类泛素化蛋白FAT10共价修饰增殖细胞核抗原PCNA及其作用的机制研究
发布时间：2018-07-14 18:47
【摘要】：背景:DNA损伤可能来自机体的内在因素的作用,例如活性氧、细胞新陈代谢的副产物以及DNA在复制过程中由于拓扑异构酶失活而导致的错配;也可能来自电离辐射(IR)、紫外光照射(UV)以及自然界中其它致癌物等外在因素的影响。DNA损伤会导致基因突变和细胞衰老。细胞发生DNA损伤并对其进行精确、高效修复的机制被称为DNA损伤应答机制(DDR),其作用是保护机体免受DNA损伤造成的不利影响。增殖细胞核抗原PCNA(proliferating cell nuclear antigen)在DNA损伤修复机制中起着核心作用。PCNA作为一个稳定的“站台”,在损伤修复过程中招募一系列与复制相关的蛋白。类泛素蛋白(ubiquitin-like proteins,UBLs),是一类与泛素类似的小蛋白家族。据报道,类泛素蛋白含有与泛素同源的结构域,其同源性大约为15%-16%。UBLs可以分为两个亚家族:类泛素结构域家族UDP和类泛素家族修饰蛋白家族ΜLM。UDP可以与泛素以及泛素修饰蛋白发生非共价结合。ΜLM具有与泛素单体或二聚体同源的结构域,可以在E1-E2-E3酶联体系的催化作用下通过C末端的双甘氨酸基团与底物蛋白质共价结合,其代表成员有ISG15、FUB1、NEDD8、SΜMO、Urm1、UBL5、Ufm1和FAT10等。据文献报道,当复制过程中的细胞发生DNA损伤时,包括泛素化以及类泛素化在内的多种翻译后修饰通过修饰PCNA,对DNA损伤修复进行调控。当表皮细胞长时间暴露在紫外线(UV)辐射中,RAD6-RAD18复合体能够介导PCNA第164位赖氨酸残基发生高度的单泛素化,从而导致复制性DNA聚合酶发生变化。FAT10(HLA-F locus adjacent transcript 10)是一种大小为18k D的蛋白,其N段和C端都含有与泛素相似的结构域,其中N端序列相似率为29%,C端序列则有36%与泛素相同,它的主要作用是编码人主要组织相容性复合体(MHC)I类基因座。人源FAT10除了在成熟树突细胞,B细胞以及在一些免疫器官,譬如胸腺和脾中表达,也可以在各种组织中通过促炎症因子(IFN-γ,TNF-α)刺激而表达,通过其C-末端结合共价修饰靶底物发挥作用。在前期工作中我们通过质谱鉴定确定增殖细胞核抗原(PCNA)是FAT10的共价修饰底物。目的:通过研究DNA损伤修复过程中类泛素蛋白FAT10与PCNA共价修饰作用机制,以及验证细胞中FAT10是否共价修饰ENO1,找到一种新的影响DNA损伤修复和癌症发生发展的调控方式,为衰老生理学研究提供新的思路。方法:(1)用UV/IR和VP-16处理细胞,通过western-blotting以及q RT-PCR实验分别检测DNA损伤对FAT10蛋白和基因水平表达的影响;通过免疫共沉淀实验检测当DNA发生损伤时FAT10蛋白是否共价修饰PCNA;通过类泛素化降解实验以及si RNA敲低FAT10实验检测PCNA能否被FAT10通过26S蛋白酶体降解;通过细胞免疫荧光实验探究FAT10和PCNA之间相互作用的亚细胞定位情况以及PCNA被FATylation对细胞形成细胞核聚集点的影响。(2)构建p Flag-CMV-eno1和p CMV-Myc-fat10两种真核表达质粒,并将这两种质粒共转入HEK293细胞内,通过免疫共沉淀的方法,探究FAT10是否在细胞中共价修饰ENO1。结果:首先,我们用UV/IR和VP-16处理细胞,western-blotting以及q RT-PCR实验检测发生DNA损伤的细胞,结果显示,DNA损伤能从基因和蛋白水平诱导FAT10表达的上调,FAT10表达量随着DNA损伤程度加重而增加。在本次实验中,当UV辐射剂量达到20J/m2、IR辐射剂量达到20Gy或VP-16剂量达到200μM时,FAT10表达量最高。通过免疫共沉淀实验检测以10J/m2和20J/m2的UV辐照强度处理后的He La细胞,发现FAT10能够共价修饰PCNA。同样,我们观察到用IR辐照以及用VP-16处理细胞时,FAT10也都能够共价修饰PCNA。通过PCNA降解实验,我们发现随着细胞因子TNF-α和IFN-γ浓度升高,诱导产生的FAT10蛋白表达量逐渐增加,而PCNA的表达水平却逐渐降低;在加入26S蛋白酶体抑制剂——MG132处理的细胞中,PCNA的表达水平不变。同样,在VP-16处理后的细胞中,PCNA和FAT10的降解也可以被MG132抑制。将FAT10 si RNA转染到被VP-16处理后的细胞中,敲低内源性FAT10,发现PCNA的降解现象明显消失,这些结果表明当DNA损伤时,FAT10可以通过26S蛋白酶体介导PCNA降解。其次,我们分离出样品的细胞质和细胞核,并通过western-blotting检测PCNA和FAT10的表达情况,发现大部分PCNA在细胞核中积累,当添加26S蛋白酶体抑制剂后,细胞质中PCNA的积累显著增加,同时,FAT10在细胞核和细胞质中都显著积累。这些数据表明在VP-16处理后的细胞中,FAT10可能在细胞质中通过26S蛋白酶体介导PCNA降解。最后,我们通过激光共聚焦显微镜检测细胞核聚集点(nuclear foci),观察到FAT10和PCNA共定位在损伤位点,并且当使用VP-16处理细胞后,细胞核聚集点的数量增加,同时,细胞质中PCNA的表达水平降低。当添加MG132后,细胞核聚集点的数量减少,并且PCNA的表达水平恢复正常。这些实验结果表明在DNA损伤修复中,FAT10在胞质中介导PCNA降解从而影响细胞核聚集点数量增多。另外,我们通过构建p Flag-CMV-eno1和p CMV-Myc-fat10两种表达质粒,并将这两种质粒共转入HEK293细胞内,利用免疫共沉淀的方法,发现在细胞中FAT10能共价修饰ENO1。结论:我们推测细胞发生DNA损伤时能够诱导FAT10表达上调并能够共价修饰PCNA蛋白使之在细胞质中被26S蛋白酶体降解,从而使细胞核损伤位点数量增加。此外,我们还发现,类泛素蛋白FAT10在细胞中能共价修饰ENO1。以上结果提示,细胞发生癌变时,FAT10可能通过共价修饰PCNA和ENO1影响和调控肿瘤的发生和转移。
[Abstract]:Background: DNA damage may come from the internal factors of the body, such as reactive oxygen species, by-products of cell metabolism, and DNA mismatch caused by the inactivation of topoisomerase during replication; it may also come from the effects of IR, ultraviolet light (UV) and other carcinogens in nature, such as the effect of.DNA damage. The mechanism of DNA damage and precision, the mechanism of efficient repair is called the DNA damage response mechanism (DDR), and its role is to protect the body from the adverse effects of DNA damage. Proliferating cell nuclear antigen PCNA (proliferating cell nuclear antigen) plays a core role in the mechanism