本文選題：Mipu1 + 鋅指蛋白　； 參考：《中南大學(xué)》2007年博士論文

【摘要】： 鋅指是一種能與DNA結(jié)合的結(jié)構(gòu)域，為鋅指蛋白中的半胱氨酸和／或組氨酸與二價(jià)的鋅離子結(jié)合形成的一種特定的二級(jí)結(jié)構(gòu)。鋅指蛋白可以通過(guò)與雙鏈DNA、單鏈DNA及RNA的結(jié)合發(fā)揮轉(zhuǎn)錄調(diào)節(jié)及RNA剪接等功能。根據(jù)與鋅離子結(jié)合的氨基酸的不同，鋅指蛋白可以分為許多亞家族，其中C_2H_2(或Kruppel)型鋅指蛋白是其最大的亞家族，它的鋅指序列的特征為CX_2CX_3FX_5LX_2HX_3H；兩個(gè)鋅指之間的保守序列為T(mén)GEKP(Y／F)X(X代表在保守氨基酸之間的任意氨基酸)。而C_2H_2型鋅指蛋白又可以根據(jù)其N端含有的結(jié)構(gòu)域分成4大類：FAX(finger-associated boxes)型，F(xiàn)AR(finger-associatedrepeats)型，POZ(pox virus and zinc fingers，also known as Zin)和KRAB(Kruppel-associated box)型。 含KRAB結(jié)構(gòu)域的鋅指蛋白也稱KRAB型鋅指蛋白(KRAB-containingzinc finger protein，KZNF)，占人類基因組中所有鋅指蛋白(799種)的約三分之一(290種)，是哺乳動(dòng)物中最大的轉(zhuǎn)錄調(diào)控因子家族，其中超過(guò)220種在胚胎發(fā)育、細(xì)胞分化、細(xì)胞轉(zhuǎn)化及細(xì)胞周期的調(diào)控中發(fā)揮重要功能，其表達(dá)水平隨時(shí)間和空間的不同而變化。 Mipu1是本室從經(jīng)歷短暫缺血-再灌注的大鼠心肌中分離克隆的新基因，Genbank登錄號(hào)為AY221750。其ORF的全長(zhǎng)為1827bp，編碼608個(gè)氨基酸。其編碼肽鏈的N-端含一KRAB結(jié)構(gòu)域，C-端含14個(gè)C_2H_2型鋅指，故為一典型的KRAB／C_2H_2型鋅指蛋白。本室近年研究發(fā)現(xiàn)，Mipu1基因在缺血-再灌注心肌中表達(dá)明顯升高；Mipu1過(guò)表達(dá)可減輕去血清對(duì)C_2C_(12)細(xì)胞的生長(zhǎng)抑制作用，在去血清應(yīng)激狀態(tài)下具有促細(xì)胞生長(zhǎng)及修復(fù)損傷的功能；Mipu1過(guò)表達(dá)可明顯抑制氧化應(yīng)激(H_2O_2處理)導(dǎo)致的LDH釋放率和細(xì)胞凋亡發(fā)生率，具有細(xì)胞保護(hù)功能。本文擬進(jìn)一步闡明Mipu1的功能及結(jié)構(gòu)-功能關(guān)系。 為了探討新基因Mipu1的DNA結(jié)合活性，我們首先構(gòu)建了Mipu1的多個(gè)原核和真核表達(dá)質(zhì)粒。利用原核表達(dá)質(zhì)粒pGEX-Mipu1，誘導(dǎo)表達(dá)并純化了Mipu1的融合蛋白GST-Mipu1。將GST-Mipul固定在Glutathione-Sepharose上后，從隨機(jī)寡核苷酸文庫(kù)中篩選到了一個(gè)Mipu1共同的DNA結(jié)合序列5’-TGTCTTATCGAA-3’。經(jīng)化學(xué)合成包含這一序列的探針，末端同位素標(biāo)記后進(jìn)行EMSA發(fā)現(xiàn)，GST-Mipu1能與探針結(jié)合，并呈劑量依賴性，，但純化的GST蛋白不能與探針結(jié)合；GST-Mipu1與探針的結(jié)合信號(hào)能被非標(biāo)記探針(冷探針)競(jìng)爭(zhēng)，但不能被含突變核心序列(CTTA)的冷探針競(jìng)爭(zhēng)。在EMSA的結(jié)合緩沖液中加入EDTA，則融合蛋白與探針的結(jié)合作用被廢除。這些結(jié)果說(shuō)明，5’-TGTCTTATCGAA-3’是Mipu1的特異性DNA結(jié)合位點(diǎn)(Mipul DNAbinding site，MDBS)，其結(jié)合作用具有鋅離子依賴性，其中CTTA為結(jié)合位點(diǎn)的核心序列。 為了進(jìn)一步揭示Mipul與DNA的結(jié)合作用，我們表達(dá)與純化了不包含KRAB結(jié)構(gòu)域及KRAB與鋅指之間的連接序列，僅包含14個(gè)鋅指的融合蛋白GST-ZF�；瘜W(xué)合成包含Mipul的特異性結(jié)合位點(diǎn)的探針，并用生物素進(jìn)行末端標(biāo)記。Target detection asssay顯示，探針能與融合蛋白GST-Mipul和GST-ZF結(jié)合，但不能與GST結(jié)合，并且上述結(jié)合作用具有鋅離子依賴性。 為探討Mipul中鋅指結(jié)構(gòu)域的功能，我們將Mipul的14個(gè)鋅指分成二大段，表達(dá)與純化了只包含部分鋅指的融合蛋白，即GST-ZFl(包含1-8個(gè)鋅指)和GST-ZF2(包含9-14個(gè)鋅指)。Target detection asssay顯示，只有其中的GST-ZF2能與探針結(jié)合。 