【摘要】：多種翻譯后修飾之間存在著復(fù)雜的聯(lián)系，，這種聯(lián)系對(duì)于細(xì)胞活動(dòng)特別是蛋白的降解和功能的調(diào)節(jié)起著重要作用。泛素化和O-GlcNAc修飾作為兩種重要的翻譯后修飾，二者之間存在聯(lián)系。其中泛素化是泛素激活酶（E1）、泛素結(jié)合酶（E2）、泛素連接酶（E3）催化的多步反應(yīng)，這些酶中有一些能被O-GlcNAc修飾，或在轉(zhuǎn)錄水平受O-GlcNAc修飾的蛋白調(diào)節(jié)。本研究發(fā)現(xiàn)了在HEK293T細(xì)胞中的蛋白泛素化和O-GlcNAc修飾的聯(lián)系。通過(guò)在HEK293T細(xì)胞中轉(zhuǎn)染OGT，使其過(guò)表達(dá)sOGT、ncOGT，以及加入PUGNAc（OGA的抑制劑）和加入GLcN三種方式來(lái)提高細(xì)胞中蛋白的O-GlcNAc的修飾水平。在三種提高O-GlcNAc修飾水平的條件下，通過(guò)RT2profiler PCR array方法，檢測(cè)人泛素化途徑相關(guān)的84個(gè)基因轉(zhuǎn)錄水平的變化。其中FBXW10蛋白是一種泛素化的靶特定蛋白F-Box蛋白，在以上增加O-GlcNAc修飾的方式中都出現(xiàn)下調(diào)表達(dá)。根據(jù)GlcN對(duì)FBXW10基因有負(fù)調(diào)節(jié)作用，在HEK293T細(xì)胞中加入GlcN，在不同時(shí)間點(diǎn)進(jìn)行real-timePCR和Western Blot檢測(cè)，本研究發(fā)現(xiàn)在不同時(shí)間點(diǎn)mRNA水平和蛋白水平都有明顯下調(diào)。另外本研究還發(fā)現(xiàn)HEK293T細(xì)胞中內(nèi)源的FBXW10被O-GlcNAc修飾，O-GlcNAc修飾可能在多重水平上調(diào)節(jié)FBXW10。這些發(fā)現(xiàn)都說(shuō)明O-GlcNAc修飾與泛素化相關(guān)基因的調(diào)節(jié)有關(guān)，對(duì)理解O-GlcNAc和泛素化之間的相互聯(lián)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.
【學(xué)位授予單位】：天津大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R329.2

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