of DNA damage repair. .PCNA is used as a stable "platform" to recruit a series of replication related proteins in the process of damage repair. Ubiquitin-like proteins (UBLs) is a class of small protein family similar to ubiquitin. It is reported that the ubiquitin protein contains the domain homologous with ubiquitin, and its homology is about 15%-16%.UBLs The two subfamilies: the ubiquitin domain family UDP and the ubiquitin family modified protein family, LM.UDP, can be non covalent with ubiquitin and ubiquitin modified proteins. LM has a homologous domain with ubiquitin monomers or two polymers, which can be catalyzed by the E1-E2-E3 enzyme system with the double glycine group at the C terminal and the substrate egg. White matter covalent binding, its representative members are ISG15, FUB1, NEDD8, S MO, Urm1, UBL5, Ufm1 and FAT10. According to the literature, a variety of post-translational modifications including ubiquitination and ubiquitination, including ubiquitination and ubiquitination, are used to regulate DNA damage repair when the cells of the replication process occur DNA damage. When epidermal cells are exposed to UV for a long time In line (UV) radiation, the RAD6-RAD18 complex can mediate the high degree of the mono ubiquitination of the PCNA 164th lysine residues, resulting in the change of the replicative DNA polymerase,.FAT10 (HLA-F locus adjacent transcript 10) is a protein of 18K D. Both the N segment and the end contain the domain similar to the ubiquitin, of which the sequence is similar. The rate is 29%, and 36% of the C end sequence is the same as ubiquitin, and its main role is the encoding human major histocompatibility complex (MHC) I gene seat. Human FAT10 can also be stimulated by the inflammatory factors (IFN- gamma, TNF- a) in various tissues except in mature dendritic cells, B cells, and some immune organs, such as thymus and spleen. Expression, through its C- terminal binding covalence to modify the target substrate. In the previous work, we identified the proliferation cell nuclear antigen (PCNA) as a covalent substrate for FAT10. Valence modified ENO1, finding a new regulation that affects the repair of DNA damage and the development of cancer, provides new ideas for the study of senescence physiology. Methods: (1) the cells were treated with UV/IR and VP-16, and the effects of DNA damage on the expression of FAT10 protein and gene level were detected by Western-blotting and Q RT-PCR. Whether FAT10 protein covalently modifies PCNA when DNA is damaged; through ubiquitination degradation experiment and Si RNA knocking low FAT10 test, PCNA can be degraded by 26S proteasome, and the subcellular localization of FAT10 and PCNA interaction between FAT10 and PCNA is investigated by cell immunofluorescence test and PCNA is considered (2) construct two eukaryotic expression plasmids of P Flag-CMV-eno1 and P CMV-Myc-fat10, and transfer these two plasmids into HEK293 cells. Through immunoprecipitation method, explore whether FAT10 covalently modifies ENO1. results in cells: first, we use UV/IR and VP-16 to treat cells and Western-blotting, Western-blotting DNA damage cells were detected by Q RT-PCR and the results showed that DNA damage could induce the up regulation of FAT10 expression from gene and protein level. The expression of FAT10 increased with