核酸酶TatDN1和磷酯酸磷酸酶PAP2d、PAP2L2的克隆与功能研究

【作者】 张勇；

【导师】 毛裕民；

【作者基本信息】 复旦大学 ， 遗传学， 2009， 博士

【摘要】 通过分析肝癌基因芯片,我们发现了一个显著上调表达的基因,确定其为TatDN1基因。保守功能域分析中,TatDN1被预测为一个核酸酶。在已有研究中表明肿瘤细胞内常存在着特定染色体区段的缺失,造成大量基因表达失调,从而将促进肿瘤的生成及发展。核酸酶是生命体核酸代谢、消化酶,可广泛或特异的作用细胞内核酸。病理条件下,过表达的核酸酶存在破坏基因结构作用的可能性。TatDN1作为核酸酶在肝癌中上调表达引起了我们对其关注。实验及信息学揭示TatDN1可广泛表达于多种组织,尤其在肿瘤组织细胞内可侦测到较高表达水平。其后,TatDN1在肝癌细胞株中的广泛上调表达被使用定量RT-PCR确证。所检测的4种不同来源肝癌细胞株中相对正常肝细胞L02有3株TatDN1是上调表达,但在HepG-2细胞中并未有显著差异,提示TatDN1基因与肿瘤的关系并不是简单的相关,可能参与癌症发生的不同发育阶段或与发病原因不同的特异型癌症有关。TatDN1在肿瘤组织和细胞内的上调高表达,使其成为潜在的肿瘤诊断的标志基因。为阐明TatDN1基因功能及理解该基因的生理、病理意义,我们成功克隆了人TatDN1基因、纯化到重组TatDN1蛋白。首次,对人TatDN1蛋白进行了较详细的酶学、生化鉴定。证实了TatDN1具有金属离子(Mg²⁺、Ca²⁺)依赖的双链和单链DNA水解活性,其最优活性在酸性pH 4.5左右,这一活性特征区别于已发现的DNaseⅠ和DNaseⅡ类型酶。此外,TatDN1的三维模型被通过Threading建模构建。基于此模型和序列比对数据,我们选取了在氨基酸序列中绝对保守且在三维空间上比邻的几个氨基酸进行了定点突变研究。当TatDN1酶的E112,H149,H174,E220,D222位点分别被突变为A后,发现活性将显著降低。表明了这些位点对维持TatDN1酶学活性至关重要性,可能来自活性位点区。TatDN1的亚细胞定位在通常情况下是全细胞分布。但在应对氧化压力刺激时,TatDN1在肝癌QGY细胞内是更多地集中到细胞核,提示可能出现有核转位现象,TatDN1可能将作用于核内DNA水解。肝癌细胞株QGY中过表达TatDN1造成S期细胞减少,而G0-G1期细胞增多,提示细胞分裂可能更多停滞在G1检测点。而TatDN1过表达却未对检测的细胞凋亡产生明显的影响,推测TatDN1在肿瘤中的上调可能是肿瘤发展中的结果。而要对肿瘤的上调表达TatDN1的生理、病理意义深入理解,仍需要大量实验研究工作来阐明。这里我们报道一个新的酸性、金属离子依赖的人类核酸酶TatDN1的起始研究。TatDN1作为DNase可能在肝癌中受到特殊的调控而发挥其生理作用。磷脂酸磷酸酶Ⅱ（PAP2）作为重要的整合膜磷酸酶,不仅在脂代谢中发挥作用,而且在生命体内对重要的脂级联信号转导起到关键的消减、转换调控作用。这里我们对先前实验室已克隆的一个新的人类PAP2d基因开展了初步的研究。对PAP2d的肿瘤细胞表达潜进行了初步的调查,以期能进一步对比正常组织细胞表达谱得到PAP2d特定病理条件下功能的提示。PAP2d在脑胶质瘤细胞U251和血癌细胞Jurkat,宫颈癌上皮Hela中表达水平要高于检测的其他细胞株,而在胶质瘤细胞C6中35个循环PCR后未能检测到PAP2d。有关PAP2d在肿瘤中表达的情况需进一步扩大样本分析规模,才可能得出有统计意义的结果。在PAP2d的亚细胞定位分析中,绿色荧光融合蛋白EGFP-PAP2d主要分布于HEK293细胞的胞质,提示了PAP2d可能在细胞质中的内质网或高尔基体内被修饰后,才将转运到细胞的质膜系统上发挥磷脂酸磷酸酶作用。为对PAP2d进行酶学和结构功能研究,获得大量提纯的蛋白是必要的。但遗憾的是,我们在所使用的原核过表达系统中并没够能获得重组蛋白的表达。除了真核PAP2磷酸酶,细菌中也发现了大量的膜结合的PAP2相似蛋白（PAP2-like protein）。作为真核PAP2蛋白的同源物,这些细菌的膜蛋白同样具有磷脂酸磷酸酶活性。膜整合PAP2磷酸酶具有重要的生物学作用。但相对而言PAP2家族酶结构和功能关系研究较少。主要的原因之一是PAP2家族酶是整合膜蛋白且低表达,妨碍了PAP2蛋白的大量提纯和相关研究。这里我们依靠筛选嗜热菌的基因组文库,成功克隆了一个热稳定的PAP2同源物编码基因,命名为PAP2L2。其后我们对这个膜结合的新的PAP2相似蛋白进行了成功表达、纯化和生化、酶学鉴定。作为PAP2的紧密同源物,提纯的PAP2L2蛋白为进一步进行PAP2家族蛋白结构和功能深入研究提供了基础。此外作为一个热稳定的膜蛋白,对PAP2L2蛋白进行深入的研究还将有助于我们对整合膜蛋白的热稳定性机理的理解。更多还原

