小鼠与人睾丸磷酸化蛋白质组学研究

发布时间：2018-02-07 13:33

本文关键词： 睾丸精子发生蛋白质组学蛋白质磷酸化磷酸化激酶　出处：《南京医科大学》2015年博士论文　论文类型：学位论文

【摘要】：背景:精子发生是一项涉及精原细胞有丝分裂,精母细胞减数分裂及精子变形的复杂生物学过程。在精子发生中,这些环节受到一系列基因的调控。其中任何一个环节发生错误,都有可能导致精子发生的异常,甚至会导致雄性不育,这已经被各种来自临床及实验动物模型的数据所证实。精子发生机制非常复杂,受到转录水平与转录后水平多层次的调控。其中磷酸化修饰,一种被广泛研究并在多种通路中发挥作用的修饰,在精子发生过程中也发挥着重要调控作用。因此,在睾丸中系统分析磷酸化修饰对深入研究精子发生的分子调控是十分必要的。之前的研究已经帮助我们对磷酸化修饰在精子发生中的作用有一定的认识。而另一方面,随着高分辨率、大规模质谱仪器的发展和应用,蛋白质组学可以帮助我们全景式的了解精子发生。构建睾丸磷酸化蛋白谱系,是通过蛋白质组学研究精子发生中磷酸化修饰的一种直接有效的方式。然而,目前为止,小鼠中仅有3周的磷酸化蛋白质谱系的研究,人睾丸磷酸化蛋白质位点的组成及调控机制亦未见报道。研究方法与结果:在本研究中,我们利用高分辨率的LTQ Orbitrap Velos质谱仪器,基于IMAC与TiO2两种磷酸化肽段富集策略,在成年小鼠睾丸中鉴定到17829个磷酸化位点,对应3955个磷酸化蛋白质。对谱系数据进行初步分析表明,富集到的磷酸化位点残基的丝氨酸(pS),苏氨酸(pT)及酪氨酸(pY)的比例约为82%,16%及2%,共有48.31%的磷酸化位点在IMAC及TiO2富集中均鉴定到。通过进一步生物信息学分析,发现在本研究的三种(Total,IMAC及TiO2)数据统计均显示为精子发生(GO:0007283)为最显著富集。蛋白的自体磷酸化(GO:0046777)也显示显著富集,这些结果说明在睾丸中,磷酸化调控是大量存在的。通过构建睾丸中激酶及其底物磷酸化位点网络进行磷酸化位点分析,我们预测了与精子发生相关的重要调控性激酶及其底物调控关系。尽管两种富集方法鉴定得到的磷酸化位点只有大约50%的重合率,但对底物对应的激酶活性程度分析结果显示,polo-like激酶在两种富集方法中都显示出显著高活性,因此,我们推测polo-like激酶在精子发生中有重要功能。通过western blot检测PLK1激酶活性位点pT210磷酸化水平确定PLK1在睾丸中和其余组织中的不同活性。实验结果与预测结果一致,PLK1在睾丸中的活性随着睾丸的成熟增加,并且比同周龄的其他组织中PLK1具有更高活性。体外实验中,在精母细胞系GC2中进行抑制实验,结果显示PLK1-3的活性调控细胞周期。在构建了小鼠睾丸磷酸化蛋白质谱系的基础上,调整了IMAC与TiO2富集方式与一维分离策略,进一步对成年人睾丸的磷酸化修饰蛋白质进行分析,共鉴定到16364个磷酸化位点,对应4683个磷酸化蛋白质。两种方法富集到的磷酸化位点残基的丝氨酸(pS),苏氨酸(pT)及酪氨酸(pY)的比例约为86%,12%及2%。GO分析显示,有丝分裂细胞周期(GO:0000278),基因表达(GO:0010467),RNA剪切(GO:0008380),有丝分裂(GO:0007067),蛋白质磷酸化(GO:0006468)及精子发生(GO:0007283)均呈现显著富集。对本研究中鉴定到的磷酸化位点构建激酶及其底物磷酸化位点网络,结果显示,大于60%的激酶对应不仅一个底物,这说明磷酸化激酶是复杂的多重调控。将小鼠睾丸磷酸化蛋白质谱系与人睾丸磷酸化蛋白质谱系进行磷酸化位点序列同源分析,结果显示,以人睾丸磷酸化位点为背景,30.1%的位点为人和小鼠同源的磷酸化位点,39.5%的基因可表达人和小鼠磷酸化位点同源的蛋白质。成年小鼠睾丸磷酸化蛋白质谱系及人睾丸磷酸化蛋白质谱系的构建,全面展现了精子发生的磷酸化调控,对研究磷酸化在精子发生中的作用提供了丰富的资源。激酶与底物网络的构建有助于更深入了解磷酸化在精子发生中的调控作用,对磷酸激酶与底物调控的机制探索提供了宝贵的线索。
[Abstract]:Background: spermatogenesis is a germ cell mitosis, complex biological processes of spermatocyte meiosis and sperm deformation. In spermatogenesis, these links are regulated by a series of genes. A part of any errors occur, are likely to lead to abnormal spermatogenesis, and even lead to male sterility and this has been confirmed by various from clinical and experimental animal model data. The mechanism of sperm is very complex and regulated by transcriptional and post transcriptional level. The multi-level phosphorylation, a widely studied and play a role in a variety of modified pathway, also plays an important regulatory role in spermatogenesis in the testis. Therefore, system analysis of phosphorylation of molecules for further study of sperm regulation is very necessary. Previous studies have helped us in the modification of phosphoric acid In spermatogenesis have a certain understanding. On the other hand, with high resolution, large-scale development and application of mass spectrometry, proteomics can help us understand the panorama of spermatogenesis. Construction of testicular protein phosphorylation by pedigree, proteomics research on spermatogenesis in a direct and effective way of phosphorylation modified. However, so far, the research of phosphorylated protein mass line only 3 weeks in mice, consisting of human testis protein phosphorylation sites and regulatory mechanisms have not been reported. The research methods and results: in this study, we use LTQ Orbitrap Velos high resolution mass spectrometer, IMAC and TiO2 two phosphate peptides enrichment strategy based on in adult mouse testes to identify 17829 phosphorylation sites, corresponding to 3955 protein phosphorylation. The pedigree data preliminary analysis showed that the enrichment