hNUDC与Mpl和LIS-1复合体的形成及其双重功能的研究

The earlier studies have revealed that hNUDC forms the complex with LIS-1, a nuclea migration protein, involved in cell mitosis and cytokineses. However, other mechanistic studies by which hNUDC and Mpl interaction has been provided by our laboratory studies recently. hNUDC has in addition been shown to act as a second naturally ligand for thrombopoietin receptor (Mpl) and it plays a biological role in maturation of megakaryocyte cells in both in vitro and in vivo. Furthermore, recent studies suggest that the co-expression of hNUDC and Mpl effectively elevates hNUDC secretion, leading us to conclude a mechanism of Mpl-dependent secretory of hNUDC. We suggest that hNUDC/Mpl and hNUDC/LIS-1 may have different funtions and locat at different sucellular compartments. For further comfirm this idea, the present investigation was undertaken to elucidate the complex formation of hNUDC/Mpl or hNUDC/LIS-1 in living cells by direct visualization of the molecular interactions between proteins using BiFC and FRET. Moreoever, the applicability of BiFC system in conjunction with co-expression of the reference subcellular location markers allow detection of BiFC fusions in various subcellular compartments. Using phage-disply ELISA and small RNA interferon techeniques, the presented study will further detecte the competition binding between Mpl ans LIS-1 for hNUDC. Correspondingly, the functional response of hNUDC in megakaryocyte maturation will be further analyzed by knowckdown expression of Mpl or LIS-1. This proposal raise an important point that hNUDC has both endo- and extra-bifunctions.

早期的研究认为NUDC与LIS-1组成复合物,调控细胞有丝分裂和胞质分裂。然而本实验室近期研究结果表明人体NUDC与血小板生成素受体（Mpl）也有结合，且在体内、体外均有促进巨核细胞成熟的功能。进一步研究证明Mpl与hNUDC在细胞的内质网、高尔基、膜均共定位，并且Mpl可以介导hNUDC向胞外释放。 因此我们推测hNUDC/Mpl和hNUDC/LIS-1之间存在不同的亚细胞共定位以及不同的生物学功能。本项将运用双分子荧光互补法（BiFC）和荧光共振能量转移技术(FRET)在活体贴壁细胞和巨核细胞中同时直观hNUDC/Mpl和hNUDC/ LIS-1复合体的共定位。另外通过噬菌体展示、基因干扰等手段验证Mpl和LIS-1之间与hNUDC竞争结合的机制。还将通过基因沉默技术分析这三种蛋白在巨核细胞的成熟分化过程中的联系。本项目首次提出了hNUDC具有内源与外源双重功能。

本项目持续四年，除了按计划完成任务以外，根据新的发现，产生新的思路, 调整一些研究方法和课题。首先研究两配体TPO或hNUDC与受体Mpl的具体结合部位，然后进一步定位Mpl-EC(102-251 aa)与两配体TPO或hNUDC结合的最小氨基酸区段，通过模建和3D结构模型，得到Mpl-EC-D1(206-251 aa)区段是TPO和hNUDC共同的有效结合微区域。后期研究发现，hNUDC对胰腺癌增殖、分化具有影响，同时也证明了hNUDC为miR-194的一个靶基因，miR-194对hNUDC基因具有靶向负调控作用，miR-194对hNUDC的靶向调控作用及其对胰腺癌细胞的影响，进而改变了细胞的一些生物学表现。对于胰腺癌细胞而言，hNUDC的下调表达可以导致细胞核的形态、分裂及迁移活动发生异常，进而影响了细胞的活性与细胞周期。与本相关的论文共4篇，国际会议论文一篇。