BAT1在小鼠晶状体上皮细胞中对Hsf4b转录活性调控的机制研究

发布时间：2018-02-21 06:45

  本文关键词： BAT1 Hsf4b 白内障 αB hsp25　出处：《河南大学》2014年硕士论文　论文类型：学位论文

【摘要】：背景 先天性白内障是导致儿童失明的主要原因，大约有1/3先天性白内障是遗传性的，其中常染色体显性遗传所占比列较高，，遗传性先天性白内障多数是由于基因突变引起的。先天性白内障主要的病理改变为晶状体的发育障碍、晶状体内纤维组织瘢块形成，最终导致晶状体浑浊。研究表明先天性白内障的发生与多种基因的突变有相关性，如晶状体细胞结构相关基因的突变（alpha-,bete-和gamma-crystallins and GJA3/A8，AQPO,BFSP2）和调控晶状体发育相关的转录因子基因突变（PITX3,PAX6，FOXE3,EYA1,MAF和Hsf4）。 热休克转录因子4（Hsf4）在新生儿晶状体发育的过程中起关键作用。Hsf4由于剪切方式不同形成不同的转录产物：Hsf4a和Hsf4b。Hsf4a为转录抑制因子，而Hsf4b则为转录激活因子。晶状体中只有Hsf4b这一种活性形式，它主要参与调控小分子热休克蛋白（Hsp25、γ-crystallin、α-crystallin）和纤维骨架蛋白（imentin、filesin）等蛋白的表达。研究发现Hsf4b基因的缺失可导致下游蛋白表达的缺失，如Hsp25、γ-晶状体蛋白中的γ-F，γ-S晶体蛋白等。这些发现都说明Hsf4b是调控晶状体早期发育的一个重要转录因子。Hsf4b受磷酸化、乙酰化、类泛素化、sumo化等的调控。但是，调控Hsf4b转录活性的信号通路以及Hsf4b调控晶状体早期发育的分子机制仍不清楚。 我们通过酵母双杂交实验,发现了一个新的与Hsf4相互作用的蛋白BAT1（又名UAP56，56KD,U2AF56-associated protein）。BAT1基因最早从猪克隆出来，它含有9个高度保守的结构域。BAT1蛋白属于DAED-box家族蛋白成员之一，DAED-box家族广泛存在于从细菌到哺乳动物的许多物种中，是一个ATP依赖的RNA解螺旋酶家族，既具有RNA激活的ATP结合和水解活性，又具有ATP依赖的RNA解螺旋活性。BAT1参与RNA的各种代谢过程如:RNA转录、mRNA前体剪接、核糖体和剪接体装配、核质运输、蛋白翻译、mRNA降解及维持mRNA的稳定性等重要生命活动。本研究在小鼠晶状上皮细胞中分析BAT1与Hsf4b的相互作用及细胞内定位，探讨BAT1对Hsf4b调控蛋白的影响，丰富先天性白内障的分子机制，为先天性白内障的早期诊断和治疗提供理论基础。 目的 探讨BAT1在小鼠晶状上皮细胞中与Hsf4b的相互作用及对Hsf4b下游蛋白表达的调控机理。 方法 (1)用pwzl-HA、pwzl-HA-Hsf4b、pbabe-HA-BAT1、pwzl-HA-Hsf4b+pbabe-HA-BAT1质粒的逆转录病毒感染hsf4-/-小鼠晶状上皮细胞（mLEC/hsf4-/-）,用blasticidin和puromycin筛选，构建细胞稳定株。突变体BAT1K95E、Hsf4b+BAT1K95E质粒的逆转录病毒感染hsf4-/-小鼠晶状上皮细胞（mLEC/hsf4-/-）,用puromycin筛选,构建细胞稳定株。 (2)应用细胞免疫荧光技术，分析Hsf4b与BAT1亚细胞定位。 (3)应用Realtime-PCR方法，分析BAT1对Hsf4b下游基因在mRNA水平的影响，应用免疫印迹（western blotting）实验，分析BAT1对Hsf4b调控蛋白αB晶状体蛋白、hsp25蛋白的表达。 (4)应用Luciferase assay实验，检测BAT1对Hsf4b下游基因aB-晶状体蛋白基因启动子的调控。 (5)应用细胞核质分离，Realtime-PCR方法，分析BAT1对Hsf4b下游蛋白的核质输出的影响，利用Pull Down实验，探讨Hsf4b是否可以促进BAT1的泛素化。 (6)利用BAT1在mRNA水平具有核输出功能，而95位点是其核输出功能的关键位点。构建pbabe-HA-BAT1K95E突变质粒，酶切测序及鉴定突变位点。 (7)应用Realtime-PCR方法，分析BAT1基因核输出位点突变对Hsf4b下游基因αΒ在mRNA水平的影响。 结果 (1)在小鼠晶状上皮细胞mLEC中，BAT1与Hsf4b在细胞核共定位，同时抑制hsf4b的下游蛋白αB基因启动子的转录活性。BAT1对Hsf4b下游蛋白αB、hsp25的表达起下调的作用。 (2)根据BAT1的生物学功能进一步分析，BAT1对Hsf4b下游基因aB在核质输出方面无调控作用。同时BAT1对Hsf4b下游蛋白αB的抑制作用不是由于BAT1 的核输出起作用，BAT1核输出功能突变后对Hsf4b的下游基因αB在mRNA水平 有所下调。同时，Hsf4b不能促进BAT1的泛素化。结论 BAT1与Hsf4b在体外相结合，同时在小鼠晶状上皮细胞内共定位于细胞核。BAT1降低Hsf4b下游基因αB、hsp25mRNA水平、减少αB、hsp25蛋白表达，且此作用与BAT1的ATP结合能力无明显关系。
[Abstract]:Background Congenital cataract is the main cause of blindness in children . Approximately 1 / 3 of congenital cataract is hereditary , in which autosomal dominant inheritance is high , hereditary congenital cataract is mostly caused by gene mutation . The main pathological changes of congenital cataract are caused by gene mutation . The main pathological changes of congenital cataract are caused by mutation of lens . The research shows that the occurrence of congenital cataract is associated with mutation of various genes ( alpha - , bete - and gamma - crystallins and GJA3 / A8 , AQPO , BFSP2 ) and transcription factor gene mutation related to regulation of lens development ( PITX3 , PAX6 , FoxE3 , eyA1 , MAF and Hsf4 ) . Hsf4 plays a key role in the process of neonatal lens development . Hsf4 forms different transcription products due to shear . Hsf4 is an active form of transcription - inhibiting factor . It is mainly involved in the regulation of the expression of downstream protein , such as Hsp25 , 纬 - crystallin , 伪 - crystallin , and so on . We found a new protein BAT1 ( also named UAP56 , 56KD , U2AF56 - associated protein ) interacting with Hsf4 through