Spindlin 1定位与功能的关键位点分析及鉴定
发布时间：2018-07-25 09:22
【摘要】： 卵母细胞的发育与成熟一直是生命研究的重点,对此过程中相关蛋白功能的研究将有助于深入了解生命起始本质。自上世纪70年代以来,越来越多的研究关注精子、卵母细胞以及受精卵早期发育相关蛋白。在这其中,一个30kD的蛋白在多个研究小组的报道中出现,1997年Bermseok小组在对小鼠卵母细胞发育过程进行研究时将其分离并命名为spindlin.研究表明,小鼠Spindlin蛋白表达具有时空特异性,在减数分裂过程中与纺锤体密切相关,表达随着卵母细胞成熟过程逐渐增高;在2细胞时期开始降低,随着受精卵发育其表达逐渐消失;鼠Spindlin蛋白可作为MOS/MAPK的作用底物在信号通路中被磷酸化修饰后发挥作用。之后文献陆续报道Spindlin在鸟类、鱼类等生殖细胞中表达,与精子发生期特异转录子Ssty (Y-linked spermiogenesis specific transcript)共同组成一个进化高度保守的与生殖细胞发育密切相关的Spin/Ssty家族。 我们研究小组最早从人卵巢癌细胞中克隆获得了与鼠spindlin基因高度同源的基因并命名为spindlin1。前期研究结果表明,Spindlin1定位于细胞核,为参与细胞周期调控的核蛋白,外源性spindlin1的表达可以引起细胞丝裂灾变以及染色体不稳定,使NIH3T3细胞发生恶性转化;进一步的分析表明spindlin1可能通过对β-catenin/ T cell factor (TCF-4)通路发挥其对细胞周期的调控作用。为更深入了解spindlin1功能和作用机制,我们原核表达spindlin1蛋白,纯化后制备晶体,并对其晶体结构进行分析,结果表明spindlin1蛋白具有三个序列上非常类似的Spin/Ssty重复结构域,每个结构域都由4～5个反向平行的β-折叠构成。那么,在Spindlin1结构分析的基础上通过构建、突变与探讨蛋白质之间的相互作用等方法确认影响蛋白定位与功能的关键氨基酸位点无疑是进一步研究的重要内容。 一.spindlin 1突变体质粒的构建 我室从人类卵巢癌样本中分离克隆获得了该家族成员——人类spindlin基因,命名为spindlin1。该基因cDNA全长4375bp,含714bp的开放读框,编码237个氨基酸的蛋白。在我们前期的研究中,我们对其结构生物学进行探讨,结果表明Spindlin1蛋白结构分为三个序列上非常类似的Spin/Ssty重复结构域。为深入阐明spindlin1的生物功能,了解其作用的分子机制,我们在生物信息学和蛋白晶体结构解析的基础上,构建了一系列突变体,将spindlin1蛋白中14、84、99位丝氨酸突变成丙氨酸,得到Ser14M,Ser84M,Ser99M,Ser14+84M,Ser14+99M,Ser84+99M,Ser14+84+99M等七种突变质粒。为下一步的研究定位和功能关键位点的确定打下基础。 二.spindlin 1突变对其定位和功能的影响 在获得野生型和突变表达载体后,我们首先以蛋白亚细胞定位作为其功能变化的直观标示,期望能从中找到在Spindlin1生物功能发挥中具有重要作用的位点。我们将野生型及七种突变质粒转染Hela细胞,观察Spindlin1-EGFP融合蛋白亚细胞定位。结果表明:野生型的Spindlin1定位于细胞核,再我们构建的七个不同突变体中,Ser14+Ser84,Ser84+Ser99,Ser14+Ser84+Ser99的联合突变均能使野生型融合蛋白在细胞核内集中分布的特性消失,成为全细胞弥散分布,而Ser14,Ser84,Ser99各位点的单独突变及Ser14+Ser99的联合突变均对Spindlin1蛋白的细胞核内分布没有影响;提示Ser84在Spindlin1蛋白的定位中发挥重要作用。 我们之前的实验初步提示Spindlin1蛋白可能通过与转录因子TCF-4结合而发挥作用,在本实验中我们利用GST-pulldown的方法证实Spindlin1和TCF-4之间在体外具有相互作用,并且证实了外源性Spindlin1表达对TCF-4荧光素报告基因的转录活性有激活作用.为了进一步验证Ser84在Spindlin1功能发挥中的作用。我们将pEGFP-spindlin1野生型表达载体或spindin1的突变载体分别与TCF-4荧光素酶报告基因载体pTOPFlash和作为转染对照的β-galactosidase载体共转染Hela细胞,荧光素酶活性分析显示与对照载体pEGFP-C1质粒相比,pEGFP-spindlin1与TCF-4荧光素酶报告基因载体pTOPFlash载体共转染后,TCF-4荧光素酶报告基因的转录活性明显上升,而Ser14+Ser84,Ser84+Ser99,Ser14+Ser84+Ser99的联合突变使Spindlin1对其活性的激活作用消失或降低。进一步证实了Ser84位点在Spindlin1功能发挥中的重要作用。 以上结果提示我们,Ser84是Spindlin1细胞核内定位和功能发挥的关键位点,但其作用的发挥需要Ser14,Ser99的协助。Spindlin1的功能发挥可能与磷酸化、核定位以及与TCF-4之间的结合密切相关。更为有意义的是,这三个丝氨酸位点所构成的结构域正是Aurora A激酶特异的作用位点,Aurora A激酶在肿瘤发生和细胞周期调控中所发挥的重要作用提示我们,Spindlin1可能作为Aurora A激酶的作用底物发挥作用。 三.Spindlin1与Aurora A激酶相互作用的初步研究 人类Aurora A定位于染色体20q13.2,由403个氨基酸组成。Aurora A不仅与纺锤体装配、胞质分裂、中心体的成熟和分离、纺锤体检查点等过程相关联,最近越来越多的证据表明Aurora A表现出成癌基因的特性。Aurora A与其众多底物相互作用,参与不同的信号通路,这些途径相互关联形成网络。我们的研究基础提示Spindlin1与Aurora A具有很多功能和特征上的关联。在本部分实验中我们构建的pGEX-T-spindlin1原核表达载体,用IPTG诱导GST- spindlin1融合蛋白表达后,利用GST-pulldown技术的初步的研究结果表明spindlin1与丝氨酸激酶Aurora A的在体外具有相互作用,有可能作为Aurora A的底物发挥功能。
[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.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R341;R737.31
本文编号：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.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R341;R737.31
【参考文献】
相关期刊论文 前1条
1 岳文,孙丽亚,李春海,张立新,裴雪涛;卵巢癌相关基因的筛选与鉴定[J];癌症;2004年02期
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