陳?ài)诖ǎ溷?,季守?/p>
軍事醫(yī)學(xué)科學(xué)院 野戰(zhàn)輸血研究所組織工程研究室,北京 100850
類(lèi)泛素蛋白FAT10對(duì)人腎上皮細(xì)胞α-烯醇酶共價(jià)修飾的初步研究
陳?ài)诖?,冷泠,季守?/p>
軍事醫(yī)學(xué)科學(xué)院 野戰(zhàn)輸血研究所組織工程研究室,北京 100850
目的:構(gòu)建帶Flag標(biāo)簽的α-烯醇酶(ENO1)基因以及帶Myc標(biāo)簽的人白細(xì)胞抗原F相關(guān)轉(zhuǎn)錄物10(FAT10)基因的真核表達(dá)質(zhì)粒載體,鑒定ENO1和FAT10在人腎上皮細(xì)胞株HEK293中的表達(dá),檢測(cè)類(lèi)泛素蛋白FAT10是否共價(jià)修飾ENO1。方法:以人宮頸癌細(xì)胞系HeLa的cDNA為模板進(jìn)行PCR,得到eno1目的片段,與分別經(jīng)EcoRⅤ和SalⅠ酶切的pFlag-CMV載體連接得到重組質(zhì)粒pFlag-CMV-eno1并進(jìn)行鑒定測(cè)序;以人腦組織cDNA文庫(kù)為模板進(jìn)行PCR,得到fat10目的片段,與分別經(jīng)EcoRⅠ和XhoⅠ酶切的pCMV-Myc載體連接得到重組質(zhì)粒pCMV-Myc-fat10并進(jìn)行鑒定并測(cè)序。將重組質(zhì)粒用脂質(zhì)體法轉(zhuǎn)染HEK293細(xì)胞,用Western印跡檢測(cè)ENO1和FAT10蛋白的表達(dá)。將重組質(zhì)粒共轉(zhuǎn)染HEK293細(xì)胞,用免疫共沉淀檢測(cè)FAT10對(duì)ENO1的修飾情況。結(jié)果:重組克隆載體內(nèi)的eno1和fat10序列與GenBank報(bào)告的序列完全一致;轉(zhuǎn)染HEK293細(xì)胞后,ENO1和FAT10蛋白過(guò)表達(dá);FAT10能夠共價(jià)修飾ENO1。結(jié)論:類(lèi)泛素蛋白FAT10共價(jià)修飾ENO1,為腫瘤的發(fā)生、發(fā)展及遷移的研究提供了新的研究思路。
α-烯醇酶(ENO1);人白細(xì)胞抗原F相關(guān)轉(zhuǎn)錄物10(FAT10);真核表達(dá);共價(jià)修飾
α-烯醇酶(α-enolase,ENO1)是由2個(gè)含433個(gè)氨基酸殘基、相對(duì)分子質(zhì)量約47 000的亞單位構(gòu)成的二聚體[1],作為糖酵解酶在細(xì)胞質(zhì)中起催化作用,催化2-磷酸-D-甘油酸在糖酵解途徑中脫水成磷酸烯醇丙酮酸[2]。在細(xì)胞膜上,ENO1可作為影響細(xì)胞遷移的纖溶酶原受體[3-4]。有文獻(xiàn)報(bào)道,α-烯醇酶在癌癥轉(zhuǎn)移中具有顯著作用[2],在膜上表達(dá)的α-烯醇酶能促進(jìn)ECM降解和癌細(xì)胞的侵襲[3,5],靶向敲除在膜表達(dá)的α-烯醇酶能夠有效抑制腫瘤的轉(zhuǎn)移[6]。
人白細(xì)胞抗原F相關(guān)轉(zhuǎn)錄物10(human leuko?cyte antigen F-associated transcript 10,F(xiàn)AT10),是相對(duì)分子質(zhì)量為18 000的類(lèi)泛素蛋白,可介導(dǎo)底物被26S蛋白酶體降解[7-8]。多項(xiàng)研究發(fā)現(xiàn)FAT10在各種癌癥如胃腸癌、肝癌、胰腺導(dǎo)管腺癌和人神經(jīng)膠質(zhì)瘤中高表達(dá)[9],F(xiàn)AT10表達(dá)的變化可誘導(dǎo)與腫瘤形成相關(guān)的異常細(xì)胞生長(zhǎng)[9-10]。這些都表明FAT10可能在癌癥的發(fā)生發(fā)展中發(fā)揮重要作用[7,11-12]。最近的研究表明,F(xiàn)AT10基因敲除后的小鼠表現(xiàn)出壽命延長(zhǎng)以及患肥胖病幾率減少等現(xiàn)象,提示FAT10在影響衰老和慢性疾病的免疫代謝調(diào)節(jié)中發(fā)揮作用[13-14],但具體機(jī)制不甚清楚。本研究旨在探討FAT10是否能夠共價(jià)修飾ENO1,從而參與腫瘤的發(fā)生發(fā)展過(guò)程。
