钙结合蛋白及作用位点的生物信息学研究

发布时间：2018-08-06 21:33

【摘要】：钙是生命活动中最重要的基本元素之一，在真核生物细胞内以离子状态充当第二信使。钙离子通过与特定钙受体或者钙结合蛋白绑定实现其生物学功能，调控细胞信号传导和细胞生命周期（包括细胞分裂、分化和凋亡）等过程。基于钙结合蛋白的功能结构域组成可将之分为两类：一类具有EF-hand结构域，其典型代表是钙调蛋白（Calmodulin）；另一类不具有EF-hand结构，即非EF-Hand钙结合蛋白。随着对钙结合蛋白研究的日趋深入，实验证据表明钙离子与蛋白质结合形式具有复杂性和不规则性。鉴定钙结合蛋白中钙离子与蛋白质的结合位点是了解生物系统中钙离子如何调控蛋白质结构和功能首要步骤。实验验证钙离子结合位点需要耗费大量的时间、精力和资金。利用生物信息学的方法开展预测，能够有效地去除潜在的假阳性结果，为进一步的实验研究提供有价值的参考信息。迄今为止，所有预测钙结合蛋白结合位点的生物信息学方法，都基于蛋白质的三级结构，从而具有较大的局限性。在本论文中，我们设计了一种基于位点特异性打分矩阵（PSSM）算法的方法，能够快速、准确的预测潜在的钙结合蛋白位点。通过阅读科学文献，搜集了实验验证的369个钙结合蛋白，包含了986个钙离子结合位点，对这些数据进行训练，并通过“除一法”(Leave-one-outvalidation)等方法评估其预测性能，，发现本方法的灵敏度（Sensitivity）和特异性（Specificity）分别为69.67%和90.00%。本方法仅需提供蛋白质的一级序列即可预测蛋白质的钙离子结合位点。与此同时，对搜集到的实验验证过的钙结合蛋白数据做了GO统计分析，从钙结合蛋白的生物学过程(Biologicalprocess）、分子功能(Molecularfunction）以及亚细胞定位（Cellularcomponent）三个层面进行了生物统计学分析。我们的结果表明：（1）钙结合蛋白主要分布在细胞质（GO:0005829）、细胞膜（GO:0005886）和细胞外区域（如组织液）（GO:0005576）等区域；（2）钙结合蛋白参与的生理过程主要有肌肉的收缩（GO:0006936）、突触信号传递（GO:0007268）和信号转导（GO:0007165）等过程；（3）钙结合蛋白具有的分子功能有与钙离子的结合（GO:0005509）、与钙依赖的蛋白结合（GO:0048306）和与其他蛋白质绑定（GO:0005515）等。另外，我们从Uniprot数据库中下载了人、小鼠、果蝇、线虫、酵母和拟南芥六个具有代表性的模式生物的序列信息，并对这六个物种进行了大规模预测，得到了与钙结合的蛋白质数目分别为人76.4%、小鼠68.6%、果蝇47.8%、线虫23.2%、酵母14.7%、拟南芥53.5%。从中可以得到两个结论：（1）钙结合蛋白在这六个物种中普遍存在；（2）物种由低等到高等，钙结合蛋白的比例逐渐增加。综上所述，我们对钙离子结合蛋白及作用位点进行了系统性分析，为进一步的实验分析提供了潜在的数据资源。我们相信，计算预测与实验验证的有机结合可将钙结合蛋白的研究带入一个新的发展阶段。
[Abstract]:Calcium is one of the most important basic elements in life activities. It acts as the second messenger in eukaryote cells. Calcium ions perform their biological functions by binding to specific calcium receptors or calcium binding proteins, and regulate cell signal transduction and cell life cycle (including cell division, differentiation and apoptosis). Based on the functional domain composition of calcium-binding protein, it can be divided into two categories: one has EF-hand domain, the typical one is calmodulin (Calmodulin); and the other is non-EF-Hand calcium-binding protein. With the development of the research on calcium binding protein, the experimental evidence shows that the form of calcium binding to protein is complex and irregular. Identification of calcium binding sites in calcium binding proteins is the first step in understanding how calcium ions regulate the structure and function of proteins in biological systems. It takes a lot of time, effort, and money to verify the calcium binding site. Using bioinformatics to carry out prediction can effectively remove the potential false positive results and provide valuable reference information for further experimental research. Up to now, all bioinformatics methods for predicting calcium-binding protein binding sites are based on the tertiary structure of proteins, so they have great limitations. In this paper, we design a method based on locus specific scoring matrix (PSSM) algorithm, which can predict the potential calcium binding protein sites quickly and accurately. By reading the scientific literature, we collected 369 calcium-binding proteins verified by experiments, including 986-calcium binding sites, trained these data, and evaluated their predictive performance by means of "divide one method" (Leave-one-outvalidation) and other methods. The sensitivity (Sensitivity) and specific (Specificity) of this method were 69.67% and 90.000%, respectively. In this method, the calcium binding sites of proteins can be predicted only by providing the first order sequence of proteins. At the same time, the data of calcium-binding protein verified by experiments were analyzed by go statistical analysis. The biostatistical analysis was carried out from three aspects of (Biologicalprocess), molecular function (Molecularfunction) and subcellular localization (Cellularcomponent) of calcium-binding protein. Our results showed that: (1) calcium binding proteins were mainly distributed in cytoplasm (GO:0005829), cell membrane (GO:0005886) and extracellular regions (such as tissue fluid) (GO:0005576). (2) the main physiological processes involved in calcium binding protein are muscle contraction (GO:0006936), synaptic signal transduction (GO:0007268) and signal transduction (GO:0007165). (3) calcium binding proteins have molecular functions such as calcium binding (GO:0005509), calcium dependent protein binding (GO:0048306) and other protein binding (GO:0005515). In addition, we downloaded from the Uniprot database the sequence information of six representative model organisms, human, mouse, Drosophila, nematode, yeast and Arabidopsis, and made large-scale predictions of these six species. The number of calcium binding proteins were 76.4am in human, 68.6 in mice, 47.8 in Drosophila, 23.2in nematode, 14.7in yeast and 53.5in Arabidopsis thaliana. Two conclusions can be drawn: (1) calcium binding protein exists widely in these six species; (2) the proportion of calcium binding protein increases gradually from low to higher species. In conclusion, we systematically analyzed calcium binding proteins and action sites, which provided potential data resources for further experimental analysis. We believe that the combination of computational prediction and experimental verification can bring the study of calcium binding protein to a new stage of development.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.04

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