利用基因敲除小鼠研究 Cdk5rap3 在肝脏发育中的功能

Cdk5rap3 is a potential tumor suppressor, which has a significant role in regulating cell cycle, proliferation, apoptosis and adherance/invasion. Genetic study in zebrafish demonstrated that Cdk5rap3 was essential for normal animal development. However, the role of Cdk5rap3 in mammalian development is still unknown. Here, we have successfully generated Cdk5rap3-deficient mouse, and found that Cdk5rap3 loss led to embryonic lethality after 16.5dpc. The defective embryos exhibited obvious growth retardation, and liver degeneration. H&E staining of E16.5 fetal liver section showed that the hepatoblasts remained undifferentiated. TUNEL staining indicated massive apoptosis. These results suggested liver degenerationwas the main reason for embryonic lethality in Cdk5rap3-deficient mouse. To investigate the molecular mechanism, we used Co-IP and Mass Spectrometry to find out the CDK5RAP3-interacting proteins in fetal livers. Ufl1, which acts as the E3 ligase in ufmylation, was captured. This links the function of Cdk5rap3 to ufmylation. In the future, we plan to further analyze the effect of Cdk5rap3 on the differentiation and apoptosis of hepatoblasts; further confirm the role of Cdk5rap3 in liver development using liver-specific knockout mice; and finally, elucidate the underlying molecular mechanism that regulates liver development in respect of ufmylation. By this study, we will have a better understanding of the essential role of Cdk5rap3 in liver development.

Cdk5rap3是一个潜在的肿瘤抑制基因, 在细胞周期、增殖、凋亡和侵袭等过程中都有重要作用。然而，Cdk5rap3在哺乳动物发育中的作用尚不明确。这里，我们建立了Cdk5rap3基因的敲除小鼠模型。我们发现Cdk5rap3基因敲除后胚胎致死，致死时间大约在胚胎期第16.5天以后；胚胎体型较小，胎肝发育迟缓；胎肝的组织学分析显示成肝细胞分化障碍、大量肝细胞凋亡，提示胎肝发育异常是胚胎致死的主要原因。为了探究其分子机制，我们捕获了胎肝中与CDK5RAP3相互作用的内源性蛋白Ufl1，它在ufmylation过程中起到连接酶作用。该结果将Cdk5rap3与ufmylation联系起来。我们将进一步检测Cdk5rap3敲除对成肝细胞分化和凋亡的影响；通过肝组织特异敲除小鼠验证Cdk5rap3在胎肝中的功能；从ufmylation的角度探索Cdk5rap3敲除后胎肝异常的分子机制。

蛋白分子的泛素和类泛素（UBLs）修饰参与调节许多的生物学功能。UFM1系统是一类新发现的类泛素修饰，它已被证实参与了小鼠的发育和造血作用，但其广泛的生物学功能和作用机制尚待深入研究。以前有报道称 CDK5RAP3 蛋白可能是 Ufm1系统的一个底物分子，而且它的缺失会严重影响斑马鱼的胚胎外包和原肠胚形成。本项目研究中，我们发现CDK5RAP3在小鼠的肝脏发育和肝脏功能完善中发挥关键作用。Cdk5rap3基因完全敲除小鼠表现为出生前致死，肝脏严重发育不全，成肝细胞增殖延迟且分化能力严重缺陷。肝细胞特异的Cdk5rap3基因敲除小鼠在离乳后陆续死亡，表现为严重的低血糖和脂代谢异常。CDK5RAP3的缺失在肝细胞中引起内质网应激，并激活了UPR（unfolded protein responses）。我们发现在肝细胞中CDK5RAP3和UFL1（Ufm1系统的E3连接酶）之间存在相互作用。CDK5RAP3的缺失会改变肝细胞中的Ufm1系统的底物谱，表明CDK5RAP3是这种类泛素修饰的一个底物适配器。总的来说，我们的研究首次报道了CDK5RAP3是Ufm1系统的一个重要的的调节者，并且首次揭示了Ufm1系统参与哺乳动物的肝脏发育。