间隙连接蛋白46基因G143R突变型致病机制的研究

发布时间：2018-05-07 20:59

本文选题：Cx46G143R + 基因突变　；参考：《兰州大学》2012年硕士论文

【摘要】：间隙连接蛋白(Connexin,Cx)是一类存在于细胞膜表面的蛋白质,是构成间隙连接通道、半通道的基本组成单位。六个间隙连接蛋白单体聚合在一起形成间隙连接子表达在细胞质膜表面,又称为半通道。两个相邻细胞膜表面的间隙连接子相互对接形成跨膜的间隙连接通道。数十个到上万个间隙连接通道能够在细胞表面聚集形成间隙连接斑。间隙连接通道和半通道允许小分子物质(分子量1kDa)在细胞之间、或细胞与细胞外基质之间进行交换传递。间隙连接蛋白介导的细胞通讯在维持晶状体的渗透性,代谢稳态以及晶状体的透明性等方面起到了重要的作用。近年来研究表明,表达在晶状体纤维细胞的间隙连接蛋白(Cx46和Cx50)基因突变与人类遗传性白内障密切相关。目前已经发现了超过20个Cx46基因突变与家族遗传性白内障相关,这些Cx46基因突变型会通过影响间隙连接通道的形成和功能,或者半通道的通透性等导致白内障的发生。 Cx46’第143位氨基酸的突变(甘氨酸突变为精氨酸,Cx46G143R)是首次在Cx46蛋白细胞内环结构上发现的基因突变,该点突变与我国一个遗传性先天性Coppock白内障(核性颗粒状白内障)的发生相关,然而其致病分子机制还不明确。本文首次对Cx46细胞内环区域突变引起遗传性白内障的发生机制进行了研究。通过多序列对比的方法分析了位于Cx46氨基酸序列第143位的甘氨酸,发现该甘氨酸在不同物种的Cx46蛋白以及人类其他的间隙连接蛋白中均高度保守,对间隙连接蛋白的形成和／或功能具有重要的意义。为了研究Cx46G143R是否影响蛋白质的转录、翻译过程,我们克隆了Cx46野生型和Cx46G143R突变型基因,构建了表达质粒并转染HeLa细胞,Western blot和免疫荧光染色结果显不Cx46G143R与Cx46野生型一样,均能够成功地表达在HeLa细胞,并且在细胞内的分布相似,都能在细胞表面形成间隙连接斑。为了确定该突变型对间隙连接通道的形成以及功能是否有影响,使用划痕染料转移实验和微注射染料转移法检测了通道功能,结果显示Cx46G143R突变型降低了间隙连接通道的染料传递能力。当Cx46G143R突变型与Cx46野生型共表达在HeLa细胞上时,该突变会呈现显性负性作用。同样,将Cx46G143R表达在配对的非洲爪蟾卵母细胞后,使用双电极电压钳技术检测间隙连接通道间的电流,结果显示该突变同样以显性负性方式使间隙连接通道丧失电耦合作用。通过Triton X-100提取实验检测了间隙连接斑的形成,发现Cx46G143R在细胞表面形成的间隙连接斑多于Cx46野生型,说明该突变不是通过影响间隙连接通道的形成而改变其功能。间隙连接子除了形成间隙连接通道,也可以在细胞表面构成有功能的半通道。本文使用染料吸收实验分析了Cx46G143R对半通道功能的作用,结果发现,不同于对间隙连接通道的作用,Cx46G143R增加了半通道的活性。通过检测细胞表面未形成间隙连接通道的连接子的表达量,发现该突变导致的半通道活性的增加并不是由于细胞表面Cx46的表达增加,相反,该突变导致了细胞表面Cx46连接子的减少。为了进一步研究Cx46G143R形成的间隙连接通道／半通道功能变化的可能机制,本文使用Swiss Model分析了Cx46G143R对蛋白质三维结构的影响,结果显示该突变可能引起Cx46蛋白细胞内环三维结构的变化,在细胞内环的第119-131位氨基酸序列上产生了一个α螺旋结构。通过制备GST-Cx46/Cx46G143R细胞内环融合蛋白、进行Pull-down实验,本文首次研究了Cx46蛋白质细胞内环区域与羧基端的相互作用,结果显示Cx46细胞内环能够与Cx46蛋白相互结合,并且与Cx46野生型相比,改变了内环结构的Cx46G143R突变型与Cx46蛋白的结合能力更强,这说明Cx46G143R可能是通过改变细胞内环与羧基端之间“球和链”的相互作用,而引起间隙连接通道／半通道功能的变化。此外,我们也检测了Cx46G143R突变型对细胞活性的影响,WST-1实验结果显示Cx46G143R能够降低细胞的生存能力。用Annexin V和PI标记细胞进行流式分析,发现Cx46G143R通过半通道功能的改变,增加了细胞凋亡,同时也丧失了Cx46野生型抵抗氧化应激的能力。综上所述,Cx46介导的细胞通讯对于晶状体纤维细胞间营养物质和代谢物质的传递,维持细胞稳态有重要的作用。Cx46G143R突变可能通过增加Cx46蛋白羧基端与细胞内环的相互作用,引起间隙连接通道功能的抑制和半通道的开放,导致细胞活性降低、凋亡增加,从而参与遗传性白内障的发病。
[Abstract]:Connexin ( Cx ) is a kind of protein present on the surface of cell membrane . It is the basic unit of gap junction channel and half - channel . The gap junction between the two adjacent cell membrane surfaces forms the gap junction channel across the membrane . The gap junction channel and the half channel allow small molecule matter ( molecular weight 1 kDa ) to exchange between cells or between the cells and the extracellular matrix .

Intergap junction protein - mediated cellular communication plays an important role in maintaining the permeability , metabolic homeostasis , and transparency of the lens . In recent years , it has been shown that the gene mutation of Cx46 and Cx50 is closely related to human genetic cataract .

The mutation of Cx46 ' s 143 amino acid ( glycine mutation is arginine , Cx46G143R ) is the first gene mutation found on the inner ring structure of Cx46 protein . The mutation of Cx46 ' is related to the occurrence of a hereditary congenital Coppock cataract in our country .

In order to study whether Cx46G143R affects the transcription and translation of protein , we cloned Cx46 wild - type and Cx46G143R mutant genes . In order to study whether Cx46G143R affects the transcription and translation process of protein , we cloned Cx46 wild - type and Cx46G143R mutant genes .

It was found that Cx46G143R had a dominant negative effect when Cx46G143R mutant was co - expressed with Cx46 wild type in HeLa cells .

The effect of Cx46G143R on the half - channel function was analyzed by dye - absorption experiments . The result showed that Cx46G143R increased the activity of the half - channel by detecting the effect of Cx46G143R on the half - channel function .

The effect of Cx46G143R on the three - dimensional structure of Cx46 protein was investigated by Swiss Model .

In addition , we also examined the effect of Cx46G143R mutation on cell activity . The results showed that Cx46G143R was able to reduce cell viability by flow cytometry . It was found that Cx46G143R increased apoptosis by half - channel function , and also lost the ability of Cx46 wild - type to resist oxidative stress .

In conclusion , Cx46 - mediated cellular communication plays an important role in the transmission of nutrients and metabolic substances between the cells . Cx46G143R mutation may increase the interaction between the carboxy terminal of Cx46 protein and the inner ring of cells , which may lead to the inhibition of the function of gap junction channel and the opening of the half - channel , resulting in decreased cell activity and increased apoptosis , thereby participating in the pathogenesis of hereditary cataract .

【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R776.1

【共引文献】