Spindlin 1定位與功能的關(guān)鍵位點分析及鑒定
發(fā)布時間：2018-07-25 09:22
【摘要】： 卵母細胞的發(fā)育與成熟一直是生命研究的重點,對此過程中相關(guān)蛋白功能的研究將有助于深入了解生命起始本質(zhì)。自上世紀70年代以來,越來越多的研究關(guān)注精子、卵母細胞以及受精卵早期發(fā)育相關(guān)蛋白。在這其中,一個30kD的蛋白在多個研究小組的報道中出現(xiàn),1997年Bermseok小組在對小鼠卵母細胞發(fā)育過程進行研究時將其分離并命名為spindlin.研究表明,小鼠Spindlin蛋白表達具有時空特異性,在減數(shù)分裂過程中與紡錘體密切相關(guān),表達隨著卵母細胞成熟過程逐漸增高;在2細胞時期開始降低,隨著受精卵發(fā)育其表達逐漸消失;鼠Spindlin蛋白可作為MOS/MAPK的作用底物在信號通路中被磷酸化修飾后發(fā)揮作用。之后文獻陸續(xù)報道Spindlin在鳥類、魚類等生殖細胞中表達,與精子發(fā)生期特異轉(zhuǎn)錄子Ssty (Y-linked spermiogenesis specific transcript)共同組成一個進化高度保守的與生殖細胞發(fā)育密切相關(guān)的Spin/Ssty家族。 我們研究小組最早從人卵巢癌細胞中克隆獲得了與鼠spindlin基因高度同源的基因并命名為spindlin1。前期研究結(jié)果表明,Spindlin1定位于細胞核,為參與細胞周期調(diào)控的核蛋白,外源性spindlin1的表達可以引起細胞絲裂災(zāi)變以及染色體不穩(wěn)定,使NIH3T3細胞發(fā)生惡性轉(zhuǎn)化;進一步的分析表明spindlin1可能通過對β-catenin/ T cell factor (TCF-4)通路發(fā)揮其對細胞周期的調(diào)控作用。為更深入了解spindlin1功能和作用機制,我們原核表達spindlin1蛋白,純化后制備晶體,并對其晶體結(jié)構(gòu)進行分析,結(jié)果表明spindlin1蛋白具有三個序列上非常類似的Spin/Ssty重復(fù)結(jié)構(gòu)域,每個結(jié)構(gòu)域都由4～5個反向平行的β-折疊構(gòu)成。那么,在Spindlin1結(jié)構(gòu)分析的基礎(chǔ)上通過構(gòu)建、突變與探討蛋白質(zhì)之間的相互作用等方法確認影響蛋白定位與功能的關(guān)鍵氨基酸位點無疑是進一步研究的重要內(nèi)容。 一.spindlin 1突變體質(zhì)粒的構(gòu)建 我室從人類卵巢癌樣本中分離克隆獲得了該家族成員——人類spindlin基因,命名為spindlin1。該基因cDNA全長4375bp,含714bp的開放讀框,編碼237個氨基酸的蛋白。在我們前期的研究中,我們對其結(jié)構(gòu)生物學(xué)進行探討,結(jié)果表明Spindlin1蛋白結(jié)構(gòu)分為三個序列上非常類似的Spin/Ssty重復(fù)結(jié)構(gòu)域。為深入闡明spindlin1的生物功能,了解其作用的分子機制,我們在生物信息學(xué)和蛋白晶體結(jié)構(gòu)解析的基礎(chǔ)上,構(gòu)建了一系列突變體,將spindlin1蛋白中14、84、99位絲氨酸突變成丙氨酸,得到Ser14M,Ser84M,Ser99M,Ser14+84M,Ser14+99M,Ser84+99M,Ser14+84+99M等七種突變質(zhì)粒。為下一步的研究定位和功能關(guān)鍵位點的確定打下基礎(chǔ)。 二.spindlin 1突變對其定位和功能的影響 在獲得野生型和突變表達載體后,我們首先以蛋白亞細胞定位作為其功能變化的直觀標(biāo)示,期望能從中找到在Spindlin1生物功能發(fā)揮中具有重要作用的位點。我們將野生型及七種突變質(zhì)粒轉(zhuǎn)染Hela細胞,觀察Spindlin1-EGFP融合蛋白亞細胞定位。結(jié)果表明:野生型的Spindlin1定位于細胞核,再我們構(gòu)建的七個不同突變體中,Ser14+Ser84,Ser84+Ser99,Ser14+Ser84+Ser99的聯(lián)合突變均能使野生型融合蛋白在細胞核內(nèi)集中分布的特性消失,成為全細胞彌散分布,而Ser14,Ser84,Ser99各位點的單獨突變及Ser14+Ser99的聯(lián)合突變均對Spindlin1蛋白的細胞核內(nèi)分布沒有影響;提示Ser84在Spindlin1蛋白的定位中發(fā)揮重要作用。 我們之前的實驗初步提示Spindlin1蛋白可能通過與轉(zhuǎn)錄因子TCF-4結(jié)合而發(fā)揮作用,在本實驗中我們利用GST-pulldown的方法證實Spindlin1和TCF-4之間在體外具有相互作用,并且證實了外源性Spindlin1表達對TCF-4熒光素報告基因的轉(zhuǎn)錄活性有激活作用.為了進一步驗證Ser84在Spindlin1功能發(fā)揮中的作用。