類泛素化蛋白FAT10共價(jià)修飾增殖細(xì)胞核抗原PCNA及其作用的機(jī)制研究
發(fā)布時(shí)間：2018-07-14 18:47
【摘要】：背景:DNA損傷可能來自機(jī)體的內(nèi)在因素的作用,例如活性氧、細(xì)胞新陳代謝的副產(chǎn)物以及DNA在復(fù)制過程中由于拓?fù)洚悩?gòu)酶失活而導(dǎo)致的錯(cuò)配;也可能來自電離輻射(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響。DNA損傷會(huì)導(dǎo)致基因突變和細(xì)胞衰老。細(xì)胞發(fā)生DNA損傷并對(duì)其進(jìn)行精確、高效修復(fù)的機(jī)制被稱為DNA損傷應(yīng)答機(jī)制(DDR),其作用是保護(hù)機(jī)體免受DNA損傷造成的不利影響。增殖細(xì)胞核抗原PCNA(proliferating cell nuclear antigen)在DNA損傷修復(fù)機(jī)制中起著核心作用。PCNA作為一個(gè)穩(wěn)定的“站臺(tái)”,在損傷修復(fù)過程中招募一系列與復(fù)制相關(guān)的蛋白。類泛素蛋白(ubiquitin-like proteins,UBLs),是一類與泛素類似的小蛋白家族。據(jù)報(bào)道,類泛素蛋白含有與泛素同源的結(jié)構(gòu)域,其同源性大約為15%-16%。UBLs可以分為兩個(gè)亞家族:類泛素結(jié)構(gòu)域家族UDP和類泛素家族修飾蛋白家族ΜLM。UDP可以與泛素以及泛素修飾蛋白發(fā)生非共價(jià)結(jié)合。ΜLM具有與泛素單體或二聚體同源的結(jié)構(gòu)域,可以在E1-E2-E3酶聯(lián)體系的催化作用下通過C末端的雙甘氨酸基團(tuán)與底物蛋白質(zhì)共價(jià)結(jié)合,其代表成員有ISG15、FUB1、NEDD8、SΜMO、Urm1、UBL5、Ufm1和FAT10等。據(jù)文獻(xiàn)報(bào)道,當(dāng)復(fù)制過程中的細(xì)胞發(fā)生DNA損傷時(shí),包括泛素化以及類泛素化在內(nèi)的多種翻譯后修飾通過修飾PCNA,對(duì)DNA損傷修復(fù)進(jìn)行調(diào)控。當(dāng)表皮細(xì)胞長時(shí)間暴露在紫外線(UV)輻射中,RAD6-RAD18復(fù)合體能夠介導(dǎo)PCNA第164位賴氨酸殘基發(fā)生高度的單泛素化,從而導(dǎo)致復(fù)制性DNA聚合酶發(fā)生變化。FAT10(HLA-F locus adjacent transcript 10)是一種大小為18k D的蛋白,其N段和C端都含有與泛素相似的結(jié)構(gòu)域,其中N端序列相似率為29%,C端序列則有36%與泛素相同,它的主要作用是編碼人主要組織相容性復(fù)合體(MHC)I類基因座。人源FAT10除了在成熟樹突細(xì)胞,B細(xì)胞以及在一些免疫器官,譬如胸腺和脾中表達(dá),也可以在各種組織中通過促炎癥因子(IFN-γ,TNF-α)刺激而表達(dá),通過其C-末端結(jié)合共價(jià)修飾靶底物發(fā)揮作用。在前期工作中我們通過質(zhì)譜鑒定確定增殖細(xì)胞核抗原(PCNA)是FAT10的共價(jià)修飾底物。目的:通過研究DNA損傷修復(fù)過程中類泛素蛋白FAT10與PCNA共價(jià)修飾作用機(jī)制,以及驗(yàn)證細(xì)胞中FAT10是否共價(jià)修飾ENO1,找到一種新的影響DNA損傷修復(fù)和癌癥發(fā)生發(fā)展的調(diào)控方式,為衰老生理學(xué)研究提供新的思路。方法:(1)用UV/IR和VP-16處理細(xì)胞,通過western-blotting以及q RT-PCR實(shí)驗(yàn)分別檢測(cè)DNA損傷對(duì)FAT10蛋白和基因水平表達(dá)的影響;通過免疫共沉淀實(shí)驗(yàn)檢測(cè)當(dāng)DNA發(fā)生損傷時(shí)FAT10蛋白是否共價(jià)修飾PCNA;通過類泛素化降解實(shí)驗(yàn)以及si RNA敲低FAT10實(shí)驗(yàn)檢測(cè)PCNA能否被FAT10通過26S蛋白酶體降解;通過細(xì)胞免疫熒光實(shí)驗(yàn)探究FAT10和PCNA之間相互作用的亞細(xì)胞定位情況以及PCNA被FATylation對(duì)細(xì)胞形成細(xì)胞核聚集點(diǎn)的影響。(2)構(gòu)建p Flag-CMV-eno1和p CMV-Myc-fat10兩種真核表達(dá)質(zhì)粒,并將這兩種質(zhì)粒共轉(zhuǎn)入HEK293細(xì)胞內(nèi),通過免疫共沉淀的方法,探究FAT10是否在細(xì)胞中共價(jià)修飾ENO1。