yeast two - hybrid experiment . BAT1 protein belongs to one of DAED - box family proteins . The DAED - box family is widely present in many species of DAED - box family . The DAED - box family is a ATP - dependent RNA helicase family , which has both RNA - activated ATP binding and hydrolytic activity and ATP - dependent RNA helicase activity . Purpose Objective To investigate the interaction between BAT1 and Hs4b and the mechanism of protein expression in the downstream protein of BAT1 . method ( 1 ) Retroviral infection of hsf4 - / - mouse crystalline epithelial cells ( mLEC / hsf4 - / - ) with the recombinant retroviral infection hsf4 - / - mouse crystalline epithelial cells ( mLEC / hsf4 - / - ) , which were transfected with the plasmid ptimiticidin and puromycin , by using the retroviral infection hsf4 - / - mouse crystalline epithelial cells ( mLEC / hsf4 - / - ) of the plasmid of pw1 - HA , pw2 - HA - HSV4b , pounce - HA - BAT1 , pw1 - HA - HSV4b + p. - HA - BAT1 , and then screened with puromycin to construct a cell - stable strain . ( 2 ) Using cellular immunofluorescence technique , we analyzed the localization of Hs4b and BAT1 sub - cells . ( 3 ) Using the method of Realtime PCR , the effect of BAT1 on mRNA level of downstream gene of HSV4b was analyzed . Western blotting was used to analyze the expression of BAT1 on the expression of 伪 - B lens protein and Hsp25 protein . ( 4 ) The regulation of the promoter of aB - lens protein in the downstream gene of BAT1 was detected by Lucifer assay . ( 5 ) To investigate the effect of BAT1 on the nuclear output of downstream protein by using nuclear separation and Realtime - PCR . Using Pull Down experiment , it was discussed whether HSF4b could promote the ubiquitination of BAT1 . ( 6 ) The expression of BAT1 has nuclear output function at mRNA level , and the 95 site is the key point of its nuclear output function . Construction of ptase - HA - BAT1K95E mutant plasmid , enzyme digestion and sequencing and identification of mutation site . ( 7 ) Using the method of Realtime PCR , the effect of the mutation of BAT1 gene nuclear output site on the mRNA level of the gene 伪尾 in the downstream gene was analyzed . Results ( 1 ) In the mouse crystalline epithelial cell mLEC , BAT1 and HSV4b were co - located in the nucleus , while inhibiting the transcription activity of the downstream protein 伪B gene promoter of hsf4b . The BAT1 could regulate the expression of downstream protein 伪B and Hsp25 . ( 2 ) According to the biological function of BAT1 , BAT1 has no regulatory effect on the nuclear output of downstream gene aB . The inhibitory effect of BAT1 on downstream protein 伪B is not due to BAT1 . The downstream gene 伪B downstream of the BAT1 nuclear output function after the mutation of the BAT1 nuclear output function is at the mRNA level There was a downregulation . At the same time , Hs4b could not promote the ubiquitination of BAT1 . Conclusion At the same time , BAT1 and HSV4b were combined in vitro . At the same time , the cells of BAT1 were located in the nucleus . BAT1 reduced the levels of the downstream gene 伪B , Hsp25 mRNA , and decreased the expression of 伪B and Hsp25 , and this effect was not significantly associated with the ATP binding ability of BAT1 .

【学位授予单位】：河南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R776.1

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