The. The Nuclease Tatdn1 And Phosphate Acid Phosphatase Pap2d, Cloning And Functional Study Of Pap2l2

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.