為了進(jìn)一步探討Mipul是否具有轉(zhuǎn)錄因子功能，將含三個(gè)MDBS的寡核苷酸序列插入報(bào)告基因載體pGL3-promoter構(gòu)建了重組報(bào)告基因載體pGL3-promoter-MDBS，并將三個(gè)MDBS核心序列突變構(gòu)建了突變的重組報(bào)告基因載體pGL3-promoter-MDBS~(mut)。將pGL3-promoter-MDBS和pGL3-promoter-MDBS~(mut)分別與Mipul的真核表達(dá)質(zhì)粒pcDNA3.1-Mipul共轉(zhuǎn)染小鼠巨噬細(xì)胞株Raw264.7或小鼠肌原細(xì)胞株C_2C_(12)，經(jīng)熒光素酶測(cè)定發(fā)現(xiàn)，Mipul的過(guò)表達(dá)能抑制pGL3-promoter-MDBS的熒光素酶活性，并呈劑量依賴性，但不能抑制pGL3-promoter-MDBS_(mut)的熒光素酶活性，表明Mipul具有轉(zhuǎn)錄抑制功能。 將Mipul開(kāi)放閱讀框的全長(zhǎng)，或KRAB結(jié)構(gòu)域或鋅指(ZF)結(jié)構(gòu)域分別與綠色熒光蛋白(GFP)融合，構(gòu)建了pEGFP-Mipul、pEGFP-KRAB及pEGFP-ZF三個(gè)真核表達(dá)質(zhì)粒。將它們導(dǎo)入C_2C_(12)，12小時(shí)后將細(xì)胞置熒光顯微鏡下觀察。結(jié)果顯示，Mipul的全長(zhǎng)和KRAB定位于核，而空載體和ZF在細(xì)胞中呈散在分布。 本文進(jìn)一步對(duì)Mipul調(diào)控的靶基因進(jìn)行了初步分析。根據(jù)Mipul的DNA結(jié)合位點(diǎn)，經(jīng)生物信息學(xué)分析發(fā)現(xiàn)，多個(gè)促凋亡基因啟動(dòng)子區(qū)含有該結(jié)合位點(diǎn)的核心序列。將其中Bax基因啟動(dòng)子的全長(zhǎng)標(biāo)上生物素后，Target detection assay顯示，生物素標(biāo)記的Bax的全長(zhǎng)啟動(dòng)子能與GST-ZF及GST-ZF2相互作用，表明Bax是Mipul的潛在靶基因之一。 綜上所述，本研究得到如下結(jié)論：①M(fèi)ipul是一個(gè)DNA位點(diǎn)特異性的核酸結(jié)合蛋白，其特異性的結(jié)合位點(diǎn)為5’-TGTCTTATCGAA-3’，核心序列為CTTA，C-末端的六個(gè)鋅指為其與DNA結(jié)合所足夠與必需；②Mipul是一個(gè)核蛋白，其核定位信號(hào)位于KRAB結(jié)構(gòu)域或KRAB與鋅指之間的連接序列中；③Mipul是一個(gè)轉(zhuǎn)錄抑制因子，可能通過(guò)抑制促凋亡基因的表達(dá)而抑制細(xì)胞凋亡，在細(xì)胞凋亡的發(fā)生過(guò)程中發(fā)揮調(diào)控作用。
[Abstract]:Zinc finger is a domain of DNA binding to DNA, a specific two grade structure formed by the binding of cysteine and / or histidine to two valent zinc ions in the zinc finger protein. The zinc finger protein can function as a transcriptional regulation and RNA splicing with the binding of two stranded DNA, single strand DNA and RNA. The zinc finger protein can be divided into many subfamilies, in which the C_2H_2 (or Kruppel) zinc finger protein is the largest subfamily, and its zinc finger sequence is characterized by CX_2CX_3FX_5LX_2HX_3H; the conservative sequence between the two zinc fingers is TGEKP (Y / F) X (X represents any amino acid between the conservative amino acids). According to the domain of its N end, the domain is divided into 4 major categories: FAX (finger-associated boxes), FAR (finger-associatedrepeats), POZ (pox virus and zinc fingers).
The zinc finger protein of the KRAB domain, also known as the KRAB zinc finger protein (KRAB-containingzinc finger protein, KZNF), accounts for about 1/3 (290 species) of all zinc finger proteins (799 species) in the human genome. It is the largest family of transcriptional regulators in mammals, of which over 220 are in embryo development, cell differentiation, cell transformation and cell cycle. It plays an important role in regulation and control, and its expression level varies with time and space.
