本文關(guān)鍵詞：一磷酸腺苷激活蛋白激酶調(diào)節(jié)孕激素受體轉(zhuǎn)錄活性的研究　出處：《華東師范大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： AMPK 酵母雙雜交 相互作用蛋白 PR 轉(zhuǎn)錄活性

【摘要】：一磷酸腺苷激活蛋白激酶(AMP-activated Protein Kinase, AMPK)是調(diào)節(jié)體內(nèi)代謝平衡的絲氨酸/蘇氨酸(Ser/Thr)蛋白激酶,是細(xì)胞能量狀態(tài)變化的感受器和能量代謝的主開(kāi)關(guān)。當(dāng)細(xì)胞內(nèi)能量缺乏、處于應(yīng)激狀態(tài)時(shí),AMP/ATP比率升高,激活A(yù)MPK。AMPK一旦被激活,會(huì)關(guān)閉消耗能量的合成代謝,而開(kāi)啟產(chǎn)生能量的分解代謝。它不僅可以通過(guò)直接磷酸化其下游底物的代謝酶類直接調(diào)控代謝步驟,而且可以通過(guò)磷酸化一些轉(zhuǎn)錄因子,對(duì)其下游信號(hào)發(fā)揮長(zhǎng)效作用。雖然這些年對(duì)于AMPK的研究越來(lái)越多,使其重要性日益突顯,但是,對(duì)AMPK整個(gè)調(diào)控網(wǎng)絡(luò)的理解還遠(yuǎn)遠(yuǎn)不夠。因此,發(fā)現(xiàn)AMPK相互作用的蛋白,并進(jìn)一步研究其與AMPK在細(xì)胞信號(hào)調(diào)控中的機(jī)制,將為我們更好的了解AMPK的功能及機(jī)制提供一個(gè)很好的平臺(tái)。 根據(jù)AMPK各亞基的表達(dá)分布及其敏感性,我們首先選取AMPKα2、β1、γ1亞基分別進(jìn)行酵母雙雜交實(shí)驗(yàn)。但由于AMPKα2本身就可以使報(bào)告基因有輕微的表達(dá)(即“自激活”),因而分別用AMPKβ1亞基和γ1亞基釣取相互作用蛋白。以AMPKγ1亞基作為“誘餌”蛋白,得到的主要是AMPKβ亞基。以AMPKβ1亞基作為“誘餌”蛋白,得到了71個(gè)與之相互作用的蛋白。其中,包括代謝酶及其它酶類30個(gè)、轉(zhuǎn)錄因子或轉(zhuǎn)錄相關(guān)蛋白9個(gè)、蛋白轉(zhuǎn)運(yùn)相關(guān)蛋白5個(gè)、GTP結(jié)合蛋白4個(gè)及支架蛋白3個(gè),此外還有細(xì)胞周期調(diào)節(jié)蛋白、RNA結(jié)合蛋白、DNA修復(fù)蛋白、伴侶蛋白等以及一些未知功能的蛋白。從酵母雙雜交的結(jié)果來(lái)看,AMPK不僅在代謝領(lǐng)域,而且在許多非代謝領(lǐng)域,如核受體及其它轉(zhuǎn)錄因子的調(diào)節(jié)、信號(hào)轉(zhuǎn)導(dǎo)、DNA修復(fù)及細(xì)胞周期調(diào)節(jié)等,可能都起到非常重要的作用。 我們進(jìn)一步從已經(jīng)得到的AMPK相互作用蛋白中,選取轉(zhuǎn)錄相關(guān)調(diào)節(jié)蛋白進(jìn)行進(jìn)一步研究。孕酮(即孕激素,progesterone)是一個(gè)調(diào)節(jié)哺乳動(dòng)物生長(zhǎng)生殖的甾醇類激素,在正常細(xì)胞的生長(zhǎng)分化以及靶組織的癌變中,起著非常重要的作用。孕酮的生物學(xué)功能是依靠孕酮受體(Progesterone Receptor, PR)實(shí)現(xiàn)的。在經(jīng)典的PR信號(hào)通路中,當(dāng)孕激素進(jìn)入細(xì)胞與PR結(jié)合后,PR構(gòu)象發(fā)生改變,與伴侶分子解離,并二聚化入核,與目標(biāo)基因啟動(dòng)子上的孕激素響應(yīng)元件(Progesterone Response Element, PRE)結(jié)合,募集轉(zhuǎn)錄共激活因子,從而啟動(dòng)下游基因的轉(zhuǎn)錄。除了PR的配體,包括其激動(dòng)劑、拮抗劑以及選擇性PR調(diào)節(jié)劑(Selective Progesterone Receptor Modulator, SPRM)外,一些細(xì)胞內(nèi)信號(hào)的調(diào)節(jié)因子,如EGF等,也可以調(diào)節(jié)PR的轉(zhuǎn)錄活性。然而,AMPK對(duì)PR轉(zhuǎn)錄活性的調(diào)節(jié),至今尚未有人報(bào)導(dǎo)。 通過(guò)免疫共沉淀方法研究發(fā)現(xiàn),PR可以與AMPK的α1、α2及β1亞基相互作用。這暗示AMPK有可能參與PR信號(hào)通路的調(diào)節(jié)。因此,我們利用AMPK的小分子調(diào)節(jié)劑檢測(cè)了其對(duì)PR轉(zhuǎn)錄活性的影響。AMPK的激活劑AICAR和metformin均可抑制PRE驅(qū)動(dòng)的熒光素酶(PRE-luciferase)的表達(dá)及PR下游調(diào)節(jié)基因的轉(zhuǎn)錄。用AMPK的選擇性抑制劑Compound C可以逆轉(zhuǎn)AICAR及metformin對(duì)PR轉(zhuǎn)錄活性的抑制。用RNA干擾的方法下調(diào)內(nèi)源性的AMPK可以提高PR的轉(zhuǎn)錄活性。這些結(jié)果均表明,AMPK激活很可能抑制PR的轉(zhuǎn)錄活性。而進(jìn)一步的研究結(jié)果表明,AMPK的激活能夠引起PR磷酸化狀態(tài)的改變。AMPK的激活不影響PR的入核,但是阻礙了SRC3對(duì)PR的轉(zhuǎn)錄調(diào)節(jié)；AMPK對(duì)PR轉(zhuǎn)錄活性的調(diào)節(jié)不改變其對(duì)孕激素響應(yīng)的閾濃度,而是降低了孕激素能引起PR轉(zhuǎn)錄活性的最高水平。 綜上所述,通過(guò)酵母雙雜交技術(shù),我們發(fā)現(xiàn)很多AMPK潛在的相互作用蛋白,它們涉及生命活動(dòng)的許多領(lǐng)域。隨后,我們以PR作為重點(diǎn)進(jìn)行了下一步研究。在細(xì)胞水平上,驗(yàn)證了PR與AMPK的相互作用。我們首次發(fā)現(xiàn),AMPK的激活可能通過(guò)改變PR的磷酸化狀態(tài)而抑制PR的轉(zhuǎn)錄活性。以上研究工作不僅擴(kuò)充了我們對(duì)AMPK信號(hào)通路的認(rèn)識(shí),也為進(jìn)一步探討能量代謝與孕激素信號(hào)通路間的關(guān)系提供了重要的線索。
[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.

