O-GlcNAc糖基化修饰对TET3蛋白核定位的影响

发布时间：2018-07-11 09:42

本文选题：TET3 + O-GlcNAc糖基化　；参考：《西北农林科技大学》2015年硕士论文

【摘要】：O-GlcNAc(O-linked N-acetylglucosamine,O-GlcNAc)糖基化修饰是存在于细胞质和细胞核蛋白质丝氨酸或苏氨酸残基的羟基基团上的一种翻译后单糖修饰。O-GlcNAc糖基化修饰通过调控蛋白质细胞定位、蛋白活性、蛋白稳定等参与多种生命过程。O-GlcNAc糖基化修饰由两个酶催化,氧连氮乙酰葡萄糖胺转移酶(O-linked N-acetylglucosamine transferase,OGT)和氧连氮乙酰葡萄胺糖苷酶(O-GlcNAcase,OGA)。其中OGT负责O-GlcNAc的添加,OGA负责O-GlcNAc的移除。TET3蛋白是TET(ten-eleven translocation)蛋白家族成员之一,属于α-酮戊二酸和Fe2+依赖的双加氧酶。精卵发生受精后,TET3蛋白能特异性介导父源基因组发生快速的主动去甲基化,在胚胎早期发育和重编程过程中发挥重要作用。近期研究发现,人OGT不仅和TET3蛋白之间存在相互作用而且能够调控TET3蛋白的亚细胞定位和酶活性。这些研究结果提示我们,OGT可能通过调控TET3蛋白的亚细胞定位和功能来影响早期胚胎的DNA去甲基化过程,进而对胚胎发育和重编程产生影响。本实验以小鼠为模型,较系统地研究了小鼠OGT对TET3蛋白亚细胞定位的调控作用,比较了小鼠、人、牛、羊和猪的TET3和OGT基因的同源性,并检测了牛体细胞核移植胚胎和体外受精胚胎早期发育过程中TET3和与O-GlcNAc糖基化修饰相关基因的表达情况。本实验的主要研究内容和结果如下:1.以小鼠为模型,分别构建小鼠TET3蛋白和OGT蛋白过表达载体。当在NIH3T3细胞中过表达TET3蛋白时,免疫荧光显示TET3蛋白主要定位在细胞核;而在NIH3T3细胞中同时过表达TET3蛋白和OGT蛋白时,大部分TET3蛋白定位于细胞质。实验结果表明OGT蛋白具有调控TET3蛋白亚细胞定位的作用。2.为了研究OGT是否依赖其O-GlcNAc转移酶活性的调控TET3蛋白的亚细胞定位,我们使用OGT抑制剂Alloxan对过表达TET3蛋白和OGT蛋白的NIH3T3细胞进行处理,结果发现处理后TET3蛋白又定位于细胞核。这表明OGT依赖其O-GlcNAc转移酶活性来调控TET3的核定位。通过使用OGA的抑制剂PUGNAc处理过表达TET3蛋白的NIH3T3细胞,我们发现PUGNAc处理促进了TET3蛋白的出核;通过改变过表达TET3蛋白的NIH3T3细胞培养液中的葡萄糖浓度,我们发现低葡萄糖培养液同样抑制TET3蛋白的出核,降低OGT对TET3蛋白亚细胞定位的调控作用。这些结果证明OGT通过影响TET3蛋白的O-GlcNAc糖基化水平来调控TET3蛋白的细胞定位。3.为了进一步探究OGT调控TET3蛋白亚细胞定位的具体机制,我们构建TET3蛋白核定位信号(nuclear localization signal,NLS)缺失的表达载体;结果显示无NLS的TET3蛋白主要定位于细胞质,这提示我们O-GlcNAc糖基化修饰可能通过影响TET3蛋白的NLS来调控TET3蛋白的亚细胞定位。4.用生物信息学软件比对了小鼠、人、牛、山羊和猪的TET3和OGT基因序列,结果显示TET3和OGT基因在这些物种间具有较高的同源性。将小鼠TET3蛋白转入牛成纤维细胞可以正常发挥其羟甲基化功能,增强牛成纤维细胞的5hmc水平。对牛体细胞核移植胚胎和体外受精胚胎早期发育过程各时期的TET3和OGT基因进行qRT-PCR检测,结果发现TET3基因在胚胎8-细胞期前高表达,桑椹胚和囊胚几乎不表达;OGT基因也在早期胚胎中表达较高。对牛核移植胚胎和体外受精胚胎发育过程中甲基化水平进行检测发现,牛核移植和体外受精胚胎在胚胎基因组激活前都发生了DNA去甲基化过程,说明TET3蛋白在早期胚胎发育的DNA去甲基化过程中发挥重要作用。以上结果表明,OGT对TET3蛋白亚细胞定位的调控作用可以用于改善牛核移植胚胎去甲基化不完全的研究。综合以上结果,本实验发现小鼠OGT通过改变蛋白的O-GlcNAc糖基化水平调控TET3蛋白的亚细胞定位;小鼠、人、牛、羊和猪的TET3和OGT基因具有较高的同源性;TET3基因和OGT基因都在早期胚胎中表达较高。这些结果提示我们OGT对TET3蛋白亚细胞定位的调控作用也可以用于改善牛核移植胚胎去甲基化不完全的研究,为提高牛核移植重编程效率的研究提供了新的思路和理论依据。
[Abstract]:O-GlcNAc (O-linked N-acetylglucosamine, O-GlcNAc) glycosylated modification is a kind of translated monosaccharide modified.O-GlcNAc glycosylation modification that exists on the cytoplasm and nuclear protein serine or threonine residues. The glycosylation modification of.O-GlcNAc is involved in a variety of life processes by regulating protein cell location, protein viability, protein stability and so on. Glycosylated modification is catalyzed by two enzymes, O-linked N-acetylglucosamine transferase, OGT, and O-GlcNAcase, OGA. OGT is responsible for the addition of O-GlcNAc. OGA responsible for the removal of O-GlcNAc is one of the members of the TET protein family, It belongs to alpha ketopamyl diacid and Fe2+ dependent dioxygenase. After fertilized sperm, TET3 protein can specifically mediate the active demethylation of the parent genome, and play an important role in early embryonic development and reprogramming. Recent studies have found that human OGT not only has interaction with TET3 protein, but also regulates TET. The subcellular localization and enzyme activity of the 3 protein suggest that OGT may affect the DNA demethylation process of early embryos by regulating the subcellular localization and function of TET3 protein, and then influence embryo development and reprogramming. In this experiment, a mouse model was used to systematically study the TET3 protein subcells of mouse OGT. The homology of TET3 and OGT genes in mice, human, cattle, sheep and pigs was compared, and the expression of TET3 and O-GlcNAc glycosylation related genes during the early development of bovine somatic cell nuclear transfer embryos and in vitro fertilized embryos were detected. The main contents and results of this experiment were as follows: 1. the model of mice was divided into mice. TET3 protein and OGT protein overexpression vector were not constructed. When the TET3 protein was