本文選題：OREBP + CK1　； 參考：《重慶醫(yī)科大學(xué)》2008年博士論文

【摘要】： 滲透壓反應(yīng)增強(qiáng)子結(jié)合蛋白(Osmolarity Response Enhancer Binding Protein,OREBP或稱Tonicity Enhancer Binding Protein,TonEBP),又名:活化T淋巴細(xì)胞核因子5 (Nuclear Factor of Activated T Cell 5,NFAT5)是迄今為止唯一已知的哺乳動物細(xì)胞滲透壓反應(yīng)調(diào)節(jié)因子。當(dāng)細(xì)胞外微環(huán)境滲透壓升高時,OREBP作為滲透壓相關(guān)的轉(zhuǎn)錄因子,從細(xì)胞漿進(jìn)入細(xì)胞核,結(jié)合到一系列滲透壓相關(guān)靶基因上游的滲透壓反應(yīng)增強(qiáng)子區(qū)域(Osmolarity Response Enhancer,ORE),啟動它們的表達(dá),提高細(xì)胞內(nèi)相應(yīng)滲透性溶質(zhì)濃度,從而降低胞內(nèi)離子濃度,維持細(xì)胞正常生理功能。當(dāng)細(xì)胞外滲透壓降低,OREBP從胞核轉(zhuǎn)運(yùn)到胞漿,相關(guān)基因的轉(zhuǎn)錄水平隨之降低。生理情況下,腎臟髓質(zhì),軟骨及消化道上皮等組織器官所處環(huán)境的滲透壓波動極大,持續(xù)時間長,滲透壓反應(yīng)機(jī)制是維持這些組織器官正常生理功能的最重要防御機(jī)制。 近來,細(xì)胞核、漿之間的運(yùn)動已成為OREBP功能研究的熱點,其具體機(jī)制有一定的發(fā)現(xiàn),但是仍然不夠詳盡。本課題組于2006年發(fā)現(xiàn)了三個控制OREBP核漿轉(zhuǎn)運(yùn)的結(jié)構(gòu)域:NLS(Nuclear Localization Signal),NES(Nuclear Export Signal)和AED(Auxiliary Export Domain,AED),NLS負(fù)責(zé)OREBP高滲、等滲下入細(xì)胞核,NES通過與CRM1(Nuclear Export Receptor Exportin 1)作用負(fù)責(zé)其等滲、低滲下出細(xì)胞核,而AED(132-156aa)可在低滲條件下,獨(dú)自攜帶OREBP出細(xì)胞核,這一過程不依賴于NES,那么AED是通過什么機(jī)制介導(dǎo)OREBP出胞核的呢?最近的研究發(fā)現(xiàn):磷酸化修飾能影響蛋白質(zhì)的分子構(gòu)象及其與核漿轉(zhuǎn)運(yùn)蛋白的相互作用,從而影響該蛋白的核漿轉(zhuǎn)運(yùn)過程。低滲刺激是否通過磷酸化AED區(qū)域的某些氨基酸殘基,改變OREBP的構(gòu)象,促使其出細(xì)胞核?本課題圍繞這一問題展開了相關(guān)研究。 1.使用丙氨酸點突變篩查方法,將AED區(qū)域9個候選氨基酸逐個突變成惰性丙氨酸,觀察突變后FLAG-OREBP_(1-581)Δ1-131融合蛋白在低滲刺激下的亞細(xì)胞定位。我們發(fā)現(xiàn):野生型融合蛋白:胞核型細(xì)胞占9%,胞漿型細(xì)胞占79%;S155A突變具有明顯抑制低滲出核效果,胞核型細(xì)胞占66%,胞漿型細(xì)胞占16.7%,提示:S155位點在低滲出胞核過程中有重要作用。 2.低滲處理后,對FLAG-OREBP_(1-581)進(jìn)行質(zhì)譜檢測發(fā)現(xiàn),S155及S158兩個位點發(fā)生磷酸化修飾。將這兩個位點突變?yōu)楸彼峄蛘咛扉T冬氨酸的實驗發(fā)現(xiàn), FLAG-OREBP_(1-581)Δ1-131S155A、S158A及S155/158A突變?nèi)诤系鞍拙荒茉诘蜐B下出胞核,S155D突變?nèi)诤系鞍啄茼樌霭?而S158D及S155/158D兩個突變?nèi)诤系鞍椎蜐B出胞核受抑。同時我們構(gòu)建GFP-OREBP_(1-581)融合蛋白及各個突變體,使用激光共聚焦顯微鏡實時觀察低滲刺激下的GFP融合蛋白出胞核進(jìn)程,結(jié)果與細(xì)胞免疫化學(xué)分析一致。因此我們認(rèn)為:只有S155和S158按先后順序依次發(fā)生磷酸化修飾,OREBP才能在低滲刺激下順利出胞核。 3.使用GST-OREBP146-167融合蛋白及各個突變體作為底物,進(jìn)行體外激酶活性實驗,發(fā)現(xiàn)細(xì)胞核內(nèi)有未知激酶在低滲刺激下迅速激活,S158位點磷酸化以S155位點磷酸化為前提,在體內(nèi)使用FLAG-OREBP_(1-581)Δ1-131及各個突變體,通過凝膠電泳滯后實驗證實了體外激酶實驗的結(jié)果。另一方面,根據(jù)生物信息學(xué)分析提示信息,采用重組人CK1α1,證實當(dāng)S155磷酸化后,S158位點成為了CK1的作用靶點,CKI-7抑制低滲刺激胞核提取物內(nèi)CK1活性后,GST-OREBP146-167S155D的磷酸化修飾明顯受抑制;使用siRNA抑制體內(nèi)CK1各亞型表達(dá),發(fā)現(xiàn)CK1α1L在OREBP低滲刺激下的出胞核過程中發(fā)揮重要作用,同時使用CK1α1L shRNA進(jìn)一步證實實驗結(jié)果。通過以上實驗,我們獲得以下結(jié)論: 低滲刺激下胞核內(nèi)一個未知的激酶迅速活化,磷酸化OREBP AED區(qū)域的S155位點,緊接著CK1α1L磷酸化S158位點,此兩個位點發(fā)生磷酸化修飾后,OREBP才能從細(xì)胞核順利進(jìn)入細(xì)胞漿。
[Abstract]:The osmotic reaction enhancer binding protein (Osmolarity Response Enhancer Binding Protein, OREBP or Tonicity Enhancer Binding Protein, TonEBP), also known as the activated T lymphocyte nuclear factor 5, is the only known regulation factor for the osmotic pressure of mammalian cells so far. When the osmotic pressure of the extracellular microenvironment is high, OREBP is a transcription factor associated with osmotic pressure, from the cytoplasm into the nucleus and binding to a series of osmotic pressure response enhancers (Osmolarity Response Enhancer, ORE) upstream of the osmotic pressure related target genes, starting their expression, improving the intracellular concentration of the corresponding osmotic solute, and thus reducing the concentration of the corresponding osmotic solute in the cell The concentrations of intracellular ions maintain normal physiological functions of the cells. When the osmotic pressure decreases, the OREBP transshipment from the nucleus to the cytoplasm, the transcriptional level of the related genes decreases. Under physiological conditions, the osmotic pressure of the tissues and organs such as the medulla, cartilage and the digestive tract of the kidneys is greatly increased, the duration of the osmotic pressure is long, and the osmotic pressure mechanism is maintained. The most important defense mechanisms of these tissues and organs are normal physiological functions.
