STAT4入核分子机制的初步研究

发布时间：2018-11-25 20:56

【摘要】：核转录因子进入细胞核是其发挥转录活性的前提。研究发现信号转导和转录活性因子(Signal transducers and activators of transcription, STATs)家族成员不具有引导蛋白入核的核定位序列(nuclear location signal, NLS),但是细胞因子的刺激可以导致STAT短暂胞核聚集现象,因此研究STAT入核机制是一个值得探讨的课题。研究发现,STAT1和STAT3的DNA结合域中一段序列具有促进其进入细胞核的功能。那么STAT4的DNA结合域中是否也存在类似的序列,能否引导STAT4入核,目前尚无报道。目的：探讨STAT4受IL-12刺激后移位入细胞核的机制。方法： 1.采用生物信息学方法将STAT1和STAT3的DNA结合域与STAT4的DNA结合域进行比对,以期找到STAT4DNA结合域上的同源序列。 2.以pEGFP-C1为表达载体、采用基因亚克隆的方法构建质粒,包括：缺失395-416位氨基酸残基序列的缺失型STAT4质粒即pEGFP-STAT4-Del、将SV40大T抗原上经典的NLS核酸序列插入表达载体的阳性对照质粒pEGFP-NLS、将NLS插入缺失型STAT4的pEGFP-NLS-STAT4-Del质粒,这些质粒经过酶切消化鉴定和DNA测序鉴定,并采用免疫印迹方法确定这些质粒在真核细胞中表达 3.将上述质粒转染Caski细胞,通过IL-12刺激,荧光显微镜观察荧光信号的亚细胞定位。结果： 1.经过生物信息学分析发现STAT4DNA结合域上395-416位氨基酸残基序列可能是STAT4潜在的二聚体特异性NLS (dimer-specific NLS, dsNLS)。 2.经过酶切鉴定和DNA序列测定证明构建了正确的质粒,包括：pEGFP-STAT4、pEGFP-STAT4-Del、pEGFP-NLS和pEGFP-NLS-STAT4-Del,将这些质粒转染真核细胞都能正确表达。 3.将pEGFP-STAT4、pEGFP-STAT4-Del分别转染Caski细胞,IL-12刺激后发现野生型STAT4进入细胞核,而缺失型STAT4在IL-12刺激前后,都主要分布在细胞浆。进一步用leptomycin B (LMB)处理转染细胞,再用IL-12刺激,发现刺激Omin时野生型STAT4分布在胞浆,45min后进入细胞核,60min后阳性信号仍然在细胞核里,表明野生型STAT4受工L-12刺激被活化,形成同源二聚体,以这种形式移入细胞核；但缺失型STAT4转染细胞用LMB处理后,再用工L-12刺激,大部分阳性信号始终分布在细胞浆,表明缺失型STAT4确实不能进入细胞核；将经典的NLS序列插入缺失型STAT4中,转染该质粒,发现含有NLS的缺失型STAT4也能进入细胞核。表明STAT4的395-416位氨基酸残基序列在引导STAT4入核中起重要作用。结论： STAT4的395-416位氨基酸序列具有dsNLS功能,在IL-12刺激下,能够引导STAT4入核。
[Abstract]:The entry of nuclear transcription factors into the nucleus is a prerequisite for their transcriptional activity. It was found that members of the (Signal transducers and activators of transcription, STATs) family of signal transduction and transcriptional activity factors do not have a nuclear localization sequence (nuclear location signal, NLS), that leads to the entry of proteins into the nucleus. However, cytokine stimulation can lead to the transient nuclear aggregation of STAT. Therefore, the study of STAT nucleation mechanism is a worthy topic. It was found that a sequence in the DNA binding domain of STAT1 and STAT3 promoted their entry into the nucleus. So whether there are similar sequences in the DNA binding domain of STAT4 and whether the STAT4 can be guided into the core is not reported. Objective: to investigate the mechanism of STAT4 translocation into nucleus after IL-12 stimulation. Methods: 1. The DNA binding domain of STAT1 and STAT3 was compared with the DNA binding domain of STAT4 by bioinformatics in order to find the homologous sequences on STAT4DNA binding domain. 2. Using pEGFP-C1 as expression vector, the plasmid was constructed by gene subcloning method, including: deletion type STAT4 plasmid (pEGFP-STAT4-Del,) with deletion of 395-416 amino acid residues. The classical NLS nucleic acid sequence on the large T antigen of SV40 was inserted into the positive control plasmid pEGFP-NLS, of the expression vector, and the NLS was inserted into the pEGFP-NLS-STAT4-Del plasmid of the deletion type STAT4. These plasmids were digested and identified by restriction endonuclease digestion and DNA sequencing. The expression of these plasmids in eukaryotic cells was determined by Western blot. The above plasmids were transfected into Caski cells. The subcellular localization of fluorescent signals was observed by IL-12 stimulation and fluorescence microscope. Results: 1. Bioinformatics analysis revealed that the amino acid residues at 395-416 on STAT4DNA binding domain may be the potential dimer specific NLS (dimer-specific NLS, dsNLS).) of STAT4. 2. The results of restriction endonuclease digestion and DNA sequencing showed that the correct plasmids were constructed, including pEGFP-STAT4,pEGFP-STAT4-Del,pEGFP-NLS and pEGFP-NLS-STAT4-Del, transfection of these plasmids into eukaryotic cells. 3. After pEGFP-STAT4,pEGFP-STAT4-Del was transfected into Caski cells, the wild type STAT4 was found to enter the nucleus after IL-12 stimulation, while the deletion type STAT4 was mainly distributed in the cytoplasm before and after IL-12 stimulation. After treated with leptomycin B (LMB) and stimulated by IL-12, it was found that the wild-type STAT4 was distributed in the cytoplasm of Omin, entered into the nucleus after 45min, and the positive signal of 60min was still in the nucleus. The results showed that wild type STAT4 was activated by L-12 to form homologous dimer and transferred into nucleus in this form. However, after treated with LMB, most of the positive signals were distributed in the cytoplasm of the deleted STAT4 transfected cells, which indicated that the deletion type STAT4 could not enter the nucleus. The classical NLS sequence was inserted into the deletion type STAT4 and transfected into the plasmid. It was found that the deletion type STAT4 containing NLS could also enter the nucleus. These results suggest that the amino acid residues at position 395-416 of STAT4 play an important role in guiding STAT4 into the nucleus. Conclusion: the amino acid sequence at position 395-416 of STAT4 has the function of dsNLS and can induce STAT4 into nucleus under the stimulation of IL-12.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392.1

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