【作者】 张璟；

【导师】 药立波； 刘新平；

【作者基本信息】 第四军医大学 ， 生物化学与分子生物学， 2005， 博士

【摘要】 NDRG2(N-myc down-stream regulated gene 2)是我们在1999年发现的一个NDRG基因家族的新成员。NDRG家族序列在进化中高度保守,成员之间高度同源,提示该基因家族具有重要的生物学功能。生物信息学分析未发现NDRG2及其家族其它成员具有任何已知的结构域或基序。目前关于NDRG2的具体功能尚不清楚。但初步的研究已经表明NDRG2在抑制肿瘤生长,参与应激反应等生命活动中有一定作用。如能找到与之相互作用的蛋白分子,会对其功能研究提供有用的线索。研究相互作用蛋白的方法有多种,应用最广泛的是酵母双杂交,尤其是对功能未知的蛋白。为深入探讨NDRG2的功能,我们以Ndrg2为诱饵用酵母双杂交方法筛选了成人脑cDNA文库,获得了12个阳性克隆,分别代表5种基因。筛选获得的5种基因片段包括①与分化相关的FHOS(forming homologue overexpression spleen)分子片段。②与肿瘤的非锚着依赖生长恶性表型关系密切的MSP58(58-KDa microspherule protein)基因片段,该分子另有两个亚型-MCRS1(Microspherule protein 1)和MCRS2(Microspherule protein 2),我们所筛到的片段为三个分子C末端共同序列。将其命名为MCRS2-C。③一个编码假想蛋白的基因片段。④一个随机的cDNA克隆。⑤编码Syntaxin 1A蛋白的mRNA3’端的一段非编码序列。 我们对酵母双杂交筛选到的阳性分子MCRS2-C与Ndrg2的相互作用进行了深入的研究。应用GST-Pulldown及免疫共沉淀方法在体外及细胞内验证了MCRS2-C与Ndrg2的物理相互作用。进一步构建了Ndrg2与红色更多还原

【Abstract】 NDRG2 ,which was first identified and cloned from normal human whole brain cDNA library in our lab in 1999, belongs to a new family of differentiation-related genes, the NDRG family. This family includes four related members: NDRG1-4 . Database search and phylogenetic analysis revealed that NDRG2 genes are highly conserved in mammals. Moreover, mammalian NDRG family members show high homology to each other, except for their C-and N-terminal regions, pointing out the important functions of this gene family. Bioinformation analysis does not indicate any known motif or domain in NDRG2 and other members of NDRG family. The precise molecular and cellular functions of NDRG2 are still unknow so far. Some preliminary researches suggest that NDRG2 may play a role in growth arrest and cell differentiation, and it may be regulated by cellular stress. So, identification of Ndrg2 interaction proteins may provide important informations for its function research. For this purpose, we performed yeast two-hybrid screening of a adult brain cDNA library using Ndrg2 as a bait protein. 96 clones were obtained after 1.65 x10~6 clones were screened by reporter genes. 12 clones were got after interactions were retested in yeast, which represents 5 different gene fragments.Of these 5 final isolates, one fragment encoded FHOS (forming homologue overexpression spleen), adifferentiation-related protein; The other fragment encoded common C-terminal part of MSP58(58-KDa microspherule protein), MCRSl (Microspherule protein 1), MCRS2(Microspherule protein 2). They are isforms of each other; we named it MCRS2-C. The remained other three fragments encoded a hypothetical protein, a one of the randomly sampled human cDNA clone and a fragment of 3 ’ terminal untranslated region of Syntaxin 1A mRNA.Among these positive clones, MCRS2-C was choosed for the further investigation. Sequence analysis revealed that fragment contains a conserved FHA domain and a coiled-coil domain. GST-pull down and co-immunoprecipitation northerly showed that Ndrg2 and MCRS2-C possessed physical interactions both in vitro and in vivo. To test the subcellular localization of Ndrg2 and MCRS2, pDsRed2-Nl-M)￡G2 and pEGFP-c3-MCRS2 recombinant plasmid was constructed and transfected into HHCC cells. The fusion proteins were expressed and observed by fluorescence microscopy, and confocal microscopy. We found that Red-Ndrg2 localized in cytoplasm while MCRS2-GFP localized in nucleus. Ndrg2 was reported to shuttle from cytoplasm to nucleus after the cell was treated by some stress stimulus. So the cells were treated by mitomycin C stress stimulus for 10 hours, and the localization of Ndrg2 and MCRS2-C were observed again. This time Ndrg2 was found to translocalize and colocalize with MCRS2 in nucleus. These results suggested that the interaction between Ndrg2 and MCRS2 (or MSP58, MCRSl) might regulated by some kind of stress stimulus. Because stress stimulus resulted in translocation of Ndrg2 from cytoplasm to nucleus. Colocalization of Ndrg2 and MCRS2 provide spatial possibility for the physical interactions of these two proteins. Todelineate the interaction region(s) of Ndrg2 and MCRS2 (or MSP58, MCRSl), various deletions of two proteins were engineered and subjected to analyze in yeast two hybrid assay. We found that the FHA domain of MCRS2-C is responsible for the interaction with Ndrg2, while amino acid residues between 100 to 257aa are responsible for its interaction with MCRS2(or MSP58, MCRSl).The two-hybrid analysis is of exceptional value for the detection of pairwise and transient associations. However, yeast two-hybrid approaches do not seem to be particularly suited for characterization of protein complexes.This supports the view that complex formation is more than the sum of binary interactions. Success of the TAP/MS approach for the characterization of protein complexes relies on its maintaining protein concentration, localization and post-translational modifications in a manner that closely approximates normal physiology. Yeast two-hybrid analysis and TAP/MS method are ideally complementary. In this research, we successfully constructed a TAP tag eukaryotic expression vector, and fused this tag to the C-terminus of Ndrg2. This recombinant was stably transfected to HHCC cells to develop an Ndrg2 sepcific mammalian TAP expression system. This work built up basis of identifying Ndrg2 interaction protein complex with TAP method.Some previous researches have demonstrated that NDRG2 is involved in the differentiation of some kinds of myelomonocytic leukemia cells. To investigate the relationship between NDRG2 and NB4 cells, a kind of myelomonocytic leukemia cells, a classic differentiation cell model was made with NB4 cell (a human APL cell line) treated by ATRA and AS2O3 at low concerntration. Expression of Ndrg2 is preliminary analized during differentiation of NB4 cells. Expression of Ndrg2 was up-regulated in the更多还原