O-GlcNAc修饰对泛素化相关基因的调控
发布时间：2019-04-28 11:43
【摘要】：多种翻译后修饰之间存在着复杂的联系，，这种联系对于细胞活动特别是蛋白的降解和功能的调节起着重要作用。泛素化和O-GlcNAc修饰作为两种重要的翻译后修饰，二者之间存在联系。其中泛素化是泛素激活酶（E1）、泛素结合酶（E2）、泛素连接酶（E3）催化的多步反应，这些酶中有一些能被O-GlcNAc修饰，或在转录水平受O-GlcNAc修饰的蛋白调节。本研究发现了在HEK293T细胞中的蛋白泛素化和O-GlcNAc修饰的联系。通过在HEK293T细胞中转染OGT，使其过表达sOGT、ncOGT，以及加入PUGNAc（OGA的抑制剂）和加入GLcN三种方式来提高细胞中蛋白的O-GlcNAc的修饰水平。在三种提高O-GlcNAc修饰水平的条件下，通过RT2profiler PCR array方法，检测人泛素化途径相关的84个基因转录水平的变化。其中FBXW10蛋白是一种泛素化的靶特定蛋白F-Box蛋白，在以上增加O-GlcNAc修饰的方式中都出现下调表达。根据GlcN对FBXW10基因有负调节作用，在HEK293T细胞中加入GlcN，在不同时间点进行real-timePCR和Western Blot检测，本研究发现在不同时间点mRNA水平和蛋白水平都有明显下调。另外本研究还发现HEK293T细胞中内源的FBXW10被O-GlcNAc修饰，O-GlcNAc修饰可能在多重水平上调节FBXW10。这些发现都说明O-GlcNAc修饰与泛素化相关基因的调节有关，对理解O-GlcNAc和泛素化之间的相互联系起到重要作用。
[Abstract]:There are complex links between a variety of post-translational modifications, which play an important role in cell activity, especially protein degradation and functional regulation. As two important post-translational modifications, ubiquitin and O-GlcNAc modification are related to each other. Ubiquitin is a multistep reaction catalyzed by ubiquitin-activating enzyme (E1), ubiquitin-binding enzyme (E2) and ubiquitin ligase (E3). Some of these enzymes can be modified by O-GlcNAc or regulated by O-GlcNAc-modified proteins at transcription level. In this study, we found the association between protein ubiquitin and O-GlcNAc modification in HEK293T cells. The modification level of protein O-GlcNAc in HEK293T cells was improved by overexpressing sOGT,ncOGT, and adding inhibitor of PUGNAc (OGA in HEK293T cells and adding GLcN in three ways. The changes of transcription levels of 84 genes related to the human ubiquitin pathway were detected by RT2profiler PCR array under three conditions of increasing the level of O-GlcNAc modification. Among them, FBXW10 protein is a ubiquitin-specific target-specific protein F-Box protein, which is down-regulated in the above-mentioned ways of increasing O-GlcNAc modification. According to the negative regulation of FBXW10 gene by GlcN, real-timePCR and Western Blot were detected by adding GlcN, into HEK293T cells at different time points. In this study, mRNA level and protein level were significantly down-regulated at different time points. In addition, we also found that endogenous FBXW10 in HEK293T cells is modified by O-GlcNAc, and O-GlcNAc modification may regulate FBXW10. at multiple levels. These findings suggest that O-GlcNAc modification is involved in the regulation of ubiquitin-related genes and plays an important role in understanding the relationship between O-GlcNAc and ubiquialization.
【学位授予单位】：天津大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R329.2
[Abstract]:There are complex links between a variety of post-translational modifications, which play an important role in cell activity, especially protein degradation and functional regulation. As two important post-translational modifications, ubiquitin and O-GlcNAc modification are related to each other. Ubiquitin is a multistep reaction catalyzed by ubiquitin-activating enzyme (E1), ubiquitin-binding enzyme (E2) and ubiquitin ligase (E3). Some of these enzymes can be modified by O-GlcNAc or regulated by O-GlcNAc-modified proteins at transcription level. In this study, we found the association between protein ubiquitin and O-GlcNAc modification in HEK293T cells. The modification level of protein O-GlcNAc in HEK293T cells was improved by overexpressing sOGT,ncOGT, and adding inhibitor of PUGNAc (OGA in HEK293T cells and adding GLcN in three ways. The changes of transcription levels of 84 genes related to the human ubiquitin pathway were detected by RT2profiler PCR array under three conditions of increasing the level of O-GlcNAc modification. Among them, FBXW10 protein is a ubiquitin-specific target-specific protein F-Box protein, which is down-regulated in the above-mentioned ways of increasing O-GlcNAc modification. According to the negative regulation of FBXW10 gene by GlcN, real-timePCR and Western Blot were detected by adding GlcN, into HEK293T cells at different time points. In this study, mRNA level and protein level were significantly down-regulated at different time points. In addition, we also found that endogenous FBXW10 in HEK293T cells is modified by O-GlcNAc, and O-GlcNAc modification may regulate FBXW10. at multiple levels. These findings suggest that O-GlcNAc modification is involved in the regulation of ubiquitin-related genes and plays an important role in understanding the relationship between O-GlcNAc and ubiquialization.
【学位授予单位】：天津大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R329.2
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