【Abstract】 Nucleases are a class of most important hydrolases in organisms.So far,they are believed to take important roles in different aspects of basic genetic mechanisms, including their participation in mutation avoidance,gene repair,DNA replication, recombinantion,and apoptotic chromatin degradation.By comparing gene expression profiles of hepatocellular carcinoma（HCC） with those of corresponding noncancerous liver in cDNA microarry,we identified an up-regulated gene-TatD domain containing 1 （TatDN1）.In following analysis,TatDN1 was predicted to be as a nuclease.Based on informatics and experiment analysis,TatDN1 is ubiquitously distributed in many human tissues and the expression is high in several tumor tissues.The up-regulated of TatDN1 in HCC cell lines was confirmed by quantitative RT-PCR.The result indicated that TatDN1 was up-regulated in 3 HCC cell lines than that in liver cell L02,but is not so in HepG-2 cell line,which implying that up-regulation of TatDN1 in HCC is depended on HCC types and HCC development.The high level expression in HCC make TatDN1 becomes a potential biomarker for HCC diagnosis.To address the TatDN1 function,we initially biochemical characterized TatDN1 by using a purified recombinant TatDN1.We confirmed the predicted DNase activity of TatDN1,which is metal-dependent and acid DNsae(Mg²⁺,Ca²⁺,opt pH 4.5) hydrolyzing single or double strand DNA.And a 3D model of TatDN1 was constructed by threading algorithm.Based on this model and sequence alignment,we analyzed several high-conserved amino acids in TatDN1 by site-directed mutation.The result showed that TatDN1 mutant protein in either of E112A, H149A,H174A,E220A,and D222A would lose its majority activity,indicating these amino acids from its activity sites.Furthermore,TatDN1 is detected ubiquitously in cell nuclear and cytoplasm by EGFP-TatDN1 expression.However,a nuclear translocation for EGFP-TatDN1 in QGY cell was found when cells were treated with 0.2 mM H₂O₂. Moreover,apoptosis of the over-expressed TatDN1 cell lines appeared to be similar to that of cells as control,implying that TatDN1 protein could have no significant effect on cell apoptosis.However,cell cycle analysis of over-expressed TatDN1 in QGY cell showed that S-phase cells was decreased and more cells entered into Gl-phase,implying over-expression of TatDN1 could partially inhibit cell proliferation.Here,we reported initial characterizations of a novel HCC up-regulated TatDN1 gene,which could be involved in hepatocarcinogenesis as a DNase.Most members in type 2 phosphatidic acid phosphatase（PAP2） superfamily are integral membrane phophatases involved in the lipid related signal transduction and metabolism.In previously,a novel PAP2 family member（named PAP2d） from Homo sapiens was reported by our lab.In this study,we further investigated PAP2d subcellular location and overexpression effect to cell cycle.Moreover,we also examined its tumor cell lines expression pattern.In order to understand PAP2 enzyme molecular mechanism, we expected to obtain a large scale purified PAP2d using E.coli expression system. Regretfully,we failed to over-express recombinant PAP2d since heterogously membrane protein expression hardness.However,a gene encoding a PAP2-like protein from thermophilic Geobacillus toebii T-85,termed PAP2L2,was successfully cloned, expressed and purified in Escherichia coli.The deduced PAP2L2 protein contains 212 amino acids and shows a limited homology to other known PAP2s,especially at conserved phosphatase motifs and similar six-transmembrane topology structure.The purified recombinant PAP2L2 is catalytically active and highly stable,making it ideal as a candidate on which to base further PAP2 structure/function studies.更多还原