在肌肉外的表达。我们已经发现，西非肺鱼Protopterus annectens窝藏至少七个不同的肌红蛋白基因，在不同的组织中差异表达。肺鱼在理解四足动物的进化和陆栖化过程中占有关键地位。肌红蛋白的意外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。本申请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肌红蛋白的O2结合动力学，分析其酶活性，并研究其组织和细胞分布。将在稳定转染的脊椎动物细胞系中进行额外的分析：将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、保护免于细胞凋亡的能力以及它们在NO产生中的作用。这些实验结果的结合将使我们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性。

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.