心肌缺血预适应上调新基因Mipu1的结构与功能研究

发布时间：2018-05-05 00:39

本文选题：Mipu1 + 锌指蛋白　；参考：《中南大学》2007年博士论文

【摘要】： 锌指是一种能与DNA结合的结构域，为锌指蛋白中的半胱氨酸和／或组氨酸与二价的锌离子结合形成的一种特定的二级结构。锌指蛋白可以通过与双链DNA、单链DNA及RNA的结合发挥转录调节及RNA剪接等功能。根据与锌离子结合的氨基酸的不同，锌指蛋白可以分为许多亚家族，其中C_2H_2(或Kruppel)型锌指蛋白是其最大的亚家族，它的锌指序列的特征为CX_2CX_3FX_5LX_2HX_3H；两个锌指之间的保守序列为TGEKP(Y／F)X(X代表在保守氨基酸之间的任意氨基酸)。而C_2H_2型锌指蛋白又可以根据其N端含有的结构域分成4大类：FAX(finger-associated boxes)型，FAR(finger-associatedrepeats)型，POZ(pox virus and zinc fingers，also known as Zin)和KRAB(Kruppel-associated box)型。含KRAB结构域的锌指蛋白也称KRAB型锌指蛋白(KRAB-containingzinc finger protein，KZNF)，占人类基因组中所有锌指蛋白(799种)的约三分之一(290种)，是哺乳动物中最大的转录调控因子家族，其中超过220种在胚胎发育、细胞分化、细胞转化及细胞周期的调控中发挥重要功能，其表达水平随时间和空间的不同而变化。 Mipu1是本室从经历短暂缺血-再灌注的大鼠心肌中分离克隆的新基因，Genbank登录号为AY221750。其ORF的全长为1827bp，编码608个氨基酸。其编码肽链的N-端含一KRAB结构域，C-端含14个C_2H_2型锌指，故为一典型的KRAB／C_2H_2型锌指蛋白。本室近年研究发现，Mipu1基因在缺血-再灌注心肌中表达明显升高；Mipu1过表达可减轻去血清对C_2C_(12)细胞的生长抑制作用，在去血清应激状态下具有促细胞生长及修复损伤的功能；Mipu1过表达可明显抑制氧化应激(H_2O_2处理)导致的LDH释放率和细胞凋亡发生率，具有细胞保护功能。本文拟进一步阐明Mipu1的功能及结构-功能关系。为了探讨新基因Mipu1的DNA结合活性，我们首先构建了Mipu1的多个原核和真核表达质粒。利用原核表达质粒pGEX-Mipu1，诱导表达并纯化了Mipu1的融合蛋白GST-Mipu1。将GST-Mipul固定在Glutathione-Sepharose上后，从随机寡核苷酸文库中筛选到了一个Mipu1共同的DNA结合序列5’-TGTCTTATCGAA-3’。经化学合成包含这一序列的探针，末端同位素标记后进行EMSA发现，GST-Mipu1能与探针结合，并呈剂量依赖性，，但纯化的GST蛋白不能与探针结合；GST-Mipu1与探针的结合信号能被非标记探针(冷探针)竞争，但不能被含突变核心序列(CTTA)的冷探针竞争。在EMSA的结合缓冲液中加入EDTA，则融合蛋白与探针的结合作用被废除。这些结果说明，5’-TGTCTTATCGAA-3’是Mipu1的特异性DNA结合位点(Mipul DNAbinding site，MDBS)，其结合作用具有锌离子依赖性，其中CTTA为结合位点的核心序列。为了进一步揭示Mipul与DNA的结合作用，我们表达与纯化了不包含KRAB结构域及KRAB与锌指之间的连接序列，仅包含14个锌指的融合蛋白GST-ZF。化学合成包含Mipul的特异性结合位点的探针，并用生物素进行末端标记。Target detection asssay显示，探针能与融合蛋白GST-Mipul和GST-ZF结合，但不能与GST结合，并且上述结合作用具有锌离子依赖性。为探讨Mipul中锌指结构域的功能，我们将Mipul的14个锌指分成二大段，表达与纯化了只包含部分锌指的融合蛋白，即GST-ZFl(包含1-8个锌指)和GST-ZF2(包含9-14个锌指)。Target detection asssay显示，只有其中的GST-ZF2能与探针结合。为了进一步探讨Mipul是否具有转录因子功能，将含三个MDBS的寡核苷酸序列插入报告基因载体pGL3-promoter构建了重组报告基因载体pGL3-promoter-MDBS，并将三个MDBS核心序列突变构建了突变的重组报告基因载体pGL3-promoter-MDBS~(mut)。将pGL3-promoter-MDBS和pGL3-promoter-MDBS~(mut)分别与Mipul的真核表达质粒pcDNA3.1-Mipul共转染小鼠巨噬细胞株Raw264.7或小鼠肌原细胞株C_2C_(12)，经荧光素酶测定发现，Mipul的过表达能抑制pGL3-promoter-MDBS的荧光素酶活性，并呈剂量依赖性，但不能抑制pGL3-promoter-MDBS_(mut)的荧光素酶活性，表明Mipul具有转录抑制功能。将Mipul开放阅读框的全长，或KRAB结构域或锌指(ZF)结构域分别与绿色荧光蛋白(GFP)融合，构建了pEGFP-Mipul、pEGFP-KRAB及pEGFP-ZF三个真核表达质粒。将它们导入C_2C_(12)，12小时后将细胞置荧光显微镜下观察。结果显示，Mipul的全长和KRAB定位于核，而空载体和ZF在细胞中呈散在分布。本文进一步对Mipul调控的靶基因进行了初步分析。根据Mipul的DNA结合位点，经生物信息学分析发现，多个促凋亡基因启动子区含有该结合位点的核心序列。将其中Bax基因启动子的全长标上生物素后，Target detection assay显示，生物素标记的Bax的全长启动子能与GST-ZF及GST-ZF2相互作用，表明Bax是Mipul的潜在靶基因之一。综上所述，本研究得到如下结论：①Mipul是一个DNA位点特异性的核酸结合蛋白，其特异性的结合位点为5’-TGTCTTATCGAA-3’，核心序列为CTTA，C-末端的六个锌指为其与DNA结合所足够与必需；②Mipul是一个核蛋白，其核定位信号位于KRAB结构域或KRAB与锌指之间的连接序列中；③Mipul是一个转录抑制因子，可能通过抑制促凋亡基因的表达而抑制细胞凋亡，在细胞凋亡的发生过程中发挥调控作用。
[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.

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R363

【引证文献】