我們將pEGFP-spindlin1野生型表達載體或spindin1的突變載體分別與TCF-4熒光素酶報告基因載體pTOPFlash和作為轉(zhuǎn)染對照的β-galactosidase載體共轉(zhuǎn)染Hela細胞,熒光素酶活性分析顯示與對照載體pEGFP-C1質(zhì)粒相比,pEGFP-spindlin1與TCF-4熒光素酶報告基因載體pTOPFlash載體共轉(zhuǎn)染后,TCF-4熒光素酶報告基因的轉(zhuǎn)錄活性明顯上升,而Ser14+Ser84,Ser84+Ser99,Ser14+Ser84+Ser99的聯(lián)合突變使Spindlin1對其活性的激活作用消失或降低。進一步證實了Ser84位點在Spindlin1功能發(fā)揮中的重要作用。 以上結(jié)果提示我們,Ser84是Spindlin1細胞核內(nèi)定位和功能發(fā)揮的關(guān)鍵位點,但其作用的發(fā)揮需要Ser14,Ser99的協(xié)助。Spindlin1的功能發(fā)揮可能與磷酸化、核定位以及與TCF-4之間的結(jié)合密切相關(guān)。更為有意義的是,這三個絲氨酸位點所構(gòu)成的結(jié)構(gòu)域正是Aurora A激酶特異的作用位點,Aurora A激酶在腫瘤發(fā)生和細胞周期調(diào)控中所發(fā)揮的重要作用提示我們,Spindlin1可能作為Aurora A激酶的作用底物發(fā)揮作用。 三.Spindlin1與Aurora A激酶相互作用的初步研究 人類Aurora A定位于染色體20q13.2,由403個氨基酸組成。Aurora A不僅與紡錘體裝配、胞質(zhì)分裂、中心體的成熟和分離、紡錘體檢查點等過程相關(guān)聯(lián),最近越來越多的證據(jù)表明Aurora A表現(xiàn)出成癌基因的特性。Aurora A與其眾多底物相互作用,參與不同的信號通路,這些途徑相互關(guān)聯(lián)形成網(wǎng)絡(luò)。我們的研究基礎(chǔ)提示Spindlin1與Aurora A具有很多功能和特征上的關(guān)聯(lián)。在本部分實驗中我們構(gòu)建的pGEX-T-spindlin1原核表達載體,用IPTG誘導(dǎo)GST- spindlin1融合蛋白表達后,利用GST-pulldown技術(shù)的初步的研究結(jié)果表明spindlin1與絲氨酸激酶Aurora A的在體外具有相互作用,有可能作為Aurora A的底物發(fā)揮功能。
[Abstract]:The development and maturation of oocytes have been the focus of life research. In this process, the study of related protein functions will help to understand the essence of life. Since 70s, more and more studies have focused on spermatozoa, oocyte and the early development of spermatozoa of fertilized eggs. In this, the protein of one 30kD is more In the report of the research group, the Bermseok group, in 1997, separated the mouse oocyte development process and named it spindlin. research. It showed that the expression of Spindlin protein in mice was spatio-temporal, and it was closely related to the spindle during meiosis, and the expression increased gradually with the maturation of oocyte. When the 2 cell period began to decrease, the expression gradually disappeared with the development of the fertilized egg, and the mouse Spindlin protein could be used as a substrate for MOS/MAPK to be phosphorylated in the signal pathway. Later, the literature reported that Spindlin was expressed in the germ cells of birds, fish and other germ cells, and Ssty (Y-linked sperm), a specific transcriptional transcript of spermatogenesis. IOGENESIS specific transcript together form a highly conserved Spin/Ssty family closely related to germ cell development.
