线虫线粒体组织特异性分离及其蛋白组学分析

Mitochondria is a key organelle in the regulation of cellular homeostasis through various functions such as energy metabolism, oxidative stress and unfolded protein response, etc., and their functional defect often leads to many diseases, such as ischemia/reperfusion injury, aging and age-related diseases, cancer, and cardiovascular diseases. While tissue specific or cell specific mitochondria display different functions, it is still not known whether the mitochondrial function differences between cell types are due to the diverse compositions of its proteins. Therefore, tissue specific isolation of mitochondria and identification of their protein components will greatly promote the understanding of mitochondrial functions. C. elegans has long term to be as an important model organism in the biological research for the mitochondria-related studies. This application would like to isolate tissue specific mitochondria and analyze its protein components through the combination of CRISPR-Cas9 based GFP endogenous gene knock in technique, GFP nanobody based high efficiency and affinity protein purification system, and mass spectrometry in C. elegans epidermis, muscle, intestine and neuron. Further function of tissue specific and novel mitochondrial protein will be investigated. This project will contribute to the understanding of mitochondrial functional specificity in different tissues, and is expected to find tissue-specific mitochondrial localization proteins, which all will provide valuable resources and lay a foundation for further study of mitochondrial functions.

线粒体通过能量代谢、氧化应激、未折叠蛋白响应等多种功能参与调控细胞稳态。其功能异常导致疾病的发生，如缺血再灌注损伤、心血管疾病、癌症、衰老相关疾病等。线粒体功能多样，在不同组织和细胞中又有其特殊性，是否与线粒体蛋白组分差异有关尚不清楚。为此，进一步分离不同组织来源的线粒体及解析其蛋白组分差异将对理解线粒体功能至关重要。秀丽线虫作为模式生物在线粒体功能研究中发挥了重要作用。本课题拟以线虫为模型，通过CRISPR-Cas9基因编辑技术构建GFP内源性标记组织特异性线粒体转基因品系；利用高效高亲和度的GFP纳米抗体，建立快速分离纯化皮肤、肌肉、肠道和神经组织线粒体的方法；并运用蛋白质质谱技术解析组织特异性的线粒体蛋白组分，探索线粒体蛋白组分差异及对其组织功能的影响。本项目的开展将有助于理解线粒体在不同组织中功能的特异性，并有望发现组织特异性的线粒体定位蛋白，为进一步研究线粒体功能奠定研究基础。

线粒体是一种细胞内重要的双层膜细胞器，在细胞能量代谢，信号转导中发挥着重要作用，参与大量的疾病发病过程。传统的线粒体提取方法是通过组织研磨后利用密度梯度离心法进行线粒体分离，耗时耗力且含量很低。最近的研究表明，利用亲和吸附标记后的线粒体可以获得纯度较高的内源性线粒体组分，且样本使用量较低。线虫是一种高效的遗传学模式生物广泛被用于研究细胞及亚细胞组织的功能及信号通路。与传统的抗体相比较，纳米抗体显示出更小的体积，更高的亲和性，更好的穿透性，有助于其与目标抗原蛋白更好的特异性结合。因此，基于别人的研究，我们开发了一种利用GFP纳米抗体对线虫线粒体表面具有GFP标记的线粒体进行分离的方法。通过直接和间接方法证明，我们分离得到的纳米抗体是完整的且具有功能的。最后，我们利用蛋白组学分析分离出来的线粒体的蛋白组分。

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