of phosphorus Acidification site residues serine threonine (pS), (pT) and tyrosine (pY) ratio is about 82%, 16% and 2%, a total of 48.31% phosphorylation sites in IMAC and TiO2 concentration were identified by bioinformatics analysis. Further, the study found in three (Total, IMAC and TiO2) statistics showed spermatogenesis (GO:0007283) is the most significant enrichment. Autologous phosphorylated proteins (GO:0046777) also showed significant enrichment, these results indicate that in the testis, phosphorylation is the existence of a large number of analyses of phosphorylation sites by constructing testis kinase and its substrate phosphorylation sites of the network, we the prediction of important regulatory kinase and its substrate regulation related with sperm. Although the phosphorylation sites were identified two enrichment methods only about 50% of the coincidence rate, but the degree of substrate kinase activity analysis results corresponding to the polo- display. Like kinase in two enrichment methods showed significantly higher activity, therefore, we speculate that polo-like kinase has an important function in spermatogenesis. Determination of PLK1 activity in different tissues of testis and the other by Western blot detection of PLK1 kinase activity sites of phosphorylation of pT210. The experimental results consistent with the prediction results, the activity of PLK1 in testis with the increase in testicular maturation, and has higher activity than other organizations with PLK1 weeks of age. In vitro inhibition in spermatocyte cell line GC2, results show that the cycle activity regulation of PLK1-3 cells. In the foundation of the mouse testis protein phosphorylation system, adjust the IMAC with the enrichment of TiO2 and one-dimensional separation strategy, further analysis of protein phosphorylation in adult testis, were identified to 16364 phosphorylation sites, 4683 phosphorylated proteins corresponding to The phosphorylation site residues. Two kinds of methods to the enrichment of serine threonine (pS), (pT) and tyrosine (pY) ratio is about 86%, 12% and 2%.GO analysis showed that the mitotic cell cycle (GO:0000278), gene expression (GO:0010467), RNA shear (GO:0008380), mitosis (GO:0007067), protein phosphorylation (GO:0006468) and spermatogenesis (GO:0007283) showed a significant enrichment of phosphorylation sites in this study were identified to construct kinase and substrate phosphorylation sites of the network, showed that more than 60% of the corresponding kinase not only a substrate, indicating that phosphorylation is regulated kinase complex the mouse testis phosphorylation protein and phosphorylated protein in human testis mass line phosphorylation site sequence analysis results showed that in human testicular phosphorylation sites as the background, the 30.1% sites of phosphorylation sites homologous to human and mouse, 39.5% base Because the expression of human and mouse homologous protein phosphorylation sites. The phosphorylation of protein construction testis and adult mouse testis protein phosphorylation system, fully demonstrated the phosphorylation regulation of spermatogenesis, provide rich resources for research on phosphorylation in spermatogenesis in vitro. Construction of kinase and substrate network contribute to a more in-depth understanding of phosphate regulation in spermatogenesis, provided valuable clues to explore the mechanism of phosphate kinase and substrate regulation.

【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R321.1

【相似文献】