人类基因hUBC16和GPx7的克隆与功能研究

【作者】 殷刚；

【导师】 毛裕民；

【作者基本信息】 复旦大学 ， 遗传学， 2006， 博士

【摘要】 本实验室大规模的cDNA克隆与测序工作积累了大量的全长基因库。借助大量的生物信息学手段，我们试图从这些库中去筛选可能与重要的生理途径及人类疾病密切相关的基因进行研究。在此基础上我们筛选到了两条感兴趣的基因分别进行研究，它们是hUBC16和GPx7。hUBC16的cDNA全长2252bp，编码一个162个氨基酸的蛋白质。蛋白序列分析显示hUBC16具有一个重要的功能结构域：泛素结合酶(Ubiquitin-conjugating enzyme，UBC)结构域。且信息学分析推测hUBC16与拟南芥(Arabidopsis thaliana)UBC16高度同源，可能为人类泛素结合酶蛋白家族的新成员。泛素结合酶(ubiquitin-conjugating enzyme)又简称为E2，是泛素系统的主要部件之一，作为一个中间桥梁般的角色，它从泛素活化酶(ubiquitin-activating enzyme，或简称E1)接受泛素，和泛素形成了高能硫脂键，再把泛素交给下游的部件：泛素连接酶(ubiquitin ligase，或简称E3)或直接底物，底物被泛素修饰后，将被降解，或生命活动受到调节。经生物信息学预测发现hUBC16可能定位于细胞核中，用NCBI数据库中进行blastn，blastp等生物信息学分析发现hUBC16的蛋白C端具有两个核定位信号，在102位的半胱氨酸可能是它的酶活中心，并且用体内体外实验验证了酶活中心的确是102位的Cys；亚细胞定位实验表明hUBC16主要定位于细胞核中与生物信息学预测的结果一致，我们也用实验证实了活性中心对于hUBC16的正确定位发挥着与核定位信号同等重要的作用；酵母双杂交实验筛选出一些潜在的可能与hUBC16相互作用的蛋白：ERAL1；UBE1v2；RNF167；KIAA1972；RNF8。细胞周期实验检测发现，hUBC16的上调表达会促使处于细胞周期S期的细胞比例增加，而下调hUBC16的表达会导致处于细胞周期S期的细胞比例减少，表明hUBC16对细胞增殖具有促进作用，但其促进细胞周期的分子机理仍然还不清楚，有待进一步实验的深入研究。谷胱甘肽过氧化物酶(GPXs)可以保护细胞免受由呼吸代谢产生的活性氧自由基的氧化伤害，也具有与细胞周期、细胞信号转导调控相关的功能，我们克隆到一个人cDNA序列，经生物信息学分析发现其编码一个长187个氨基酸的蛋白具有GSHPx的结构域和BtuE的基序，并且通过基因和编码蛋白的结构信息分析表明它就是NPGPx基因。通过生物信息学EST分析，以及组织表达谱分析，证实它在正常组织中广谱表达，具有肿瘤组织表达特异性。我们克隆了全长GPx7的cDNA，亚细胞定位预测与亚细胞定位实验表明GPx7主要定位于细胞质中。酵母双杂交实验筛选出一些潜在的可能与GPx7相互作用的蛋白：RAN and EIF3S12。序列分析发现RAN是原癌基因Ras超家族中的成员，对细胞增殖起到促进作用，而EIF3S12目前仅知道它是真核转录促进因子3的一个亚基，可能参与真核基因的转录。细胞周期实验证明GPx7的上调表达会使细胞周期G1期细胞所占比例增加，同时S期细胞所占比例急剧减少，此外同时上调GPx7与RAN的结果发现比单独上调表达RAN而引起的S期细胞增加比例要小，上述结果看出GPx7可能对细胞周期进程具有抑制作用。综合而言，我们克隆了一条新的人类泛素结合酶基因hUBC16和一条已报道的谷胱甘肽过氧化物酶基因GPx7并对它们的功能进行了研究。结果显示hUBC16与GPx7都与细胞周期有关，hUBC16可能对细胞增殖起到促进作用，而GPx7的上调可能对细胞周期进程起到抑制作用，但具体的分子机理还不明确。更多还原