Mipu1 is a new gene isolated from rat myocardium undergoing transient ischemia-reperfusion. The full length of ORF is 1827bp and 608 amino acids are encoded by Genbank. The N- terminal of the peptide chain contains a KRAB domain, and the C- end contains 14 C_2H_2 type zinc fingers. Therefore, it is a typical KRAB / C_2H_2 type zinc finger protein. It was found that the expression of Mipu1 gene was significantly higher in the ischemic reperfusion myocardium, and the overexpression of Mipu1 could reduce the inhibitory effect of the serum on the growth of C_2C_ (12) cells, the function of promoting cell growth and repairing the damage in the stress state of the serum, and the overexpression of Mipu1 could obviously inhibit the LDH release rate and finer induced by oxidative stress (H_2O_2 treatment). The incidence of apoptosis has the function of cell protection. This paper intends to further elucidate the function and structure function relationship of Mipu1.
In order to explore the DNA binding activity of the new gene Mipu1, we first constructed multiple prokaryotic and eukaryotic expression plasmids of Mipu1. Using the prokaryotic expression plasmid pGEX-Mipu1, we induced and purified the fusion protein GST-Mipu1. of Mipu1 to immobilization of GST-Mipul on Glutathione-Sepharose, and screened a Mipu1 from the random oligonucleotide library. The common DNA binding sequence 5 '-TGTCTTATCGAA-3. It is chemically synthesized with the probe of this sequence, EMSA found after the end isotope labeling, that GST-Mipu1 can be combined with the probe and is dose-dependent, but the purified GST protein can not be combined with the probe; the binding signal of the GST-Mipu1 and the probe can be competitive with the unlabeled probe (cold probe). But it is not competitive with the cold probe containing the mutant core sequence (CTTA). The binding of the fusion protein with the probe is abolished in the EMSA binding buffer. These results indicate that 5 '-TGTCTTATCGAA-3' is a specific DNA binding site of Mipu1 (Mipul DNAbinding site, MDBS), and its binding has a zinc ion dependence, which is CT. TA is the core sequence of the binding site.