the severity of DNA damage. In this experiment, when UV radiation dose reached 20J/m2, IR radiation dose reached 20Gy or VP-16 doses reached 200 mu. He La cells treated with UV irradiation intensity of 10J/m2 and 20J/m2 were detected by immunoprecipitation experiment. It was found that FAT10 could covalently modify PCNA.. We observed that when IR irradiated and the cells were treated with VP-16, FAT10 can also covalently modify the PCNA. through PCNA degradation experiment. The expression level of the induced FAT10 protein increased gradually, but the expression level of PCNA decreased gradually; the expression level of PCNA was unchanged in the cells treated with the 26S proteasome inhibitor - MG132 treatment. Also, the degradation of PCNA and FAT10 could also be inhibited by MG132 in the cells treated with VP-16. FAT10 Si RNA was transfected to the region. In the after cells, the endogenous FAT10 was knocked down and the degradation of PCNA disappeared. These results showed that when DNA was damaged, FAT10 could be degraded by 26S proteasome. Secondly, we isolated the cytoplasm and nucleus of the samples and detected the expression of PCNA and FAT10 by Western-blotting, and found that most PCNA were fine. Accumulation in the nucleus, when the 26S proteasome inhibitor is added, the accumulation of PCNA in the cytoplasm increases significantly, and FAT10 is accumulated in the nucleus and cytoplasm. These data indicate that in the cells after VP-16 treatment, FAT10 may be degraded by the 26S proteasome in the cytoplasm. Finally, we use laser confocal microscopy. The nucleus aggregation point (nuclear foci) was detected by the microscope, and both FAT10 and PCNA were found to be located at the damage site. When VP-16 was used to treat the cells, the number of nuclear aggregation points increased. At the same time, the expression level of PCNA in the cytoplasm decreased. When MG132 was added, the number of nuclear aggregation points decreased and the level of PCNA expression returned to normal. The experimental results showed that in DNA damage repair, FAT10 was degraded by cytoplasmic mediated PCNA degradation and increased the number of nuclear aggregation points. In addition, we transformed the two plasmids into HEK293 cells by constructing two expression plasmids of P Flag-CMV-eno1 and P CMV-Myc-fat10, and found that FAT10 can be shared in the cells by the method of co immunoprecipitation. ENO1. conclusion: we speculate that DNA damage can induce up regulation of FAT10 expression and can covalently modify PCNA protein to be degraded by 26S proteasome in cytoplasm, thus increasing the number of nuclear damage sites. Furthermore, we also found that ubiquitin protein FAT10 can covalently modify ENO1. in cells. When cells are cancerous, FAT10 may influence and regulate the occurrence and metastasis of tumors through covalent modification of PCNA and ENO1.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q75
，
本文编号：2122621
