摘要
目的:PJS综合征(Peutz-Jeghers syndrome,PJS)是一种常染色体显性遗传疾病,发病率为1/10000-12000。临床表现主要为多发性错构瘤样胃肠息肉以及皮肤粘膜色素沉着,其致病基因STK11位于人类常染色体19p13-3,跨度为23kb,包含10个外显子,由433个氨基酸残基组成,具有调控细胞周期、调节细胞极性以及细胞基础能量代谢等重要功能。虽然大多数PJS患者的致病原因是由于STK11催化活性域的突变导致的激酶功能丧失,但是在部分PJS患者和散发肿瘤中,却存在STK11羧基端的突变,STK11羧基端共123个氨基酸残基,包括多个翻译后修饰位点,对STK11生物学功能的实现起着调节作用,机制尚未明确。
方法:本研究利用GST(Glutathione S transferase)融合蛋白表达系统,构建了pGEX4T-2-STK11羧基端重组载体,在低温的条件下诱导了GST-STK11羧基端融合蛋白的表达,并利用Glutathione Sepharose获得高度可溶性GST-STK11羧基端融合蛋白,采用GST-pull down结合质谱分析的方法,筛选到STK11羧基端的可能互作蛋白LOH12CR1。LOH12CR1定位于人12号染色体上,为抑瘤基因的候选基因,且进化上的高度保守。
结果:我们通过免疫共沉淀技术证实STK11激酶与LOH12CR1的互作关系;并利用哺乳动物双杂交系统显示两者之间非直接互作,需第三方参与。免疫荧光共定位实验发现STK11与LOH12CR1共定位。
结论:STK11与LOH12CR1互作的发现为研究STK11的功能提供新的切入点。
Objective:Peutz-Jeghers(PJ)syndrome is an autosomal-dominant disorder characterized by melanocytic macules of the lips,multiple gastrointestinal hamartomatous polyps and an increased risk for various neoplasms,including gastrointestinal cancer. The incidence frequency of PJS is varied greatly from 1/10000 to 1/12000.Recently the disease gene STK11 was mapped to chromosome 19p13.3,extending over 23 kb of genomic DNA and composed of 9 exons,encoding the serine/threonine kinase which is consisited of 433 amino acid residues and highly conserved in the evolution process.It can regulate cell cycle,cell polarity and cell basic energy metabolism.Although the mutation in STK11 catalytic domain is responsible for the loss of its function,many mutations in the C-terminal region of STK11 have been identified in PJS as well as in other tumors.Since the C-terminal including 123 amino acid residue has multiple post-translational modification locus,it maybe play an important role in tumorigeness,but the mechanism is unclear.
Methods:In order to further investigate the function of the C-terminal of STK11,we cloned and inserted it into the prokaryotic expression system of pGEX4T-2,and induced the expression of the fusion protein GST-STK11,then purified the highly soluble fusion protein with the Glutathione Sepharose,then we screened the LOH12CR1 which can be interacted with the C-terminal region of STK11 by GST-pull down and mass chromatographic analysis. LOH12CR1,located on the 12p,is the candidate gene of suppressor tumor gene and highly reserved in evolution.
Results:The LOH12CR1 can co-localize with STK11 by immunofluorescence test,also we proved that they can interact by co-immunoprecipitation,however they do not directly affect in mammalian two-hybrid assay system.
Conclusion:Our research found the novel interaction protein LOH12CR1 of STK11,therefore provided a new clue for the research of its function.
引文
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