Our team first cloned from human ovarian cancer cells to obtain a highly homologous gene from the mouse spindlin gene and named it as a preliminary study of spindlin1.. The results showed that Spindlin1 was located in the nucleus and involved in the nuclear proteins involved in the regulation of cell cycle. The expression of exogenous spindlin1 could cause the calamity of cell filaments and the instability of chromosomes. Further analysis shows that spindlin1 may play a role in regulating the cell cycle through the beta -catenin/ T cell factor (TCF-4) pathway. In order to further understand the function and mechanism of spindlin1, we prokaryotic spindlin1 egg white, purified the crystal after purification, and carry out its crystal structure. The results show that spindlin1 protein has a very similar Spin/Ssty duplication domain on three sequences, each domain is composed of 4~5 reverse parallel beta folds. Then, on the basis of the structure analysis of Spindlin1, the protein localization and function can be confirmed by the method of construction, mutation and phase interaction between proteins. The key amino acid locus is undoubtedly an important part of further research.
Construction of a.Spindlin 1 mutant plasmid
We isolated and cloned from human ovarian cancer samples to obtain the family member, human spindlin gene, named spindlin1. cDNA full length 4375bp, 714bp open reading frame, and encoding 237 amino acid proteins. In our previous study, we explored its structural biology and showed the structure of Spindlin1 protein. In order to further clarify the biological function of spindlin1 and understand the molecular mechanism of its role, a series of mutants were constructed on the basis of bioinformatics and protein crystal structure analysis, and a series of mutants were constructed on the basis of bioinformatics and protein crystal structure analysis. The 14,84,99 bit serine in the spindlin1 protein was mutated into alanine, and Ser14M, Se was obtained, in order to further clarify the biological function of the Spin/Ssty. R84M, Ser99M, Ser14+84M, Ser14+99M, Ser84+99M, Ser14+84+99M and other seven mutant plasmids, which lay the foundation for further research and location of functional key loci.
The effect of two.Spindlin 1 mutation on its location and function
After obtaining the wild type and the mutant expression vector, we first use the protein subcellular location as a visual indication of its functional changes. We expect to find the site that plays an important role in the Spindlin1 function. We transfect the wild type and seven mutant plasmids to the Hela cell, and observe the Spindlin1-EGFP fusion protein subcell The results showed that the wild type Spindlin1 was located in the nucleus, and the joint mutation of Ser14+Ser84, Ser84+Ser99 and Ser14+Ser84+Ser99 in the seven different mutants we constructed could dissolve the concentration distribution of the wild type fusion protein in the nucleus, and become the dispersed distribution of the whole cell, and the individual process of Ser14, Ser84 and Ser99 points. The combined mutation of Ser14+Ser99 and Spindlin1 had no effect on the nuclear distribution of Ser84 protein, suggesting that Ser84 plays an important role in the localization of Spindlin1 protein.
Our previous experiments preliminarily suggested that Spindlin1 protein may play a role in binding with the transcription factor TCF-4. In this experiment, we used GST-pulldown method to confirm the interaction between Spindlin1 and TCF-4 in vitro, and confirmed the activation of the exogenous Spindlin1 expression to the transcriptional activity of the TCF-4 luciferase reporter gene. In order to further verify the role of Ser84 in Spindlin1 function, we have co transfected Hela cells with the TCF-4 luciferase reporter vector pTOPFlash and the beta -galactosidase vector as the transfection control, respectively, with the pEGFP-spindlin1 wild type expression vector or the mutation vector of spindin1, and the luciferase activity analysis showed and compared with the control. After CO transfection of pEGFP-spindlin1 and TCF-4 luciferase reporter vector pTOPFlash vector, the transcriptional activity of TCF-4 luciferase reporter gene was significantly increased, while the joint mutation of Ser14+Ser84, Ser84+Ser99, Ser14+Ser84+Ser99 caused Spindlin1 to activate the activation of TCF-4, and further confirmed the Se, and further confirmed the Se. R84 locus plays an important role in Spindlin1 function.