[Abstract]:Background: DNA damage may come from the internal factors of the body, such as reactive oxygen species, by-products of cell metabolism, and DNA mismatch caused by the inactivation of topoisomerase during replication; it may also come from the effects of IR, ultraviolet light (UV) and other carcinogens in nature, such as the effect of.DNA damage. The mechanism of DNA damage and precision, the mechanism of efficient repair is called the DNA damage response mechanism (DDR), and its role is to protect the body from the adverse effects of DNA damage. Proliferating cell nuclear antigen PCNA (proliferating cell nuclear antigen) plays a core role in the mechanism of DNA damage repair. .PCNA is used as a stable "platform" to recruit a series of replication related proteins in the process of damage repair. Ubiquitin-like proteins (UBLs) is a class of small protein family similar to ubiquitin. It is reported that the ubiquitin protein contains the domain homologous with ubiquitin, and its homology is about 15%-16%.UBLs The two subfamilies: the ubiquitin domain family UDP and the ubiquitin family modified protein family, LM.UDP, can be non covalent with ubiquitin and ubiquitin modified proteins. LM has a homologous domain with ubiquitin monomers or two polymers, which can be catalyzed by the E1-E2-E3 enzyme system with the double glycine group at the C terminal and the substrate egg. White matter covalent binding, its representative members are ISG15, FUB1, NEDD8, S MO, Urm1, UBL5, Ufm1 and FAT10. According to the literature, a variety of post-translational modifications including ubiquitination and ubiquitination, including ubiquitination and ubiquitination, are used to regulate DNA damage repair when the cells of the replication process occur DNA damage. When epidermal cells are exposed to UV for a long time In line (UV) radiation, the RAD6-RAD18 complex can mediate the high degree of the mono ubiquitination of the PCNA 164th lysine residues, resulting in the change of the replicative DNA polymerase,.FAT10 (HLA-F locus adjacent transcript 10) is a protein of 18K D. Both the N segment and the end contain the domain similar to the ubiquitin, of which the sequence is similar. The rate is 29%, and 36% of the C end sequence is the same as ubiquitin, and its main role is the encoding human major histocompatibility complex (MHC) I gene seat. Human FAT10 can also be stimulated by the inflammatory factors (IFN- gamma, TNF- a) in various tissues except in mature dendritic cells, B cells, and some immune organs, such as thymus and spleen. Expression, through its C- terminal binding covalence to modify the target substrate. In the previous work, we identified the proliferation cell nuclear antigen (PCNA) as a covalent substrate for FAT10. Valence modified ENO1, finding a new regulation that affects the repair of DNA damage and the development of cancer, provides new ideas for the study of senescence physiology. Methods: (1) the cells were treated with UV/IR and VP-16, and the effects of DNA damage on the expression of FAT10 protein and gene level were detected by Western-blotting and Q RT-PCR. Whether FAT10 protein covalently modifies PCNA when DNA is damaged; through ubiquitination degradation experiment and Si RNA knocking low FAT10 test, PCNA can be degraded by 26S proteasome, and the subcellular localization of FAT10 and PCNA interaction between FAT10 and PCNA is investigated by cell immunofluorescence test and PCNA is considered (2) construct two eukaryotic expression plasmids of P Flag-CMV-eno1 and P CMV-Myc-fat10, and transfer these two plasmids into HEK293 cells. Through immunoprecipitation method, explore whether FAT10 covalently modifies