結(jié)果:首先,我們用UV/IR和VP-16處理細(xì)胞,western-blotting以及q RT-PCR實(shí)驗(yàn)檢測(cè)發(fā)生DNA損傷的細(xì)胞,結(jié)果顯示,DNA損傷能從基因和蛋白水平誘導(dǎo)FAT10表達(dá)的上調(diào),FAT10表達(dá)量隨著DNA損傷程度加重而增加。在本次實(shí)驗(yàn)中,當(dāng)UV輻射劑量達(dá)到20J/m2、IR輻射劑量達(dá)到20Gy或VP-16劑量達(dá)到200μM時(shí),FAT10表達(dá)量最高。通過免疫共沉淀實(shí)驗(yàn)檢測(cè)以10J/m2和20J/m2的UV輻照強(qiáng)度處理后的He La細(xì)胞,發(fā)現(xiàn)FAT10能夠共價(jià)修飾PCNA。同樣,我們觀察到用IR輻照以及用VP-16處理細(xì)胞時(shí),FAT10也都能夠共價(jià)修飾PCNA。通過PCNA降解實(shí)驗(yàn),我們發(fā)現(xiàn)隨著細(xì)胞因子TNF-α和IFN-γ濃度升高,誘導(dǎo)產(chǎn)生的FAT10蛋白表達(dá)量逐漸增加,而PCNA的表達(dá)水平卻逐漸降低;在加入26S蛋白酶體抑制劑——MG132處理的細(xì)胞中,PCNA的表達(dá)水平不變。同樣,在VP-16處理后的細(xì)胞中,PCNA和FAT10的降解也可以被MG132抑制。將FAT10 si RNA轉(zhuǎn)染到被VP-16處理后的細(xì)胞中,敲低內(nèi)源性FAT10,發(fā)現(xiàn)PCNA的降解現(xiàn)象明顯消失,這些結(jié)果表明當(dāng)DNA損傷時(shí),FAT10可以通過26S蛋白酶體介導(dǎo)PCNA降解。其次,我們分離出樣品的細(xì)胞質(zhì)和細(xì)胞核,并通過western-blotting檢測(cè)PCNA和FAT10的表達(dá)情況,發(fā)現(xiàn)大部分PCNA在細(xì)胞核中積累,當(dāng)添加26S蛋白酶體抑制劑后,細(xì)胞質(zhì)中PCNA的積累顯著增加,同時(shí),FAT10在細(xì)胞核和細(xì)胞質(zhì)中都顯著積累。這些數(shù)據(jù)表明在VP-16處理后的細(xì)胞中,FAT10可能在細(xì)胞質(zhì)中通過26S蛋白酶體介導(dǎo)PCNA降解。最后,我們通過激光共聚焦顯微鏡檢測(cè)細(xì)胞核聚集點(diǎn)(nuclear foci),觀察到FAT10和PCNA共定位在損傷位點(diǎn),并且當(dāng)使用VP-16處理細(xì)胞后,細(xì)胞核聚集點(diǎn)的數(shù)量增加,同時(shí),細(xì)胞質(zhì)中PCNA的表達(dá)水平降低。當(dāng)添加MG132后,細(xì)胞核聚集點(diǎn)的數(shù)量減少,并且PCNA的表達(dá)水平恢復(fù)正常。這些實(shí)驗(yàn)結(jié)果表明在DNA損傷修復(fù)中,FAT10在胞質(zhì)中介導(dǎo)PCNA降解從而影響細(xì)胞核聚集點(diǎn)數(shù)量增多。另外,我們通過構(gòu)建p Flag-CMV-eno1和p CMV-Myc-fat10兩種表達(dá)質(zhì)粒,并將這兩種質(zhì)粒共轉(zhuǎn)入HEK293細(xì)胞內(nèi),利用免疫共沉淀的方法,發(fā)現(xiàn)在細(xì)胞中FAT10能共價(jià)修飾ENO1。結(jié)論:我們推測(cè)細(xì)胞發(fā)生DNA損傷時(shí)能夠誘導(dǎo)FAT10表達(dá)上調(diào)并能夠共價(jià)修飾PCNA蛋白使之在細(xì)胞質(zhì)中被26S蛋白酶體降解,從而使細(xì)胞核損傷位點(diǎn)數(shù)量增加。此外,我們還發(fā)現(xiàn),類泛素蛋白FAT10在細(xì)胞中能共價(jià)修飾ENO1。以上結(jié)果提示,細(xì)胞發(fā)生癌變時(shí),FAT10可能通過共價(jià)修飾PCNA和ENO1影響和調(diào)控腫瘤的發(fā)生和轉(zhuǎn)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.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q75
，
本文編號(hào)：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.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q75
，
本文編號(hào)：2122621
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