PRB与PIAS3的相互作用及其功能研究

发布时间：2018-05-30 10:28

本文选题：PRB + PIAS3　；参考：《中国人民解放军军事医学科学院》2005年硕士论文

【摘要】：孕激素是一种甾体类激素,在细胞生长、增殖功能中发挥重要作用。孕激素功能的发挥主要通过激素与细胞内特异的孕激素受体相互作用而产生,而孕激素受体PR主要有两种亚型,即孕激素受体PRA和PRB。孕激素受体是一个结构相似基因家族-核受体亚家族的成员,这个家族的蛋白都是配基激活转录调节子。孕激素受体这两种亚型的不同主要在于PRB在其N端的BUS区包含一段164个氨基酸的AF3区。一般来说,PRB存在于细胞浆中,在孕激素作用下,PRB会进入细胞核发挥其转录活性。 PR和其他核受体的转录活性与蛋白的转录后共价修饰有关,它会影响到蛋白质的结构和蛋白质与蛋白质之间的相互作用。这些修饰包括蛋白质的磷酸化、乙酰化、泛素化和SUMO化。其中SUMO化与共价结合SUMO(small ubiauitin-like modifier protein)蛋白有关。一些重要的转录因子,比如GR、AR、P53、c-Jun和c-Myc都与SUMO-1共价结合。与泛素化一样,SUMO化有一组激活酶-E1、E2、E3酶,SUMO化修饰会引起蛋白质亚细胞定位、稳定性或者是基因的转录活性的改变。本文结果显示,我们在用激光扫描共聚焦显微镜(LSCM)观察PRB的亚细胞定位时发现,在PIAS3的存在下,即使加入孕激素,也有大部分PRB并不进入细胞核。这提示在PRB与PIAS3之间可能存在某种功能上的相互作用。因此,我们在人胚胎肾细胞293T中建立了一套PRB转录激活荧光报告测试系统,检测到PIAS3对PRB转录激活活性有很强的抑制作用,并且这种抑制作用是孕激素依赖的。为了验证PRB与PIAS3之间是否可以相互结合,我们将PRB与PIAS3构建到酵母表达载体上。实验证明PRB与PIAS3在酵母细胞中可以相互结合,并且我们找出PRB与PIAS3相互作用的区域主要是PRB的DBD结构域。为进一步确认PRB与PIAS3之间存在特异性的蛋白质相互作用,本文进行了体外结合实验(in vitro: GST pull-down)和体内结合实验(in vivo: IP),并获得了与酵母中的发现相一致的实验结果。除此之外,本文还应用激光扫描共聚焦
[Abstract]:Progesterone is a steroid hormone, which plays an important role in cell growth and proliferation. The function of progesterone is mainly produced by hormone interacting with specific progesterone receptor in cells, and PR has two subtypes: progesterone receptor (PRA) and progesterone receptor (PRB). Progesterone receptors are members of a structurally similar gene family nuclear receptor subfamily whose proteins are ligand-activated transcriptional regulators. The difference between the two subtypes of progesterone receptor is that PRB contains a 164 amino acid AF3 region in its N-terminal BUS region. Generally speaking, PRB exists in the cytoplasm, and PRB enters the nucleus to play its transcriptional activity under the action of progesterone. The transcriptional activity of PR and other nuclear receptors is related to the post-transcriptional modification of proteins, which affects the structure of proteins and the interaction between proteins. These modifications include protein phosphorylation, acetylation, ubiquification and SUMO. Among them, SUMO is related to covalent binding SUMO(small ubiauitin-like modifier protein. Some important transcription factors, such as GRARN P 53 C Jun and c-Myc, are covalently bound to SUMO-1. Similar to Ubiquitin, there is a group of activase-E _ (1) E _ (2) E _ (2) E _ (3) modified to cause subcellular localization, stability, or transcriptional activity of the gene. The results showed that when we observed the subcellular localization of PRB by laser scanning confocal microscope (LSCM), we found that in the presence of PIAS3, most of PRB did not enter the nucleus even if progesterone was added. This suggests that there may be some functional interaction between PRB and PIAS3. Therefore, we have established a PRB transcription activation fluorescence report system in human embryonic kidney cells 293T. We found that PIAS3 has a strong inhibitory effect on the activation of PRB transcription, and this inhibition is progesterone-dependent. To verify whether PRB and PIAS3 can be combined with each other, PRB and PIAS3 were constructed into yeast expression vector. The results showed that PRB and PIAS3 could bind to each other in yeast cells, and we found that the main domain of interaction between PRB and PIAS3 was the DBD domain of PRB. In order to further confirm the specific protein interaction between PRB and PIAS3, in vitro binding test (GST pull-down) and in vivo binding experiment (in Vivo: IPN) were carried out, and the results were in agreement with those found in yeast. In addition, laser scanning confocal technique is also used in this paper.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：Q504

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