p120ctn、Kaiso及matrilysin在非小细胞肺癌中的表达及其意义

目的探讨p120catenin(p120ctn)、Kaiso和matrilysin在非小细胞肺癌(non-small cell lung cancer,NSCLC)发生发展中的作用。方法采用免疫组织化学法检测98例NSCLC(52例肺鳞状细胞癌和46例肺腺癌)以及16例支气管扩张症组织中p120ctn、Kaiso和matrilysin的表达情况,分析NSCLC中p120ctn、Kaiso和matrilysin与临床病理特征的关系及三者表达的相关性。结果在NSCLC组织和支气管扩张症组织中,p120ctn蛋白异常表达率分别为74.5%、6.3%,两组之间表达率比较有统计学差异(P<0.001);在肺癌细胞、杯状细胞、纤毛细胞、基底细胞中Kaiso蛋白异位表达率分别为63.3%、6.3%、6.3%、37.5%,肺癌细胞与杯状细胞、纤毛细胞表达率比较有统计学差异(P<0.001)。p120ctn蛋白异常表达、matrilysin蛋白细胞质表达均与NSCLC组织学类型密切相关(P<0.05),Kaiso蛋白异位表达与NSCLC分化程度密切相关(P<0.05)。在NSCLC组织中,Kaiso细胞核表达与matrilysin细胞质表达存在显著负相关(r=-0.23,P=0.02)。结论 p120ctn可能通过与Kaiso在胞质内异位表达,解除了Kaiso对靶基因的抑制作用,促进matrilysin的细胞质表达,而参与肺癌的发生发展。     

On how mammalian transcription factors recognize methylated DNA

DNA methylation is an epigenetic mark that is essential for the development of mammals; it is frequently altered in diseases ranging from cancer to psychiatric disorders. The presence of DNA methylation attracts specialized methyl-DNA binding factors that can then recruit chromatin modifiers. These methyl-CpG binding proteins (MBPs) have key biological roles and can be classified into three structural families: methyl-CpG binding domain (MBD), zinc finger, and SET and RING finger-associated (SRA) domain. The structures of MBD and SRA proteins bound to methylated DNA have been previously determined and shown to exhibit two very different modes of methylated DNA recognition. The last piece of the puzzle has been recently revealed by the structural resolution of two different zinc finger proteins, Kaiso and ZFP57, in complex with methylated DNA. These structures show that the two methyl-CpG binding zinc finger proteins adopt differential methyl-CpG binding modes. Nonetheless, there are similarities with the MBD proteins suggesting some commonalities in methyl-CpG recognition across the various MBP domains. These fresh insights have consequences for the analysis of the many other zinc finger proteins present in the genome, and for the biology of methyl-CpG binding zinc finger proteins.     

Engineered zinc finger proteins that respond to DNA modification by HaeIII and HhaI methyltransferase enzymes

Zinc finger modules are capable of specifically interacting with DNA that contains 5-methylcytosine (5-mC) in place of cytosine, suggesting that zinc finger-DNA binding could be regulated by extrinsic methylation of DNA. Here, we have used phage display to engineer zinc finger proteins that detect and discriminate DNA methylation by the prokaryotic enzymes HaeIII and HhaI. In these systems, zinc finger-DNA complexes are induced by DNA modification using the appropriate enzyme, which can therefore act as a switch. To further develop the specificity of the switch, zinc finger discrimination between 5-mC and thymine in DNA sequences is demonstrated despite the presence of the characteristic major groove methyl group that is common to both bases. Specificity was achieved using a DNA-binding strategy involving synergy between adjacent zinc fingers. We propose that engineered zinc fingers that recognise particular DNA modifications, such as sequence-specific DNA methylation, could be integrated into artificial regulatory circuits for the control of gene expression and other biological processes.     

DNA-Binding ability of GAGA zinc finger depends on the nature of amino acids present in the beta-hairpin

The GAGA factor of Drosophila melanogaster uses a single Cys2-His2-type zinc finger for specific DNA binding. Comparative sequence alignment of the GAGA zinc finger core with other structurally characterized zinc fingers reveals that the beta-hairpin of the GAGA zinc finger prefers amino acids with an aliphatic side-chain different from those of other zinc fingers. To probe the substitution effect of aromatic amino acids in the beta-hairpin on the DNA binding, three mutant peptides were designed by substituting consensus phenylalanine, an aromatic amino acid, at key positions in the beta-hairpin region. The metal-binding and the overall fold of the mutant peptides are very similar to those of the wild-type as shown by UV-vis absorption spectroscopy and circular dichroism spectroscopy. However, the gel mobility shift assay and isothermal calorimetric studies demonstrated that none of the mutants are able to bind the cognate DNA substrate, although the mutation is confined only to the beta-hairpin region. The present results suggest that the nature of the amino acids in the beta-hairpin plays an important role in the DNA-binding of the GAGA factor protein.