一磷酸腺苷激活蛋白激酶调节孕激素受体转录活性的研究

发布时间：2017-12-31 13:29

本文关键词：一磷酸腺苷激活蛋白激酶调节孕激素受体转录活性的研究　出处：《华东师范大学》2011年硕士论文　论文类型：学位论文

【摘要】：一磷酸腺苷激活蛋白激酶(AMP-activated Protein Kinase, AMPK)是调节体内代谢平衡的丝氨酸/苏氨酸(Ser/Thr)蛋白激酶,是细胞能量状态变化的感受器和能量代谢的主开关。当细胞内能量缺乏、处于应激状态时,AMP/ATP比率升高,激活AMPK。AMPK一旦被激活,会关闭消耗能量的合成代谢,而开启产生能量的分解代谢。它不仅可以通过直接磷酸化其下游底物的代谢酶类直接调控代谢步骤,而且可以通过磷酸化一些转录因子,对其下游信号发挥长效作用。虽然这些年对于AMPK的研究越来越多,使其重要性日益突显,但是,对AMPK整个调控网络的理解还远远不够。因此,发现AMPK相互作用的蛋白,并进一步研究其与AMPK在细胞信号调控中的机制,将为我们更好的了解AMPK的功能及机制提供一个很好的平台。根据AMPK各亚基的表达分布及其敏感性,我们首先选取AMPKα2、β1、γ1亚基分别进行酵母双杂交实验。但由于AMPKα2本身就可以使报告基因有轻微的表达(即“自激活”),因而分别用AMPKβ1亚基和γ1亚基钓取相互作用蛋白。以AMPKγ1亚基作为“诱饵”蛋白,得到的主要是AMPKβ亚基。以AMPKβ1亚基作为“诱饵”蛋白,得到了71个与之相互作用的蛋白。其中,包括代谢酶及其它酶类30个、转录因子或转录相关蛋白9个、蛋白转运相关蛋白5个、GTP结合蛋白4个及支架蛋白3个,此外还有细胞周期调节蛋白、RNA结合蛋白、DNA修复蛋白、伴侣蛋白等以及一些未知功能的蛋白。从酵母双杂交的结果来看,AMPK不仅在代谢领域,而且在许多非代谢领域,如核受体及其它转录因子的调节、信号转导、DNA修复及细胞周期调节等,可能都起到非常重要的作用。我们进一步从已经得到的AMPK相互作用蛋白中,选取转录相关调节蛋白进行进一步研究。孕酮(即孕激素,progesterone)是一个调节哺乳动物生长生殖的甾醇类激素,在正常细胞的生长分化以及靶组织的癌变中,起着非常重要的作用。孕酮的生物学功能是依靠孕酮受体(Progesterone Receptor, PR)实现的。在经典的PR信号通路中,当孕激素进入细胞与PR结合后,PR构象发生改变,与伴侣分子解离,并二聚化入核,与目标基因启动子上的孕激素响应元件(Progesterone Response Element, PRE)结合,募集转录共激活因子,从而启动下游基因的转录。除了PR的配体,包括其激动剂、拮抗剂以及选择性PR调节剂(Selective Progesterone Receptor Modulator, SPRM)外,一些细胞内信号的调节因子,如EGF等,也可以调节PR的转录活性。然而,AMPK对PR转录活性的调节,至今尚未有人报导。通过免疫共沉淀方法研究发现,PR可以与AMPK的α1、α2及β1亚基相互作用。这暗示AMPK有可能参与PR信号通路的调节。因此,我们利用AMPK的小分子调节剂检测了其对PR转录活性的影响。AMPK的激活剂AICAR和metformin均可抑制PRE驱动的荧光素酶(PRE-luciferase)的表达及PR下游调节基因的转录。用AMPK的选择性抑制剂Compound C可以逆转AICAR及metformin对PR转录活性的抑制。用RNA干扰的方法下调内源性的AMPK可以提高PR的转录活性。这些结果均表明,AMPK激活很可能抑制PR的转录活性。而进一步的研究结果表明,AMPK的激活能够引起PR磷酸化状态的改变。AMPK的激活不影响PR的入核,但是阻碍了SRC3对PR的转录调节；AMPK对PR转录活性的调节不改变其对孕激素响应的阈浓度,而是降低了孕激素能引起PR转录活性的最高水平。综上所述,通过酵母双杂交技术,我们发现很多AMPK潜在的相互作用蛋白,它们涉及生命活动的许多领域。随后,我们以PR作为重点进行了下一步研究。在细胞水平上,验证了PR与AMPK的相互作用。我们首次发现,AMPK的激活可能通过改变PR的磷酸化状态而抑制PR的转录活性。以上研究工作不仅扩充了我们对AMPK信号通路的认识,也为进一步探讨能量代谢与孕激素信号通路间的关系提供了重要的线索。
[Abstract]:Adenosine monophosphate activated protein kinase (AMP-activated Protein, Kinase, AMPK) to regulate the metabolic balance of serine / threonine protein kinase (Ser/Thr), is the main switch sensor and energy cell energy metabolism status change. When cells lack of energy, stressed, AMP/ATP ratio increased, the activation of AMPK.AMPK once activated synthesis and metabolism of energy consumption, will be closed, and open the catabolism of generating energy. It can not only through direct phosphorylation of its downstream substrates directly regulate the metabolic enzymes and metabolic steps through phosphorylation of transcription factors, play a long-term effect on the downstream signal. Although these years more and more studies on AMPK, the with its increasing importance, however, the whole AMPK regulatory network understanding is not enough. Therefore, AMPK interacting protein, and further study with A The mechanism of MPK in the regulation of cellular signals will provide a good platform for us to better understand the functions and mechanisms of AMPK.
According to the expression and distribution of sensitivity of AMPK subunits, we first select the AMPK alpha 2 beta 1 gamma 1 subunit of the yeast two hybrid experiment. But because the AMPK alpha 2 itself can make a slight expression of the reporter gene ("self activation"), which were used AMPK beta 1 subunit and gamma 1 subunit obtained interacting protein. AMPK gamma 1 subunit as bait protein, are mainly AMPK beta subunit. AMPK beta 1 subunit as bait protein, we obtained 71 interacting proteins. Among them, including metabolic enzymes and other enzymes 30 the transcription factor, or transcription associated protein 9, protein transport related protein 5, GTP binding protein 4 and 3 in addition to the scaffold protein, cell cycle regulatory proteins, RNA binding protein, DNA repair proteins, chaperones and other unknown proteins from yeast two hybrid results, not only AMPK in metabolism In many areas, such as nuclear receptor and other transcription factors, signal transduction, DNA repair and cell cycle regulation may play a very important role.
