类泛素蛋白Nedd8对PDX-1蛋白稳定性的调控研究

发布时间：2018-07-12 15:17

本文选题：Nedd8 + PDX-1　；参考：《南京医科大学》2015年硕士论文

【摘要】：PDX-1(pancreatic duodenal homobox-1,胰腺-十二指肠同源盒基因-1)是调节胰岛素合成和分泌功能最重要的转录因子之一,参与成熟胰岛β细胞功能的维持。PDX-1的蛋白水平受到转录和翻译后修饰等方式的调控,维持其在胰岛β细胞中的高丰度。在氧化应激状态下,PDX-1通过泛素-蛋白酶体途径快速降解,造成胰岛β细胞损伤。已有的研究发现,Cullin3-pcif1复合物是PDX-1的E3泛素连接酶,然而该复合物受何种信号激活仍然不得而知。类泛素蛋白Nedd8(neural precursor cell-expressed developmentally down-regulated 8,神经发育下调因子8)通过对蛋白质进行Neddylation修饰来调控靶蛋白的功能,其作用底物主要是Cullin家族成员,包括Cullin3;Cullin蛋白被Neddylation修饰后激活其E3泛素连接酶的活性。本论文的研究目的是探讨Nedd8是否是Cullin3-pcif1介导的PDX-1蛋白降解的关键因子。借助于大鼠胰岛β细胞系INS1细胞,我们发现,高脂或者炎症因子处理都能使细胞内PDX-1的蛋白水平下调,而Neddylation修饰水平显著上升。外源性过表达Nedd8可诱导细胞内Neddylation修饰增强,与此同时,PDX-1蛋白水平下降;PDX-1蛋白水平与Neddylation修饰的程度呈相反的趋势。过表达Nedd8并不会影响PDX-1的基因表达。给予放线菌酮(CHX)处理后发现,Neddylation修饰的增强会降低PDX-1蛋白的稳定性。MG132处理能够逆转PDX-1的蛋白水平,说明Nedd8介导的PDX-1蛋白的降解与泛素-蛋白酶体途径相关。反之,用MLN4924抑制Nedd8的活性可使细胞内PDX-1蛋白的水平显著升高。为了明确Nedd8与PDX-1之间的关系,我们使用了激光共聚焦显微镜和免疫共沉淀(CO-IP)技术。结果显示,Nedd8与PDX-1之间存在共定位和相互作用。萤光素酶报告基因技术显示,过表达Nedd8能抑制胰岛素基因启动子的活性,并且主要是减少了胰岛素启动子A3/A4区域PDX-1的结合。定量RT-PCR实验显示,Neddylation修饰增强也能减少胰岛素基因的转录。钾刺激的胰岛素分泌功能实验(KSIS)显示,Neddylation修饰增强下调了胰岛β细胞的基础胰岛素分泌以及胰岛素的含量。总而言之,本项研究发现Nedd8能够加速PDX-1蛋白的泛素化降解。在氧化应激情况下,Nedd8的修饰活性增强,通过对PDX-1的E3泛素连接酶复合物(Cullin3-pcif1)进行修饰,加速了PDX-1蛋白的降解,从而抑制了胰岛素基因的转录,降低了胰岛素的含量和基础胰岛素的分泌。反之,抑制Nedd8的修饰活性可以增加细胞内PDX-1蛋白的水平。本研究揭示生理及病理情况下Nedd8都参与了PDX-1蛋白水平的调控,为糖尿病的防治提供潜在的靶点。
[Abstract]:PDX-1 (pancreatic duodenal homobox-1 (pancreaticoduodenal homobox gene -1) is one of the most important transcription factors regulating insulin synthesis and secretion. The protein level of PDX-1 involved in the maintenance of the function of mature islet 尾 cells is regulated by transcription and posttranslational modification. Maintain its high abundance in islet 尾 cells. Under oxidative stress, PDX-1 was rapidly degraded by ubiquitin proteasome pathway, resulting in islet 尾 cell damage. It has been found that the Cullin3-pcif1 complex is the E3 ubiquitin ligase of PDX-1, but the signal activation of the complex remains unknown. Ubiquitin like protein Nedd8 (neural precursor cell-expressed developmentally down-regulated 8, neurodevelopmental downregulation factor 8) regulates the function of target proteins by modifying them with Neddylation, the main substrates of which are members of the Cullin family. The activity of E 3 ubiquitin ligase was activated by the modification of Cullin 3 by Neddylation. The purpose of this study was to investigate whether Nedd8 is a key factor in cullin3-pcif1 mediated degradation of PDX-1 protein. With the aid of rat islet 尾 cell line INS1, we found that both hyperlipidemia and inflammatory factor treatment could down-regulate the protein level of PDX-1, while the Neddylation level increased significantly. Exogenous overexpression of Nedd8 could induce the enhancement of Neddylation modification, while the level of PDX-1 protein decreased and the level of PDX-1 protein decreased. Overexpression of Nedd8 does not affect the expression of PDX-1 gene. After treatment with actinomycin (CHX), it was found that the enhancement of Neddylation could decrease the stability of PDX-1 protein. MG132 treatment could reverse the protein level of PDX-1, indicating that the degradation of PDX-1 protein mediated by Nedd8 was related to the ubiquitin proteasome pathway. On the contrary, inhibition of Nedd8 activity by MLN4924 significantly increased the level of PDX-1 protein. In order to clarify the relationship between Nedd8 and PDX-1, we used laser confocal microscopy and immunoprecipitation (CO-IP) technique. The results showed that there was colocalization and interaction between Nedd8 and PDX-1. Luciferase reporter gene technique showed that overexpression of Nedd8 inhibited the activity of insulin gene promoter and mainly reduced the binding of A3/A4 region of insulin promoter to PDX-1. Quantitative RT-PCR showed that the enhancement of Neddylation could also reduce the transcription of insulin gene. Potassium stimulated insulin secretion assay (KSIS) showed that Neddylation enhanced the basal insulin secretion and insulin content of islet 尾 cells. All in all, this study found that Nedd8 can accelerate the ubiquification degradation of PDX-1 protein. The modification activity of Nedd8 was enhanced under oxidative stress. By modifying the E3 ubiquitin ligase complex of PDX-1 (Cullin3-pcif1), the degradation of PDX-1 protein was accelerated and the transcription of insulin gene was inhibited. Decreased insulin content and basal insulin secretion. In contrast, inhibition of the modification activity of Nedd8 increased the level of PDX-1 protein in cells. This study revealed that both physiologically and pathologically Nedd8 is involved in the regulation of PDX-1 protein level and provides a potential target for the prevention and treatment of diabetes.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.1

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