Recently, the movement between the nucleus and the plasma has become a hot spot in the research of OREBP function, and its specific mechanism has been found, but it is still not exhaustive. In 2006, we found three domains that control the OREBP nuclear plasma transport: NLS (Nuclear Localization Signal), NES (Nuclear Export Signal) and AED (Auxiliary). LS is responsible for OREBP hyperosmotic, isosmotic into the nucleus, NES is responsible for its isosmotic and hypotonic nucleus through the action of CRM1 (Nuclear Export Receptor Exportin 1), and AED (132-156aa) can carry the OREBP nucleus of the nucleus under the hypotonic condition, this process is not dependent on NES, then what mechanism has been used to mediate the nucleus? The study found that phosphorylation can affect the molecular conformation of protein and its interaction with the nucleoplasma transporter, which affects the nucleoplasma transport process of the protein. Whether or not the hypotonic stimulation changes the conformation of OREBP through some amino acid residues in the phosphorylated AED region, and promotes its nucleus out of the nucleus. Research.
1. using the alanine point mutation screening method, 9 candidate amino acids in the AED region were mutated to inert alanine one by one, and the subcellular localization of FLAG-OREBP_ (1-581) Delta 1-131 fusion protein was observed under the hypotonic stimulation. We found that the wild type fusion protein was 9%, the cytoplasm cells accounted for 79%, and the S155A mutation was obviously inhibited. The effect of low exudate nucleus was 66% and cytoplasmic cells accounted for 16.7%, suggesting that the S155 locus plays an important role in the process of low exudate nucleus.
After 2. hypotonic treatment, FLAG-OREBP_ (1-581) was detected by mass spectrometry, and two sites of S155 and S158 were phosphorylated. The two sites were mutated to alanine or aspartic acid, and the FLAG-OREBP_ (1-581) Delta 1-131S155A, S158A and S155/158A mutant fusion proteins were not low permeable nuclei and S155D mutation fusion. The protein can smooth out the nucleus, while the low exudation nuclei of the two fusion proteins of S158D and S155/158D are suppressed. We construct the GFP-OREBP_ (1-581) fusion protein and the various mutants. The process of the GFP fusion protein exocytosis under the hypotonic stimulation is observed by laser confocal microscopy. The result is consistent with the cell immunocytochemical analysis. We believe that only S155 and S158 can undergo phosphorylation in sequence, and OREBP can smooth out the nucleus under hypotonic stimulation.
3. using the GST-OREBP146-167 fusion protein and the various mutants as substrates, the activity of kinase activity in vitro was carried out. It was found that the unknown kinase in the nucleus was activated rapidly under the hypotonic stimulation. The phosphorylation of the S158 site was based on the phosphorylation of the S155 site, and the FLAG-OREBP_ (1-581) Delta 1-131 and the various mutants were used in the body, and the gel electrophoresis was delayed. On the other hand, on the other hand, according to the information of bioinformatics analysis, the recombinant human CK1 alpha 1 was used to confirm that when S155 phosphorylation, the S158 site became the target of CK1, and the phosphorylation of GST-OREBP146-167S155D was obviously inhibited after CKI-7 inhibited the CK1 activity in the nucleus extracts of the hypotonic stimulation; and siRNA was used. Inhibiting the expression of CK1 subtypes in vivo, it is found that CK1 alpha 1L plays an important role in the process of nucleus exocytosis under OREBP hypotonic stimulation. Meanwhile, CK1 alpha 1L shRNA is used to further confirm the results of the experiment.
An unknown kinase in the nucleus of the cell is activated rapidly under the hypotonic stimulation, and the S155 site of the phosphorylated OREBP AED region is followed by the CK1 alpha 1L phosphorylated S158 site. After phosphorylation of the two sites, the OREBP can enter the cytoplasm smoothly from the nucleus.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R341

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