The above results suggest that Ser84 is the key site for the localization and function of Spindlin1 in the nucleus, but its role is Ser14. Ser99's assistance in.Spindlin1's function may be closely related to phosphorylation, nuclear localization and the binding to TCF-4. More intentionally, the structure of these three serine sites Domain is a specific site of action of Aurora A kinase, and the important role of Aurora A kinase in tumorigenesis and cell cycle regulation suggests that Spindlin1 may play a role as a substrate for Aurora A kinase.
Preliminary study on the interaction between three.Spindlin1 and Aurora A kinase
Human Aurora A is located at chromosome 20q13.2, and the.Aurora A consisting of 403 amino acids is not only associated with the spindle assembly, cytokinesis, the maturation and separation of the centrosome and the spindle checkpoint, but more and more evidence suggests that Aurora A has shown that the specific.Aurora A of the oncogene is interacted with many substrates and participates in the difference. Our research basis suggests that Spindlin1 and Aurora A have many functional and characteristic associations. In this part of the experiment, we constructed the pGEX-T-spindlin1 prokaryotic expression vector, using IPTG to induce the expression of GST- spindlin1 fusion protein and using the preliminary research of GST-pulldown technology. The results showed that spindlin1 had interaction with serine kinase Aurora A in vitro and might act as a substrate for Aurora A.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R341;R737.31
本文編號：2143365
[Abstract]:The development and maturation of oocytes have been the focus of life research. In this process, the study of related protein functions will help to understand the essence of life. Since 70s, more and more studies have focused on spermatozoa, oocyte and the early development of spermatozoa of fertilized eggs. In this, the protein of one 30kD is more In the report of the research group, the Bermseok group, in 1997, separated the mouse oocyte development process and named it spindlin. research. It showed that the expression of Spindlin protein in mice was spatio-temporal, and it was closely related to the spindle during meiosis, and the expression increased gradually with the maturation of oocyte. When the 2 cell period began to decrease, the expression gradually disappeared with the development of the fertilized egg, and the mouse Spindlin protein could be used as a substrate for MOS/MAPK to be phosphorylated in the signal pathway. Later, the literature reported that Spindlin was expressed in the germ cells of birds, fish and other germ cells, and Ssty (Y-linked sperm), a specific transcriptional transcript of spermatogenesis. IOGENESIS specific transcript together form a highly conserved Spin/Ssty family closely related to germ cell development.
Our team first cloned from human ovarian cancer cells to obtain a highly homologous gene from the mouse spindlin gene and named it as a preliminary study of spindlin1.. The results showed that Spindlin1 was located in the nucleus and involved in the nuclear proteins involved in the regulation of cell cycle. The expression of exogenous spindlin1 could cause the calamity of cell filaments and the instability of chromosomes. Further analysis shows that spindlin1 may play a role in regulating the cell cycle through the beta -catenin/ T cell factor (TCF-4) pathway. In order to further understand the function and mechanism of spindlin1, we prokaryotic spindlin1 egg white, purified the crystal after purification, and carry out its crystal structure. The results show that spindlin1 protein has a very similar Spin/Ssty duplication domain on three sequences, each domain is composed of 4~5 reverse parallel beta folds. Then, on the basis of the structure analysis of Spindlin1, the protein localization and function can be confirmed by the method of construction, mutation and phase interaction between proteins. The key amino acid locus is undoubtedly an important part of further research.
