晶状体发育过程中间隙连接组成蛋白Connexin 50断裂的机制及相关生物学功能的研究

发布时间：2018-04-13 08:28

本文选题：间隙连接 + 半通道　；参考：《南京大学》2012年博士论文

【摘要】：Connexin50(Cx50)是晶状体中最为重要的间隙连接组成蛋白,并且在晶状体发育过程中自然断裂,而以前的研究认为晶状体Connexin的剪切与细胞死亡和白内障形成有关。数年之前,本课题组的研究结果发现鸡Cx50可以被类似Caspase-3的蛋白酶剪切。在本文研究中,我们采用最新的核磁共振技术,首次在晶状体内鉴定出了两个断裂位点：Glu-368和Asp-379。这两个位点位于Cx50的羧基端。根据之前的研究结果,Glu-368是Caspase-3所识别的酶切位点；通过计算机分析,Asp-379被确认为Caspase-1的剪切位点。Caspase-1和Caspase-3的活性只能够在晶状体的皮质层中被检测到,而与之相反的是：剪切生成的Cx50片段几乎都集中于晶状体核内。经紫外辐射处理后的年轻晶状体可以再现年龄较大的晶状体中出现的Cx50断裂情形,并且这种断裂可以被Caspase-1/3的特异性抑制剂所抑制。与全长的Cx50相比,由Caspase剪切而成的Cx50片段所组成的间隙连接通道的活性显著降低、而半通道的功能则完全丧失。此外,表达Cx50片段的细胞在紫外照射后表现出较高的细胞存活率。以上结果显示Cx50断裂可能是晶状体纤维细胞进行自我保护的一种机制。在第一章中,我们采用核磁共振的方法在体内鉴定出了Connexin50的断裂位点,并且在ExPASy Proteomics Sever数据库中搜寻到了与该断裂位点所关联的蛋白酶。此外,我们还引入体外酶切的方法来确认Cx50是否为Caspase-1和Caspase-3的底物,并且通过构建多种Cx50的点突变克隆来验证Glu-368和Asp-379是否为真正的酶切位点。结果显示,Cx50不仅是Caspase-3的底物而且可以被Caspase-1切割,位于羧基端的位点Glu-368和Asp-379分别是这两个蛋白酶的关键识别位点。在第二章中,我们首先确定了Caspase-1和Caspase-3在晶状体不同时期发育的表达区域以及表达水平,并且与Cx50断裂的情形进行了对应比较,验证了之前关于Cx50在晶状体皮质层中被剪切的假设。我们通过引入紫外辐射的技术手段,在年轻晶状体组织中再现了Cx50的断裂,并且用体内的实验证据再次验证了断裂位点存在的真实性。划痕染色实验和液滴刺激实验揭示了Cx50片段的生物学功能,即间隙连接活性大幅降低、半通道功能彻底丧失。借助流式细胞仪和Annexin V染色的技术,我们将Caspase切割所产生的Cx50片段特有的生物功能与细胞凋亡联系在一起,首次揭示了晶状体Connexin在保护细胞,抗氧化方面的积极作用。在最后一章的研究中,我们将研究对象拓展到了哺乳动物。通过体外酶切实验,我们确认了Caspase-1同样可以识别和切割小鼠Cx50,尽管其氨基酸序列上不存在保守的Caspase-1底物序列。为了准确定位酶切位点,我们克隆并且表达了包含小鼠Cx50全长羧基端或膜内环的GST融合蛋白,通过体外酶切实验,排除了该位点在羧基端的可能性,初步确定小鼠Cx50序列上与Caspase-1相关的断裂位点位于膜内环上。综上所述,本文通过研究Cx50的断裂机制,准确定位了断裂位点,找到并且证实了相关的蛋白酶,揭示了Cx50断裂的生物学功能和晶状体纤维自我保护的一种全新机制,为以后对晶状体疾病的研究提供了新的思路。
[Abstract]:Connexin50 ( Cx50 ) is the most important gap junction constituent protein in the lens , and naturally breaks during the development of the lens . In the past few years , we have found that the chicken Cx50 can be cleaved by the protease like Caspase - 3 . In this study , we have identified two cleavage sites : Glu - 368 and Asp - 379 in the lens for the first time . According to the previous study , Glu - 368 is the cleavage site recognized by Caspase - 3 .
By computer analysis , Asp - 379 was identified as the cleavage site of Caspase - 1 . The activity of Caspase - 1 and Caspase - 3 could only be detected in the cortical layer of the lens . In contrast , the Cx50 fragment produced by shearing could be inhibited by the specific inhibitor of Caspase - 1 / 3 .

In chapter 1 , we identified the cleavage site of Connexin50 in vivo by nuclear magnetic resonance method , and searched for the protease associated with the cleavage site in the ExPASY Proteomics Sever database . In addition , we introduced in vitro enzyme digestion method to confirm whether Cx50 is the substrate of Caspase - 1 and Caspase - 3 . The results show that Cx50 is not only the substrate of Caspase - 3 but also can be cleaved by Caspase - 1 , and Glu - 368 and Asp - 379 at the carboxy terminal are the key recognition sites of these two proteases .

In chapter 2 , we first determined the expression region and the expression level of Caspase - 1 and Caspase - 3 during different periods of lens , and compared with Cx50 ' s fracture , we verified the hypothesis that Cx50 was sheared in the lens cortex .

In the last chapter , we extended the research object to mammal . Through in vitro enzyme digestion experiment , we confirmed that Caspase - 1 can also recognize and cut the mouse Cx50 , although there is no conserved Caspase - 1 substrate sequence on its amino acid sequence . In order to locate the cleavage site accurately , we cloned and expressed the possibility of the site at the carboxy terminus by in vitro enzyme digestion experiment , and preliminarily determined that the cleavage site related to Caspase - 1 on the Cx50 sequence of mice is located on the inner ring of the membrane .

In conclusion , by studying the fracture mechanism of Cx50 , we locate the cleavage site accurately , find out and confirm the relevant protease , reveal the biological function of Cx50 cleavage and a new mechanism of lens fiber self - protection , and provide a new idea for the study of lens disease later .

【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R3416

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