ENO1. results in cells: first, we use UV/IR and VP-16 to treat cells and Western-blotting, Western-blotting DNA damage cells were detected by Q RT-PCR and the results showed that DNA damage could induce the up regulation of FAT10 expression from gene and protein level. The expression of FAT10 increased with the severity of DNA damage. In this experiment, when UV radiation dose reached 20J/m2, IR radiation dose reached 20Gy or VP-16 doses reached 200 mu. He La cells treated with UV irradiation intensity of 10J/m2 and 20J/m2 were detected by immunoprecipitation experiment. It was found that FAT10 could covalently modify PCNA.. We observed that when IR irradiated and the cells were treated with VP-16, FAT10 can also covalently modify the PCNA. through PCNA degradation experiment. The expression level of the induced FAT10 protein increased gradually, but the expression level of PCNA decreased gradually; the expression level of PCNA was unchanged in the cells treated with the 26S proteasome inhibitor - MG132 treatment. Also, the degradation of PCNA and FAT10 could also be inhibited by MG132 in the cells treated with VP-16. FAT10 Si RNA was transfected to the region. In the after cells, the endogenous FAT10 was knocked down and the degradation of PCNA disappeared. These results showed that when DNA was damaged, FAT10 could be degraded by 26S proteasome. Secondly, we isolated the cytoplasm and nucleus of the samples and detected the expression of PCNA and FAT10 by Western-blotting, and found that most PCNA were fine. Accumulation in the nucleus, when the 26S proteasome inhibitor is added, the accumulation of PCNA in the cytoplasm increases significantly, and FAT10 is accumulated in the nucleus and cytoplasm. These data indicate that in the cells after VP-16 treatment, FAT10 may be degraded by the 26S proteasome in the cytoplasm. Finally, we use laser confocal microscopy. The nucleus aggregation point (nuclear foci) was detected by the microscope, and both FAT10 and PCNA were found to be located at the damage site. When VP-16 was used to treat the cells, the number of nuclear aggregation points increased. At the same time, the expression level of PCNA in the cytoplasm decreased. When MG132 was added, the number of nuclear aggregation points decreased and the level of PCNA expression returned to normal. The experimental results showed that in DNA damage repair, FAT10 was degraded by cytoplasmic mediated PCNA degradation and increased the number of nuclear aggregation points. In addition, we transformed the two plasmids into HEK293 cells by constructing two expression plasmids of P Flag-CMV-eno1 and P CMV-Myc-fat10, and found that FAT10 can be shared in the cells by the method of co immunoprecipitation. ENO1. conclusion: we speculate that DNA damage can induce up regulation of FAT10 expression and can covalently modify PCNA protein to be degraded by 26S proteasome in cytoplasm, thus increasing the number of nuclear damage sites. Furthermore, we also found that ubiquitin protein FAT10 can covalently modify ENO1. in cells. When cells are cancerous, FAT10 may influence and regulate the occurrence and metastasis of tumors through covalent modification of PCNA and ENO1.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q75
，
本文编号：2122621
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