Construction of a.Spindlin 1 mutant plasmid
We isolated and cloned from human ovarian cancer samples to obtain the family member, human spindlin gene, named spindlin1. cDNA full length 4375bp, 714bp open reading frame, and encoding 237 amino acid proteins. In our previous study, we explored its structural biology and showed the structure of Spindlin1 protein. In order to further clarify the biological function of spindlin1 and understand the molecular mechanism of its role, a series of mutants were constructed on the basis of bioinformatics and protein crystal structure analysis, and a series of mutants were constructed on the basis of bioinformatics and protein crystal structure analysis. The 14,84,99 bit serine in the spindlin1 protein was mutated into alanine, and Ser14M, Se was obtained, in order to further clarify the biological function of the Spin/Ssty. R84M, Ser99M, Ser14+84M, Ser14+99M, Ser84+99M, Ser14+84+99M and other seven mutant plasmids, which lay the foundation for further research and location of functional key loci.
The effect of two.Spindlin 1 mutation on its location and function
After obtaining the wild type and the mutant expression vector, we first use the protein subcellular location as a visual indication of its functional changes. We expect to find the site that plays an important role in the Spindlin1 function. We transfect the wild type and seven mutant plasmids to the Hela cell, and observe the Spindlin1-EGFP fusion protein subcell The results showed that the wild type Spindlin1 was located in the nucleus, and the joint mutation of Ser14+Ser84, Ser84+Ser99 and Ser14+Ser84+Ser99 in the seven different mutants we constructed could dissolve the concentration distribution of the wild type fusion protein in the nucleus, and become the dispersed distribution of the whole cell, and the individual process of Ser14, Ser84 and Ser99 points. The combined mutation of Ser14+Ser99 and Spindlin1 had no effect on the nuclear distribution of Ser84 protein, suggesting that Ser84 plays an important role in the localization of Spindlin1 protein.
Our previous experiments preliminarily suggested that Spindlin1 protein may play a role in binding with the transcription factor TCF-4. In this experiment, we used GST-pulldown method to confirm the interaction between Spindlin1 and TCF-4 in vitro, and confirmed the activation of the exogenous Spindlin1 expression to the transcriptional activity of the TCF-4 luciferase reporter gene. In order to further verify the role of Ser84 in Spindlin1 function, we have co transfected Hela cells with the TCF-4 luciferase reporter vector pTOPFlash and the beta -galactosidase vector as the transfection control, respectively, with the pEGFP-spindlin1 wild type expression vector or the mutation vector of spindin1, and the luciferase activity analysis showed and compared with the control. After CO transfection of pEGFP-spindlin1 and TCF-4 luciferase reporter vector pTOPFlash vector, the transcriptional activity of TCF-4 luciferase reporter gene was significantly increased, while the joint mutation of Ser14+Ser84, Ser84+Ser99, Ser14+Ser84+Ser99 caused Spindlin1 to activate the activation of TCF-4, and further confirmed the Se, and further confirmed the Se. R84 locus plays an important role in Spindlin1 function.
The above results suggest that Ser84 is the key site for the localization and function of Spindlin1 in the nucleus, but its role is Ser14. Ser99's assistance in.Spindlin1's function may be closely related to phosphorylation, nuclear localization and the binding to TCF-4. More intentionally, the structure of these three serine sites Domain is a specific site of action of Aurora A kinase, and the important role of Aurora A kinase in tumorigenesis and cell cycle regulation suggests that Spindlin1 may play a role as a substrate for Aurora A kinase.
Preliminary study on the interaction between three.Spindlin1 and Aurora A kinase
Human Aurora A is located at chromosome 20q13.2, and the.Aurora A consisting of 403 amino acids is not only associated with the spindle assembly, cytokinesis, the maturation and separation of the centrosome and the spindle checkpoint, but more and more evidence suggests that Aurora A has shown that the specific.Aurora A of the oncogene is interacted with many substrates and participates in the difference. Our research basis suggests that Spindlin1 and Aurora A have many functional and characteristic associations. In this part of the experiment, we constructed the pGEX-T-spindlin1 prokaryotic expression vector, using IPTG to induce the expression of GST- spindlin1 fusion protein and using the preliminary research of GST-pulldown technology. The results showed that spindlin1 had interaction with serine kinase Aurora A in vitro and might act as a substrate for Aurora A.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R341;R737.31
【參考文獻】
相關(guān)期刊論文 前1條
1 岳文,孫麗亞,李春海,張立新,裴雪濤;卵巢癌相關(guān)基因的篩選與鑒定[J];癌癥;2004年02期
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