In order to further reveal the combination of Mipul and DNA, we express and purify the connection sequences that do not contain the KRAB domain and the KRAB and the zinc finger. Only 14 zinc finger fusion proteins, GST-ZF., are chemically synthesized by the probe of the specific binding site of Mipul, and the detection of.Target detection asssay display by biotin. The needle can bind to fusion protein GST-Mipul and GST-ZF, but can not bind to GST, and the binding effect is zinc dependent.
In order to explore the function of zinc finger domain in Mipul, we divide 14 zinc fingers of Mipul into two large segments, and express and purify a fusion protein containing only part of the zinc finger, that is, GST-ZFl (including 1-8 zinc fingers) and GST-ZF2 (including 9-14 zinc fingers).Target detection asssay, only GST-ZF2 of which can be combined with the probe.
In order to further explore whether Mipul has the function of transcription factor, the recombinant reporter vector pGL3-promoter-MDBS was constructed by inserting three MDBS oligonucleotide sequences into the reporter gene carrier pGL3-promoter, and three MDBS core sequence mutations were constructed for the mutant recombinant report based pGL3-promoter-MDBS~ (MUT). PGL3-promote R-MDBS and pGL3-promoter-MDBS~ (MUT) Co transfected mouse macrophage strain Raw264.7 or murine myogenic cell line C_2C_ (12) with eukaryotic expression plasmid pcDNA3.1-Mipul of Mipul, respectively. Through luciferase determination, the overexpression of Mipul can inhibit the luciferase activity of pGL3-promoter-MDBS, and it is dose-dependent, but it can not inhibit pGL3-promoter-. The luciferase activity of MDBS_ (MUT) indicates that Mipul has the function of transcriptional inhibition.
The full length of the Mipul open reading frame, or the KRAB domain or the zinc finger (ZF) domain and the green fluorescent protein (GFP) domain, respectively, were fused with the green fluorescent protein (GFP). The three eukaryotic expression plasmids of pEGFP-Mipul, pEGFP-KRAB and pEGFP-ZF were constructed. They were introduced into C_2C_ (12), and the cells were observed under fluorescent microscopes for 12 hours. The results showed that the length of Mipul and KRAB were located in the nucleus. The space carrier and ZF were scattered in the cells.
In this paper, the target gene of Mipul regulation is further analyzed. According to the DNA binding site of Mipul, it is found that the core sequences of the binding sites are contained in the promoter region of multiple apoptotic genes. After the full length of the Bax promoter is labeled with biotin, Target detection assay shows the Bax of the biotin. The full-length promoter can interact with GST-ZF and GST-ZF2, indicating that Bax is one of the potential target genes of Mipul.
To sum up, the following conclusions are obtained: (1) Mipul is a specific nucleic acid binding protein of DNA site, whose specific binding site is 5 '-TGTCTTATCGAA-3, the core sequence is CTTA, and the six zinc fingers at the end of C- are sufficient and necessary for their binding with DNA; and Mipul is a nuclear protein, and its nuclear location signal is located in the KRAB domain. Or the connection between KRAB and zinc finger; (3) Mipul is a transcriptional inhibitor, which may inhibit apoptosis by inhibiting the expression of apoptotic genes and play a regulatory role in the process of apoptosis.

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R363

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 王智;谷良標(biāo);劉鴻慧;王丹;涂自智;王慷慨;蔣碧梅;肖獻(xiàn)忠;;Mipu1在人腦星形細(xì)胞瘤中的表達(dá)及臨床意義初步探討[J];中華神經(jīng)外科疾病研究雜志;2013年01期

本文編號(hào)：1845430