Def-Capn3蛋白降解新途径调控肝脏发育和再生的分子机制研究

Def (Digestive organ expansion factor) was first identified by our lab as a pan-endoderm enriched factor that is essential for the growth of liver and other digestive organs in zebrafish. Def belongs to a novel protein family evolutionally conserved from yeast to human and is a component of the ribosomal small subunit (SSU) complex in the nucleolus. In zebrafish, loss-of-function of def (e.g. the defhi429 null mutant) confers a small liver phenotype. We recently reported that loss-of-function of def led to up-regulation of the level of the p53 protein, a well-known tumor suppressor, which was notablely specifically accumulated in the nucleolus in the mutant cells. Consequently, the p53 pathway was activated to selectively upregulate the transcriptional expression of Δ113p53, which encodes an N-terminal truncated p53 isoform. The transcriptional expression of Δ113p53 is initiated by an alternative promoter contained in the intron 4 of the p53 gene which is totally p53-dependent. We also reported that both zebrafish Def and its human counterpart hu-Def (c1orf107, 1q32.2) teamed up with Calpain3, a cystein proteinase, to mediate the degradation of the p53 protein. In additon, our unpublished data showed that defhi429 heterozygous mutant was defective in liver regeneration. This research proposal plans a detailed research program on this novel finding with the purpose to elucidate the molecular mechanism of the Def-Calpain 3 protein degradation pathway and to define the biological role of this pathway in the processes of liver development and liver regeneration. Our findings will not only help us to understand more about the liver biology but to define a novel function for the nucleolus in the context of cell biology as well.

Def是我们首先研究并命名的一种新型核仁蛋白。在斑马鱼中def基因失活导致肝脏变小。我们最近发现Def与半胱氨酸蛋白酶Calpain3形成复合体介导p53的降解,从而解释了为什么def突变体中p53蛋白水平增加，且富集于核仁中。def 突变体中p53的激活诱导其异构体Δ113p53转录表达。Δ113p53能促进p53抑制细胞周期的功能，但拮抗p53促细胞凋亡功能，这解释了为什么def突变体肝脏变小是由于细胞增殖受阻而不是由于细胞凋亡异常导致的。最近我们观察到def杂合突变体的肝脏切除后不能正常再生。但目前对Def-Capn3蛋白降解途径如何行使其功能、如何调节肝脏发育和再生仍是未知数。本项目旨在利用斑马鱼和小鼠为模式深入研究以Def-Capn3为轴心的蛋白降解途径的作用机理以及Def蛋白本身受调控的机理，以期揭示核仁中是否存在一个新的途径来调节细胞的增殖和死亡并进而调控肝脏的发育和再生。

本项目的研究目标是以斑马鱼及小鼠为模式生物，利用多种分析手段来确定Def-Capn3蛋白降解途径是否为核仁中通过调控蛋白稳定性来调控细胞增殖与死亡的新途径，并揭示Def调控肝脏发育和再生的分子机理。在项目执行期间，我们证明Def蛋白N末端有五个位点发生磷酸化修饰，该修饰促进Def招募Capn3进入核仁。Def-Capn3复合体组成的蛋白降解途径位于核仁，该途径通过降解p53、Δ113p53及Chek2等蛋白来调控细胞周期及细胞分化，进而调控器官（如肝脏）的发育和再生。但p53突变蛋白p53-R175H不被降解，有可能说明为何p53-R175H在p53突变关联肿瘤中概率最高。该途径还通过降解Mpp10来调控核糖体小亚基的正常组装，由此保证器官发育时蛋白质合成的需求，而Sas10保护Mpp10不被Def-Capn3途径降解。此外，在成体器官如肝脏（细胞基本不增殖）中Def-Capn3途径很可能通过参与调控关键因子如Nkap等的稳定性来调控先天性免疫反应，特别是在后期弱化这些关键因子的功能时起重要作用。我们已取得的研究结果成功揭示核仁中存在一个以Def-Capn3为轴心的具重要生物学功能的蛋白降解途径，成果具有明显的原始创新性。项目执行期间共发表标注论文7篇，其中与本研究目标直接相关的研究论文5篇，还有4项重要研究成果有待发表。我们的研究成果完全达到并超过了预期研究目标。