本文關(guān)鍵詞： CTSD 相互作用蛋白 免疫共沉淀 質(zhì)譜分析 生物信息學(xué)分析　出處：《南華大學(xué)》2014年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的： 篩選并鑒定鼻咽癌細(xì)胞中組織蛋白酶D (Cathepsin D, CTSD)的相互作用蛋白，初步了解CTSD在鼻咽癌侵襲轉(zhuǎn)移中的作用及機(jī)制。方法： 收集高轉(zhuǎn)移鼻咽癌5-8F細(xì)胞的總蛋白，采用免疫共沉淀結(jié)合凝膠電泳分離CTSD相互作用蛋白，應(yīng)用電噴霧四級(jí)桿飛行時(shí)間串聯(lián)質(zhì)譜鑒定相互作用蛋白，結(jié)合基因本體論、功能聚類(lèi)、信號(hào)通路、蛋白質(zhì)相互作用網(wǎng)絡(luò)分析等生物信息學(xué)方法綜合分析CTSD相互作用蛋白，采用免疫共沉淀結(jié)合Western blot方法對(duì)CTSD相互作用蛋白EGFR、HSP90A進(jìn)行驗(yàn)證。結(jié)果1、采用免疫共沉淀結(jié)合質(zhì)譜方法篩選并鑒定了143個(gè)CTSD相互作用蛋白。2、將143個(gè)CTSD相互作用蛋白進(jìn)行功能聚類(lèi)分析顯示：其中70個(gè)CTSD相互作用蛋白根據(jù)功能聚為12類(lèi)，主要涉及到以下幾個(gè)方面：跨膜運(yùn)輸、細(xì)胞骨架、氧化磷酸化、蛋白質(zhì)合成、細(xì)胞凋亡、信號(hào)轉(zhuǎn)到、氧化還原酶活性、分子伴侶、糖代謝等。另外73個(gè)CTSD相互作用蛋白未參與聚類(lèi)。3、將143個(gè)CTSD相互作用蛋白進(jìn)行GO分析，結(jié)果如下：GO_BP分析顯示CTSD相互作用蛋白生物學(xué)過(guò)程主要涉及到應(yīng)激反應(yīng)、負(fù)調(diào)控、代謝過(guò)程、運(yùn)輸過(guò)程、蛋白定位及轉(zhuǎn)運(yùn)過(guò)程；GO_MF分析顯示CTSD相互作用蛋白分子功能主要涉及到蛋白結(jié)合、催化活性、嘌呤核苷酸綁定、核苷酸結(jié)合、細(xì)胞骨架、氧化還原酶活性、結(jié)構(gòu)分子活性等；GO_CC分析顯示CTSD相互作用蛋白亞細(xì)胞定位于膜、細(xì)胞器、囊泡、高分子復(fù)合物中等。4、KEGG和Biocarta信號(hào)通路分析顯示：143個(gè)CTSD相互作用蛋白涉及2條Biocarta信號(hào)通路包括低氧誘導(dǎo)因子相關(guān)信號(hào)通路，端粒、端粒酶、細(xì)胞老化；9條KEGG信號(hào)通路：氧化磷酸化、丙酮酸代謝、糖酵解信號(hào)通路、粘附連接、抗原處理和遞呈、丙酸代謝、核糖體信號(hào)通路、磷酸戊糖信號(hào)通路。5、蛋白質(zhì)相互作用網(wǎng)絡(luò)分析顯示：EGFR、HSP90A蛋白與CTSD形成相互作用組。6、應(yīng)用免疫共沉淀結(jié)合Western blot顯示EGFR、HSP90A與CTSD結(jié)合，驗(yàn)證了質(zhì)譜結(jié)果的可靠性。 結(jié)論1、本研究采用免疫共沉淀結(jié)合質(zhì)譜分析在鼻咽癌細(xì)胞中鑒定了143個(gè)CTSD的相互作用蛋白，為進(jìn)一步研究CTSD蛋白的功能提供了實(shí)驗(yàn)依據(jù)。2、生物信息學(xué)綜合分析發(fā)現(xiàn)，，EGFR、HSP90A蛋白與CTSD在鼻咽癌細(xì)胞中形成相互作用組，可能在鼻咽癌侵襲轉(zhuǎn)移中發(fā)揮重要作用。3、應(yīng)用免疫共沉淀結(jié)合Western blot對(duì)其中2個(gè)CTSD相互作用蛋白質(zhì)EGFR、HSP90A進(jìn)行了驗(yàn)證，與質(zhì)譜結(jié)果一致，提示質(zhì)譜結(jié)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.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R739.63

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