大腸桿菌DH5α感受態(tài)菌株購(gòu)于天根生物技術(shù)公司;限制性?xún)?nèi)切酶EcoRⅤ和SalⅠ、T4DNA連接酶購(gòu)于NEB公司;ExTaq酶購(gòu)于TaKaRa公司;PCR產(chǎn)物回收試劑盒購(gòu)于威格拉斯公司;質(zhì)粒提取試劑盒購(gòu)于OmegaBiotek公司;Myc標(biāo)簽抗體和Flag標(biāo)簽抗體購(gòu)于CST公司。
根據(jù)GenBank數(shù)據(jù)庫(kù)提供的ENO1氨基酸序列,分別在上、下游引物中引入EcoRⅤ和SalⅠ酶切位點(diǎn)。上游引物為5'-CGGATATCTTCTTCAGG TCTGGGAAGTATG-3',下游引物為5'-CCGGTCG ACGGTTGCAGGACTTCTCGTTC-3'。以人宮頸癌細(xì)胞系HeLa的cDNA為模板進(jìn)行PCR(94℃預(yù)變性3 min;94℃ 30 s,65℃ 45 s,72℃ 1.5 min,30個(gè)循環(huán);72℃延伸10 min)。PCR擴(kuò)增產(chǎn)物行1%瓊脂糖凝膠電泳,紫外燈下觀察結(jié)果并拍照。用試劑盒回收擴(kuò)增的目的片段,并與經(jīng)EcoRⅤ和SalⅠ雙酶切的pFlag-CMV載體連接,轉(zhuǎn)化大腸桿菌DH5α菌株,陽(yáng)性克隆經(jīng)測(cè)序正確后提取質(zhì)粒。
根據(jù)人類(lèi)蛋白質(zhì)參考數(shù)據(jù)庫(kù)(human protein reference database,HPRD)提供的FAT10氨基酸序列,分別在上、下游引物中引入EcoRⅠ和XhoⅠ酶切位點(diǎn)。上游引物為5'-CTCTCGAGTCAATGGCT CCCAATGCTTCC-3',下游引物為5'-GGCGAATT CTCTCACCCTCCAATACAATAA-3'。以人腦組織cDNA文庫(kù)為模板進(jìn)行PCR(94℃預(yù)變性3 min;94℃ 30 s,65℃ 45 s,72℃ 1.5 min,35 個(gè)循環(huán);72℃延伸10 min)。PCR 擴(kuò)增產(chǎn)物行1%瓊脂糖凝膠電泳,紫外燈下觀察結(jié)果并拍照。用試劑盒回收擴(kuò)增的目的片段,并與經(jīng)EcoRⅠ和XhoⅠ雙酶切的pCMV-Myc載體連接,轉(zhuǎn)化大腸桿菌DH5α菌株,陽(yáng)性克隆經(jīng)測(cè)序正確后提取質(zhì)粒。
將培養(yǎng)的HEK293細(xì)胞接種到細(xì)胞培養(yǎng)皿中,加入含10%胎牛血清和雙抗的新鮮DMEM培養(yǎng)基培養(yǎng)24 h,細(xì)胞融合度為70%時(shí)更換新鮮無(wú)血清無(wú)雙抗培養(yǎng)基,將質(zhì)粒與脂質(zhì)體按體積比1∶3混合,每個(gè)培養(yǎng)皿中轉(zhuǎn)入質(zhì)粒3 μg,6 h后換上新鮮的含10%胎牛血清和雙抗的DMEM培養(yǎng)基,48 h后加入細(xì)胞裂解液,收集細(xì)胞,提取蛋白,SDS-PAGE分離,隨后進(jìn)行免疫印跡分析。
重組載體共轉(zhuǎn)染48 h后,將HEK293細(xì)胞用PBS洗滌3次,加入細(xì)胞裂解液裂解細(xì)胞,裂解產(chǎn)物在4℃下與目的抗體孵育4 h后離心(4℃,2500 r/min,5 min);將蛋白A/G瓊脂糖加入裂解產(chǎn)物,于4℃進(jìn)一步孵育4 h;收集和洗滌結(jié)合的蛋白質(zhì),將樣品煮沸10 min,SDS-PAGE分離,隨后進(jìn)行免疫印跡分析。
以人宮頸癌細(xì)胞系HeLa的cDNA為模板進(jìn)行PCR,擴(kuò)增產(chǎn)物的1%瓊脂糖凝膠電泳結(jié)果見(jiàn)圖1,擴(kuò)增片段約為1305 bp,與GenBank所示的分子質(zhì)量完全吻合。用試劑盒回收擴(kuò)增的目的片段,并與經(jīng)EcoRⅤ和SalⅠ雙酶切的pFlag-CMV載體連接。對(duì)重組質(zhì)粒pFlag-CMV-eno1進(jìn)行序列檢測(cè),其部分測(cè)序結(jié)果見(jiàn)圖2。