【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R346

【共引文獻(xiàn)】

相關(guān)期刊論文 前3條

1 胡淑國(guó);宋光耀;;腺苷酸活化蛋白激酶與胰島素抵抗[J];國(guó)際內(nèi)科學(xué)雜志;2009年03期

2 周鴻雁;;AMPK與Ⅱ型糖尿病的研究進(jìn)展[J];貴州體育科技;2014年03期

3 常洪霞;張紅學(xué);;AMPK與胰島素抵抗[J];哈爾濱師范大學(xué)自然科學(xué)學(xué)報(bào);2012年04期

相關(guān)博士學(xué)位論文 前1條

1 莊靜靜;AMPK及LXR通路新型天然調(diào)節(jié)劑的發(fā)現(xiàn)及相應(yīng)作用機(jī)制研究[D];華東理工大學(xué);2013年

相關(guān)碩士學(xué)位論文 前4條

1 龔豪杰;運(yùn)動(dòng)對(duì)AMPK不同基因型小鼠骨骼肌MEF2-GLUT4的影響[D];北京體育大學(xué);2011年

2 景志強(qiáng);運(yùn)動(dòng)對(duì)鏈脲佐菌素致糖尿病大鼠脂質(zhì)代謝的影響[D];揚(yáng)州大學(xué);2006年

3 宋帥召;高糖血脂喂養(yǎng)妊娠大鼠脂聯(lián)素和AMPK基因表達(dá)與胰島素抵抗關(guān)系研究[D];鄭州大學(xué);2012年

4 王寶明;運(yùn)動(dòng)與二甲雙胍聯(lián)合干預(yù)對(duì)2型糖尿病大鼠骨骼肌LKB1-AMPK-GLUT4信號(hào)通路的影響[D];沈陽(yáng)體育學(xué)院;2013年

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本文編號(hào)：1359995