We have been further from the AMPK interacting protein, transcription related regulatory proteins were selected for further study. Progesterone (i.e. progesterone, progesterone) is a growth regulating mammalian reproductive steroid hormone in normal cell growth, differentiation and carcinogenesis in the target tissue, plays a very important role in the biological function of progesterone. Is to rely on progesterone receptor (Progesterone Receptor, PR). In the implementation of the canonical PR signaling pathway, when progesterone into cells when combined with PR, PR undergoes conformational changes, and chaperone dissociation, and two poly into nuclear, and the target gene promoter on the progesterone response element (Progesterone Response, Element, PRE) with raising the transcriptional coactivator, which initiates transcription of downstream genes. In addition to PR ligands, including its agonists, antagonists and selective PR modulators (Selective Progester One, Receptor, Modulator, SPRM, and some intracellular signal regulatory factors, such as EGF, can also regulate PR transcriptional activity. However, the regulation of AMPK on PR transcriptional activity has not been reported yet.
The co immunoprecipitation method study found that PR and AMPK alpha 1, alpha 2 and beta 1 subunit interaction. It suggested that AMPK may regulate PR signaling pathway. Therefore, we use small molecule modulators AMPK detected its effect on transcriptional activity of PR activator of.AMPK AICAR and metformin can inhibition of PRE driven luciferase (PRE-luciferase) and the expression of PR downstream regulated gene transcription by AMPK. The selective Compound inhibitor C can inhibit the reversal of AICAR and metformin on the transcriptional activity of PR by RNA interference method. Downregulation of endogenous AMPK can provide high transcriptional activity of PR. These results indicate that AMPK activation of transcription activity may inhibit PR. Further study results show that the activation of AMPK can activate PR phosphorylation state changes in the.AMPK does not affect the PR into the nucleus, but hindered the transcriptional regulation of SRC3 on PR AMP; The regulation of K's transcriptional activity of PR does not alter the threshold concentration of the response to progestin, but reduces the highest level of PR transcriptional activity by progestin.
To sum up, through the yeast two hybrid system, we found many potential interacting proteins of AMPK, which relates to many fields of life activities. Then, we use PR as the focus of the next step of the research. At the cellular level, verified the interaction between PR and AMPK. For the first time we found that the transcriptional activity of AMPK activation may inhibition of PR phosphorylation by PR and the change of state. The above research work not only expands our understanding of the AMPK signaling pathway, but also to further explore the relationship between energy metabolism and progesterone signaling pathways provide important clues.

【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

【共引文献】