圖1 eno1片段PCR產(chǎn)物瓊脂糖電泳圖
圖2 重組質(zhì)粒pFlag-CMV-eno1部分測(cè)序圖
以人腦組織cDNA庫(kù)為模板進(jìn)行PCR,擴(kuò)增產(chǎn)物的1%瓊脂糖凝膠電泳結(jié)果見(jiàn)圖3,擴(kuò)增片段約為971 bp,與GenBank所示分子質(zhì)量完全吻合。用試劑盒回收擴(kuò)增的目的片段,并與經(jīng)EcoRⅠ和XhoⅠ雙酶切的pFlag-CMV載體連接。對(duì)重組質(zhì)粒pCMV-Myc-fat10進(jìn)行序列檢測(cè),其部分測(cè)序結(jié)果見(jiàn)圖4。
圖3 fat10片段PCR產(chǎn)物瓊脂糖電泳圖
圖4 重組質(zhì)粒pCMV-Myc-fat10部分測(cè)序圖
將構(gòu)建的質(zhì)粒pFlag-CMV-eno1和pCMVMyc-fat10用脂質(zhì)體法分別轉(zhuǎn)入HEK293細(xì)胞,48 h后加入細(xì)胞裂解液,收集細(xì)胞,提取蛋白。Western印跡結(jié)果顯示,質(zhì)粒組pFlag-CMV-eno1在相對(duì)分子質(zhì)量約47 000處有單一條帶(圖5),pCMV-Myc-fat10在相對(duì)分子質(zhì)量約17 000處有單一條帶(圖6)。
圖5 Western印跡檢測(cè)重組質(zhì)粒pFlag-CMV-eno1在293細(xì)胞中的表達(dá)
圖6 Western印跡檢測(cè)重組質(zhì)粒pCMV-Myc-fat10在293細(xì)胞中的表達(dá)
將構(gòu)建的質(zhì)粒pFlag-CMV-eno1和pCMVMyc-fat10用脂質(zhì)體法共轉(zhuǎn)入HEK293細(xì)胞,48 h后加入細(xì)胞裂解液,裂解產(chǎn)物在4℃與Flag抗體孵育4 h后2500 r/min離心5 min,將蛋白A/G瓊脂糖加入裂解產(chǎn)物,4℃進(jìn)一步孵育4 h,收集和洗滌結(jié)合的蛋白質(zhì),將樣品煮沸10 min,行SDSPAGE分離,隨后進(jìn)行免疫印跡分析。結(jié)果見(jiàn)圖7,在相對(duì)分子質(zhì)量約65 000處有一條帶,而ENO1-FAT10共軛體大小也為65 000。提示FAT10能夠共價(jià)修飾ENO1。
圖7 免疫共沉淀檢測(cè)蛋白FAT10共價(jià)修飾ENO1
ENO1在癌癥轉(zhuǎn)移中有顯著作用。ENO1從細(xì)胞質(zhì)轉(zhuǎn)移到細(xì)胞表面結(jié)合纖溶酶原(PLG)以增強(qiáng)細(xì)胞周質(zhì)纖維蛋白溶酶的產(chǎn)生和細(xì)胞運(yùn)動(dòng)性。在肺癌、結(jié)腸癌和急性髓細(xì)胞樣白血病中,ENO1表達(dá)上調(diào)。FAT10能介導(dǎo)靶蛋白的降解及細(xì)胞凋亡,其表達(dá)能夠被p53負(fù)調(diào)控,多項(xiàng)研究表明FAT10在各種癌癥中高表達(dá),并可能在癌癥中發(fā)揮重要作用。我們構(gòu)建了pFlag-CMV-eno1和pCMV-Myc-fat10表達(dá)質(zhì)粒,并將這2種質(zhì)粒共轉(zhuǎn)入HEK293細(xì)胞,通過(guò)免疫沉淀發(fā)現(xiàn)在細(xì)胞中FAT10能夠共價(jià)修飾ENO1。這提示我們FAT10可能通過(guò)共價(jià)修飾ENO1影響和調(diào)控腫瘤的發(fā)生和轉(zhuǎn)移。目前,尚有許多問(wèn)題待闡明,比如,類(lèi)泛素蛋白FAT10共價(jià)修飾ENO1如何調(diào)控腫瘤的遷徙能力,其中是否依賴(lài)蛋白酶體水解系統(tǒng);FAT10和ENO1共同參與的信號(hào)通路有哪些等等。以上具體的作用機(jī)制還不清楚,值得進(jìn)行深入探究,為癌癥的發(fā)生與發(fā)展提供新的研究方向。
