NIRF泛素化p53作用过程中NIRF与p53结合域的测定
发布时间：2018-11-18 18:20
【摘要】：目的 Np95/ICBP90-like RING finger protein(NIRF)是新近发现的一种核蛋白，其包括802个氨基酸残基，蛋白分子内部含有UBL(ubiquitin-like domain)、PHD、YDG/SRA和RING finger结构域。随着其研究的深入，NIRF功能已涉及细胞增殖调节、蛋白多聚泛素化降解、细胞癌变进程控制以及更多未知的领域。已有研究报道，NIRF能与Cdk2-cyclin E复合体和let-7a结合，过表达的NIRF能阻止细胞周期于G1期。另外NIRF还具有泛素化酶E3的作用，即泛素化和自我泛素化功能，表明NIRF介导重要的蛋白降解途径。p53是细胞内重要的抑癌基因，p53的功能活动被认为是细胞对细胞异常、损伤、压力的一种应激反应，通过启动自我修复程序抑制细胞分裂诱导其进入衰老程序，或启动细胞程序性死亡清除异变的细胞。已有研究报道NIRF能与p53相互作用, NIRF本身也是一个高度调节蛋白，在细胞正常的生理状态下发挥泛素化E3连接酶的作用结合p53并将其降解，但NIRF与p53结合的蛋白结合域目前尚不清楚。本文研究证明NIRF能与p53结合成复合体参与泛素化蛋白降解途径，并测定出NIRF与p53结合的区域，为下一步研究蛋白泛素化降解和肿瘤的治疗提供实验基础和依据。 方法 （1）采用DMEM培养基培养HEK293细胞。培养基中补充含10%小牛血清和双抗。 （2） RIPA蛋白裂解液收集HEK293细胞总蛋白。 （3）细胞转染按照转染试剂说明说进行，转染48小时之后收获细胞，细胞收获前10小时添加蛋白酶体抑制剂MG-132。 （4）将NIRF突变体在细胞内全部表达。在HEK293细胞内，分别转染四个突变体质粒，同时转染pCMV-3×FLAG空质粒和pCMV-FLAG-NIRF全长质粒，作为阴阳性对照．细胞裂解液经Western印迹，anti-NIRF, anti-FLAG和anti-β-Actin抗体检测分析。 （5）用免疫蛋白共沉淀技术和蛋白质印迹分析技术鉴定NIRF与p53结合的蛋白结合域。HEK293细胞分别同时转染p53和各突变体质粒，细胞裂解液经p53抗体IP纯化，产物经WB分析。NIRF全长能与p53稳定结合，同时转染NIRF全长质粒作为阳性对照。 结果 结果显示，所有的突变体都能在细胞中表达，并且两种抗体检测结果完全一致。在免疫共沉淀试验中，，ΔUBL、ΔYDG/SRA和ΔRING缺失突变体蛋白依然能被p53沉淀出，而ΔPHD却未能被p53沉淀出。 结论 本研究表明，NIRF通过PHD区域与p53形成复合体.该复合体可能参与蛋白分选、蛋白降解、DNA修复以及细胞凋亡等一系列重要的细胞活动，从而形成与细胞增殖相关的新的信号通路，在肿瘤的发生发展中可能发挥着某种程度上作用。
[Abstract]:Objective Np95/ICBP90-like RING finger protein (NIRF) is a newly discovered nuclear protein, which contains 802 amino acid residues and contains UBL (ubiquitin-like domain), PHD,YDG/SRA and RING finger) domains. With the development of its research, the function of NIRF has been involved in the regulation of cell proliferation, the degradation of protein polyubiquitin, the control of cell carcinogenesis and more unknown fields. It has been reported that NIRF can bind to Cdk2-cyclin E complex and let-7a, and overexpression of NIRF can block cell cycle in G1 phase. In addition, NIRF also has the function of ubiquiding enzyme E3, that is, ubiquitization and self-ubiquification, which indicates that NIRF mediates important protein degradation pathway. P53 is an important tumor suppressor gene in cells, and p53 function is thought to be abnormal and damaged by cells. A stress response by initiating a self-repair program that inhibits cell division and induces it to enter the aging process, or activates programmed cell death to clear the metamorphosis of the cells. It has been reported that NIRF can interact with p53, and NIRF itself is a highly regulated protein, which acts as a Ubiquitin E3 ligase to bind and degrade p53 in normal physiological state of cells. However, the binding domain of NIRF to p53 protein is unclear. In this paper, it is proved that NIRF can bind to p53 complex to participate in the degradation pathway of ubiquitin, and the region of NIRF binding to p53 is determined, which provides the experimental basis and basis for further study on the degradation of protein ubiquitin and the treatment of tumor. Methods (1) HEK293 cells were cultured on DMEM medium. The medium was supplemented with 10% calf serum and double antibodies. (2) the total protein of HEK293 cells was collected by RIPA protein lysate. (3) Cell transfection was carried out according to the instructions of transfection reagent. After 48 hours of transfection, the cells were harvested and the proteasome inhibitor MG-132. was added 10 hours before harvest. (4) all the NIRF mutants were expressed in the cells. Four mutants were transfected into HEK293 cells, and pCMV-3 脳 FLAG empty plasmids and pCMV-FLAG-NIRF full-length plasmids were transfected respectively. The cell lysate was detected by Western blot, anti-NIRF, anti-FLAG and anti- 尾-Actin antibodies. (5) the protein binding domains of NIRF and p53 were identified by immunoprecipitation and Western blot analysis. HEK293 cells were transfected with p53 and mutant granulocytes respectively, and the cell lysate was purified by p53 antibody IP. The product was analyzed by WB. The full length of NIRF could bind to p53 stably, and the full-length plasmid of NIRF was transfected as positive control at the same time. The results showed that all the mutants could be expressed in the cells, and the results of the two antibodies were identical. In immunoprecipitation assay, 螖 UBL, 螖 YDG/SRA and 螖 RING deletion mutants could still be precipitated by p53, while 螖 PHD could not be precipitated by p53. Conclusion this study suggests that NIRF forms a complex with p53 via the PHD region. The complex may be involved in a series of important cellular activities, such as protein sorting, protein degradation, DNA repair and apoptosis, thus forming a new signal pathway related to cell proliferation. It may play a role in the occurrence and development of tumor to some extent.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363