overexpressed in NIH3T3 cells, the immunofluorescence showed that the TET3 protein was mainly located in the nucleus; and when the TET3 protein and OGT protein were overexpressed in NIH3T3 cells, most of the TET3 protein was located in the cytoplasm. The experimental results showed that the OGT protein had the regulation of TET3 protein. The role of subcellular localization.2. in order to study whether OGT depends on the subcellular localization of the TET3 protein that is dependent on its O-GlcNAc transferase activity, we use OGT inhibitor Alloxan to process NIH3T3 cells expressing the TET3 protein and OGT protein, and the results show that the TET3 protein is located in the nucleus after treatment. This indicates that OGT depends on its O-GlcNAc transfer. Enzyme activity was used to regulate the nuclear location of TET3. By using the OGA inhibitor PUGNAc to treat NIH3T3 cells expressing TET3 protein, we found that PUGNAc treatment promoted the nucleation of TET3 protein; by changing the glucose concentration in the NIH3T3 cell culture medium that overexpressed the TET3 protein, we found that the low glucose culture also inhibited the TET3 protein. Nucleation, reducing the regulatory role of OGT on the subcellular localization of TET3 protein. These results show that OGT regulates the cell localization of TET3 protein by affecting the O-GlcNAc glycosylation level of TET3 protein to further explore the specific mechanism of OGT regulating TET3 protein subcellular localization. We construct TET3 protein nuclear localization signal (nuclear localization) Nal, NLS) missing expression vector; the results show that the TET3 protein without NLS is mainly located in the cytoplasm, which suggests that the O-GlcNAc glycosylation modification may regulate the subcellular localization of TET3 protein by the NLS of the TET3 protein to regulate the.4. using bioinformatics software to compare the TET3 and OGT gene sequences of mice, cattle, goats and pigs. The results show TET The 3 and OGT genes have high homology among these species. The transfer of mouse TET3 protein into bovine fibroblast cells can normally play its hydroxyl methylation and enhance the 5hmc level of bovine fibroblasts. QRT-PCR detection of TET3 and OGT genes in the early development period of bovine and in vitro fertilized embryos. It was found that the TET3 gene was highly expressed before the 8- cell stage of the embryo, and the morula and blastocysts were almost not expressed, and the OGT gene was also highly expressed in the early embryos. The detection of the methylation level in the bovine embryo and the development of in vitro fertilized embryos found that the bovine and in vitro fertilized embryos were all DNA before the activation of the embryo genome. The methylation process indicates that TET3 protein plays an important role in the DNA demethylation of early embryonic development. The above results show that the regulatory role of OGT on the subcellular localization of TET3 protein can be used to improve the study of the incomplete demethylation of bovine nuclear transplants. The results of the above results show that the mouse OGT can be used to change the O-Gl of the protein in the mouse. The level of cNAc glycosylation regulates the subcellular localization of TET3 protein; the TET3 and OGT genes of mice, human, cattle, sheep and pigs have high homology; both the TET3 gene and the OGT gene are highly expressed in the early embryos. These results suggest that the regulation of OGT on the subcellular localization of TET3 protein can also be used to improve the demethylation of bovine nuclear transplantation embryos. Incomplete research provides a new idea and theoretical basis for improving the reprogramming efficiency of bovine nuclear transfer.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.23

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