【Abstract】 The large-scale cDNA library cloning and sequencing analysis in our lab lead to the accumulation of plenty of full-length human genes. Using various kinds of bioinformatics analysis, we are trying to screen from the library and study the function of potential genes closely related to important physiological pathways and human diseases. On this basis, the two genes we have screened out are hUBC16 and GPx7.The hUBC16 gene is 2252 base pairs in length, encoding a putative 162 amino acids protein. Protein sequence analysis reveals that hUBC16 has an important functional domain: the ubiquitin-conjugating enzyme domain (UBC domain). This cDNA sequence has been submitted in GeneBank, and the accession number is AY948289. Bioinformatics analysis also predicts that hUBC16 shares high homology to Arabidopsis thaliana ubiquitin-conjugating enzyme 16, suggesting its role as a new member of human ubiquitin-conjugating enzyme protein family.The ubiquitin-conjugating enzyme (E2) is an essential part of the ubiquitination machinery. As a bridging element, E2s receive activated ubiquitin from ubiquitin-activating enzymes E1s, form a thiol ester with the C terminus of the ubiquitin and transfer it to down stream ubiquitin-ligases E3s or the immediate substrate proteins. When the target protein is modified by ubiquitin, it will undergo degradation or protein activity modification. Bioinformatics prediction found out that hUBC16 may be localized in the nucleus. Blast n and blast p analysis by NCBI database showed two nuclear localization signals (NLSs) in the C terminus of hUBC16 protein sequence and a putative enzymatic site at the 102 amino acid cysteine, which is further proved through in vitro and in vivo experiments. The result in subcellular localization accords with the bioinformatics prediction that hUBC16 is mainly localized in the nucleus. We experimentally proved that the active site and NLSs are both important components for the normal localization of hUBC16. Yeast two-hybrid analysis screened out several potential interacting proteins with hUBC16, which are ERAL1, UBE1v2, RNF167, KIAA1972 and RNF8.Cell cycle test found out that up regulation of hUBC16 expression leads to an increase in the S phase of the cell cycle, and down regulation causesdecrease in the S phase cells. All of the cell cycle test results show that hUBC16 can promotes cell cycle progression, while the molecular mechanism of which is still unclear and needs further study.Glutathione peroxidases (GPxs) are believed to protect cells against constitutive oxidative damages induced by reactive oxygen species (ROS) generated from aerobic metabolism, and play a role in the regulation of cell cycle and signaling pathways. Here we cloned a human cDNA sequence, encoding a 187 amino acids protein containing a GSHPx domain and a BtuE motif using bioinformatics analysis. Through gene analysis and analysis of the structure of the encoded protein, this sequence turns out to be an NPGPx gene. Using EST and tissue expression pattern analysis, this gene is proved to be widely expressed in normal tissues while specifically expressed in tumor tissues. We cloned the full-length GPx7 cDNA. Subcellular localization prediction and experiment proved the cytoplasmic localization of GPx7.Two of the yeast two-hybrid analysis screen-outs are RAN and EIF3S12. Sequence analysis shows that RAN is a member of the Ras oncogene super family and promotes cell proliferation. EIF3S12 is reported as a subunit of human eukaryotic translation initiation factor 3 and may have a potential function in the transcription of eukaryotic genes. Cell cycle experiments proved that up regulation of GPx7 leads to the shrink in S phase and increase in G1 phase in cell cycle, and the S phase cells are less when RAN and GPx7 are both expressed than RAN alone, suggesting a role of GPx7 in cell cycle inhibition.In a word, we cloned a novel human ubiquitin-conjugating enzyme gene hUBC16 and a reported glutathione peroxidase gene GPx7. we conducted functional studies on them and found out both of them play a role in cell cycle. While hUBC16 might promote cell cycle progression, the up regulation of GPx7 might inhibit cell proliferation, while the molecular mechanisms of both cases need further study.更多还原