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A Preliminary Study on the Covalent Modification of α-Enolase in Human Renal Epithelial Cells by Ubiquitin-Like Protein FAT10
CHEN Zhen-Chuan,LENG Ling,JI Shou-Ping*
Tissue Engineering Lab,Beijing Institute of Transfusion Medicine,Beijing 100850,China
Objective:Through co-express two eukaryotic plasmids carrying Flag-tagged α-enolase(ENO1) gene and Myc-tagged F-related transporter 10(FAT10) gene to detect modification of ENO1 by FAT10 in HEK293 cell lines.Methods:Theeno1target fragment was obtained by PCR from the cDNA of HeLa cells and inserted into pFlag-CMV vector,and the constructed plasmid pFlag-CMV-eno1 was identified by sequencing.Thefat10frag?ment was obtained by PCR from human brain tissue cDNA library as template,and cloned into pCMV-Myc vec?tor,and the constucted plasmid pCMV-Myc-fat10 was identified by sequencing.The two recombinant plasmids were co-transfected into HEK293 cell lines by liposome method,and Western blotting was used to detect the ex?pression of ENO1 and FAT10 protein,and the co-immunoprecipitation method was used to detect whether ENO1 and FAT10 proteins could interact in cells.Results:Theeno1sequence andfat10sequence in the recombinant cloning vector were identical with those reported data by GenBank.ENO1 and FAT10 proteins significantly ex?pressed in HEK293 cell lines,and ENO1 was covalently modified by Ubiquitin protein FAT10.Conclusion:ENO1 is covalently modified by Ubiquitin protein FAT10 in HEK293 cell lines,which is a clue to study the de?velopment and immigration of tumor cells.
α-enolase(ENO1);human leukocyte antigen F-associated transcript 10(FAT10);eukaryotic expres?sion;covalent modification
Q78
A
1009-0002(2017)05-0614-04
10.3969/j.issn.1009-
*Corresponding author,E-mail:jishouping@yahoo.com
2017-03-02
陳?ài)诖ǎ?992- ),男,碩士研究生,(E-mail)14789866055@163.com
季守平,(E-mail)jishouping@yahoo.com