组织蛋白酶D在鼻咽癌细胞中相互作用蛋白的筛选与鉴定

发布时间：2018-02-16 02:01

本文关键词： CTSD 相互作用蛋白免疫共沉淀质谱分析生物信息学分析　出处：《南华大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：筛选并鉴定鼻咽癌细胞中组织蛋白酶D (Cathepsin D, CTSD)的相互作用蛋白，初步了解CTSD在鼻咽癌侵袭转移中的作用及机制。方法：收集高转移鼻咽癌5-8F细胞的总蛋白，采用免疫共沉淀结合凝胶电泳分离CTSD相互作用蛋白，应用电喷雾四级杆飞行时间串联质谱鉴定相互作用蛋白，结合基因本体论、功能聚类、信号通路、蛋白质相互作用网络分析等生物信息学方法综合分析CTSD相互作用蛋白，采用免疫共沉淀结合Western blot方法对CTSD相互作用蛋白EGFR、HSP90A进行验证。结果1、采用免疫共沉淀结合质谱方法筛选并鉴定了143个CTSD相互作用蛋白。2、将143个CTSD相互作用蛋白进行功能聚类分析显示：其中70个CTSD相互作用蛋白根据功能聚为12类，主要涉及到以下几个方面：跨膜运输、细胞骨架、氧化磷酸化、蛋白质合成、细胞凋亡、信号转到、氧化还原酶活性、分子伴侣、糖代谢等。另外73个CTSD相互作用蛋白未参与聚类。3、将143个CTSD相互作用蛋白进行GO分析，结果如下：GO_BP分析显示CTSD相互作用蛋白生物学过程主要涉及到应激反应、负调控、代谢过程、运输过程、蛋白定位及转运过程；GO_MF分析显示CTSD相互作用蛋白分子功能主要涉及到蛋白结合、催化活性、嘌呤核苷酸绑定、核苷酸结合、细胞骨架、氧化还原酶活性、结构分子活性等；GO_CC分析显示CTSD相互作用蛋白亚细胞定位于膜、细胞器、囊泡、高分子复合物中等。4、KEGG和Biocarta信号通路分析显示：143个CTSD相互作用蛋白涉及2条Biocarta信号通路包括低氧诱导因子相关信号通路，端粒、端粒酶、细胞老化；9条KEGG信号通路：氧化磷酸化、丙酮酸代谢、糖酵解信号通路、粘附连接、抗原处理和递呈、丙酸代谢、核糖体信号通路、磷酸戊糖信号通路。5、蛋白质相互作用网络分析显示：EGFR、HSP90A蛋白与CTSD形成相互作用组。6、应用免疫共沉淀结合Western blot显示EGFR、HSP90A与CTSD结合，验证了质谱结果的可靠性。结论1、本研究采用免疫共沉淀结合质谱分析在鼻咽癌细胞中鉴定了143个CTSD的相互作用蛋白，为进一步研究CTSD蛋白的功能提供了实验依据。2、生物信息学综合分析发现，，EGFR、HSP90A蛋白与CTSD在鼻咽癌细胞中形成相互作用组，可能在鼻咽癌侵袭转移中发挥重要作用。3、应用免疫共沉淀结合Western blot对其中2个CTSD相互作用蛋白质EGFR、HSP90A进行了验证，与质谱结果一致，提示质谱结果的可靠性。
[Abstract]:Objective:. To screen and identify the interacting proteins of cathepsin D Cathepsin D (CTSDs) in nasopharyngeal carcinoma cells, and to understand the role and mechanism of CTSD in the invasion and metastasis of nasopharyngeal carcinoma. The total proteins of 5-8F cells with high metastasis were collected. The CTSD interacting proteins were separated by immunoprecipitation and gel electrophoresis. The interacting proteins were identified by electrospray four-step time-of-flight mass spectrometry. Bioinformatics methods, such as functional clustering, signal pathway and protein interaction network analysis, are used to analyze CTSD interacting proteins. The CTSD interaction protein EGFRN HSP90A was verified by immunoprecipitation and Western blot. Results 1. 143 CTSD interacting proteins were screened and identified by immunoprecipitation mass spectrometry and 143 CTSD interacting proteins were inserted into it. The results of functional cluster analysis showed that 70 CTSD interacting proteins were clustered into 12 clusters according to their functions. Transmembrane transport, cytoskeleton, oxidative phosphorylation, protein synthesis, apoptosis, signal transfer, redox enzyme activity, molecular chaperone, Glucose metabolism. Another 73 CTSD interaction proteins were not involved in cluster. 143 CTSD interaction proteins were analyzed by go. The results showed that the biological process of CTSD interaction proteins was mainly involved in stress response and negative regulation. The analysis of metabolic process, transport process, protein localization and transport process showed that the molecular functions of CTSD interaction proteins were mainly related to protein binding, catalytic activity, purine nucleotide binding, nucleotide binding, cytoskeleton, redox enzyme activity. Structural molecular activity analysis showed that CTSD interaction protein subcells were located in membrane, organelle and vesicle. An analysis of the intermediate. 4kEGG and Biocarta signaling pathways of polymer complexes showed that 143 CTSD interacting proteins were involved in two Biocarta signaling pathways, including hypoxia inducible factor-related signaling pathways, telomere, telomerase, cellular aging and 9 KEGG signaling pathways: oxidative phosphorylation. Pyruvate Metabolism, glycolysis signal Pathway, Adhesion Junction, Antigen processing and presentation, Propionic Acid Metabolism, ribosomal signaling Pathway, The protein-protein interaction network analysis of pentose phosphate pathway showed that the interaction group of CTSD with CTSD was composed of 1: EGFR- HSP90A protein. The result of mass spectrometry was proved to be reliable by immunoprecipitation combined with Western blot to display the binding of EGFR- HSP90A to CTSD. Conclusion 1. 143 interacting proteins of CTSD were identified by immunoprecipitation and mass spectrometry in nasopharyngeal carcinoma cells. In order to further study the function of CTSD protein, the bioinformatics analysis showed that EGFR HSP90A protein interacted with CTSD in nasopharyngeal carcinoma cells. It may play an important role in the invasion and metastasis of nasopharyngeal carcinoma. Two of them, EGFR HSP90A, were verified by immunoprecipitation combined with Western blot, which were consistent with the results of mass spectrometry, suggesting the reliability of the results of mass spectrometry.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.63

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