本文编号：2340777
[Abstract]:Objective Np95/ICBP90-like RING finger protein (NIRF) is a newly discovered nuclear protein, which contains 802 amino acid residues and contains UBL (ubiquitin-like domain), PHD,YDG/SRA and RING finger) domains. With the development of its research, the function of NIRF has been involved in the regulation of cell proliferation, the degradation of protein polyubiquitin, the control of cell carcinogenesis and more unknown fields. It has been reported that NIRF can bind to Cdk2-cyclin E complex and let-7a, and overexpression of NIRF can block cell cycle in G1 phase. In addition, NIRF also has the function of ubiquiding enzyme E3, that is, ubiquitization and self-ubiquification, which indicates that NIRF mediates important protein degradation pathway. P53 is an important tumor suppressor gene in cells, and p53 function is thought to be abnormal and damaged by cells. A stress response by initiating a self-repair program that inhibits cell division and induces it to enter the aging process, or activates programmed cell death to clear the metamorphosis of the cells. It has been reported that NIRF can interact with p53, and NIRF itself is a highly regulated protein, which acts as a Ubiquitin E3 ligase to bind and degrade p53 in normal physiological state of cells. However, the binding domain of NIRF to p53 protein is unclear. In this paper, it is proved that NIRF can bind to p53 complex to participate in the degradation pathway of ubiquitin, and the region of NIRF binding to p53 is determined, which provides the experimental basis and basis for further study on the degradation of protein ubiquitin and the treatment of tumor. Methods (1) HEK293 cells were cultured on DMEM medium. The medium was supplemented with 10% calf serum and double antibodies. (2) the total protein of HEK293 cells was collected by RIPA protein lysate. (3) Cell transfection was carried out according to the instructions of transfection reagent. After 48 hours of transfection, the cells were harvested and the proteasome inhibitor MG-132. was added 10 hours before harvest. (4) all the NIRF mutants were expressed in the cells. Four mutants were transfected into HEK293 cells, and pCMV-3 脳 FLAG empty plasmids and pCMV-FLAG-NIRF full-length plasmids were transfected respectively. The cell lysate was detected by Western blot, anti-NIRF, anti-FLAG and anti- 尾-Actin antibodies. (5) the protein binding domains of NIRF and p53 were identified by immunoprecipitation and Western blot analysis. HEK293 cells were transfected with p53 and mutant granulocytes respectively, and the cell lysate was purified by p53 antibody IP. The product was analyzed by WB. The full length of NIRF could bind to p53 stably, and the full-length plasmid of NIRF was transfected as positive control at the same time. The results showed that all the mutants could be expressed in the cells, and the results of the two antibodies were identical. In immunoprecipitation assay, 螖 UBL, 螖 YDG/SRA and 螖 RING deletion mutants could still be precipitated by p53, while 螖 PHD could not be precipitated by p53. Conclusion this study suggests that NIRF forms a complex with p53 via the PHD region. The complex may be involved in a series of important cellular activities, such as protein sorting, protein degradation, DNA repair and apoptosis, thus forming a new signal pathway related to cell proliferation. It may play a role in the occurrence and development of tumor to some extent.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363
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相关期刊论文 前3条
1 段昌柱;蒲淑萍;TSUTOMU MORI;HIDEO KOCHI;邱宗荫;;NIRF对P53蛋白泛素化作用的研究[J];生物化学与生物物理进展;2006年02期
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