基于单个活细胞实时影像技术对B23蛋白感应核仁应激发生定位和功能改变机制的研究

The nucleolus has recently been shown as a sensor for cellular stress. The translocation of B23, among other nucleolar proteins, from the nucleolus to the nucleoplasm or the cytoplasm, is an indicator of the nucleolar stress. However, how the transloation of the nucleolar proteins is regulated by the stress, which is actually how the nucleolus senses and responds to the stress, is poorly known. B23 plays a role in ribosome biogenesis, the fundamental function of the nucleolus, when it locates within the nucleolus, while it can, in the outside of the nucleolus, regulate the stress signaling by activation of p53 and gene expression. Therefore, to explore the mechanisms underlying B23 translocation and their functional implications will help a better understanding to cell biology of the nucleolus. Our preliminary study showed that a variety of cellular stress induced oxidation of nucleolar compartments, and the cysteines to serine mutagenesis could block B23 translocation upon stress, indicating that oxidative modification of B23 within an oxidized nucluolar environment may be the cause of its translocation. Based on these findings, we planed to conduct the present study. We will first clarify whether there exists generally oxidation of the nucleolar compartments under the nucleolar stress. We then reveal the molecular mechanism underlying B23 translocation under the nucleolar stress, focusing on the sites for oxidative modification, the effects of oxidation and the consequent changes in the interacting molecules. We will finally examine the functional alterations induced by B23 translocation at two events, ribosome biogenesis and p53 activation. Overall this study will increase our knowledge about how the nucleolus senses and responds to the stress. Owing to the differential redox states of cells responding to same stress factors and differential dynamic of protein localization following stress time-course, it is extremely necessary to investigate the above events in single live cell and in real-time tracing manner. Therefore, the single live cell real-time imaging is the basic technique in the present study.

近年研究显示核仁是细胞应激的感受器，核仁蛋白B23等分子在应激下向核质/胞质移位被看作是核仁应激标志，但是，核仁蛋白移位如何受应激调控尚无报导，该问题的实质是核仁如何感知和应答细胞应激。B23在核仁内参与核糖体生物合成，在核仁外则调节p53活化等应激信号，因此对其在应激下定位和功能改变的机制研究有助于全面认识核仁的细胞生物学。我们预初实验提示核仁在应激下发生氧化导致B23分子氧化修饰可能是其移位的原因。因此，本项目拟首先阐明核仁应激是否普遍存在核仁环境氧化，然后从B23分子上氧化修饰位点、效应和相互作用分子改变入手，探索B23在核仁应激条件下移位的分子机制，并考察B23移位对其在核糖体加工和p53激活两方面功能的影响，从而增进对核仁感受和应答应激的机制的理解。由于各个细胞应答应激时氧化状态的差异性和应激因素的时间动态性，本项目以激光共聚焦扫描显微镜下的单个活细胞实时影像技术为基本技术。

核仁是细胞核内合成和装配核糖体亚基的场所。近十多年来亚细胞的蛋白组学发现大量蛋白质在核仁和核质之间穿梭移位，原因不明。同时，很多文献报导多种类型的应激刺激都会引起核仁蛋白的移位并激活著名的应激信号通路p53通路，造成所谓的“核仁应激”。关于核仁应激激活p53的缘由曾有多种解释，而为什么各种细胞应激都会表现为核仁应激、表现为核仁蛋白移位出核仁则尚无人回答。本项目针对这一科学问题，用活细胞中同时表达氧化还原荧光探针ro-GFP和荧光标记的著名核仁蛋白NPM1（B23），并在同一细胞实时观察等方法，发现各种不同刺激如饥饿、辐射、低氧、高温和化疗药物放线菌素D等均像双氧水一样引起核仁区室的氧化，进而触发NPM1从核仁移位到核质。该研究通过在同一个活细胞观察野生型和突变型的NPM1移位模式以及观察NPM1单独或与氧化修饰催化酶融合的蛋白的移位模式，明确了NPM1上一个半胱氨酸残基C275的氧化（类型为谷胱甘肽化）修饰导致该蛋白与核仁的rDNA和rRNA解离，离开核仁进入核质。最后证明，尽管前人提出的激活p53的核仁应激相关蛋白Arf和核糖体蛋白L23确实参与了该事件，但是，如果NPM1滞留于核仁不发生移位（如突变体NPM1那样），则核仁应激无法引起p53激活，从而厘清了前人所述多个分子与NPM1在介导p53激活中的角色关系。最近报导化疗药物放线菌素D治疗白血病的作用需要与NPM1的C275结合。因此，核仁应激感应的氧化还原机制不仅为理解核仁的新功能提供线索，还可能解释化疗药物治疗相关白血病的作用原理。这些发现发表于Nature Communications。课题实施期间，课题组还发表了核仁应激的综述一篇，对引起核仁应激的因素以及核仁形态和结构的改变作了分类，对领域贡献了新的知识。课题实施期间还进行了对另一个核仁蛋白SENP3的研究。该项目的科学发现已在国内外学术会议上进行过交流，也用科普的方式对公众进行过介绍。毕业博士研究生4名，团队的青年教师得到晋升成长。