利用泛素结合结构域UBAN探针鉴定线性泛素化底物
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摘要
目的根据已知线性泛素化底物NF-κB必需调节蛋白(NEMO)的泛素结合结构域UBAN构建蛋白探针,在目的细胞中富集线性泛素化底物并应用质谱技术进行鉴定,探索线性泛素化在不同生物学事件中发挥调控功能的分子机制。方法 (1)首先通过大肠杆菌表达系统表达纯化特异性识别线性泛素链的蛋白UBAN,通过谷胱甘肽S-转移酶(GST)标签将其与GST琼脂糖微珠连接制成UBAN探针。随后在293T细胞中共转染阳性底物NEMO和线性泛素化链组装复合体(LUBAC)使之过表达,利用UBAN探针免疫共沉淀富集NEMO上的线性泛素链,并通过Western蛋白印迹法验证UBAN探针与线性泛素化链的结合能力,从而确定体系是否建立成功。(2)在293T细胞裂解液中免疫共沉淀富集线性泛素化底物并对其进行质谱鉴定和生物信息学分析,得到潜在线性泛素化底物。在293T细胞中共转染潜在线性泛素化底物极光激酶A(Aurora-A)和LUBAC,用Western蛋白印迹法鉴定。(3)用siRNA干扰Hela细胞LUBAC,并使细胞周期同步化,用Western蛋白印迹法观察线性泛素化对潜在底物Aurora-A生物学功能的调控。结果 (1) Western蛋白印迹法结果表明,UBAN探针能够结合NEMO上的线性泛素链,从而确定体系建立成功。(2)质谱鉴定结果显示,免疫共沉淀法富集到包括极光激酶A(Aurora-A)在内的403个潜在线性泛素化底物;在293T细胞中共转染Aurora-A和LUBAC后,用Western蛋白印迹法测定结果发现,Aurora-A具有线性泛素链特征性条带,具有与线性泛素化链结合的能力。(3) Western蛋白印迹实验结果显示,与对照组相比,干涉LUBAC后Aurora-A苏氨酸288位磷酸化水平上调,表明其被激活。结论运用泛素结合结构域UBAN探针可以实现线性泛素化底物的有效捕捉与富集,在293T细胞中鉴定到底物Aurora-A的自激活可能受线性泛素化调控。本研究建立的线性泛素化底物鉴定策略为进一步探索蛋白质线性泛素化修饰的更多生物学功能提供了新的思路和方法。
OBJECTIVE In this research, the ubiquitin binding in ABIN and NEMO proteins(UBAN)domain of NF-κB essential modulator(NEMO) was used as a protein probe to screen potent linear ubiquitination substrates. Adopting the proteomics strategy, the putative substrates could be enriched and identified. This work will greatly expand our knowledge on the molecular mechanism of linear ubiquitination regulation in various biological events. METHODS(1) The glutathione S-transferase(GST)-UBAN protein was overexpressed by Escherichia Coli and connected with glutathione agarose beads by GST tag to be used as the UBAN probe. Then, the function of the probe was verified in linear ubiquitination chain binding ability test by immunoprecipitation and Western blotting in 293 T cells.(2) Using the UBAN probe,linear ubiquitination substrates were enriched and identified by mass spectrometry in 293 T cell lysate and analyzed by bioinformatics. Among them, the potential linear ubiquitination substrates Aurora kinase A(Aurora-A) and linear ubiquitin chain assembly complex(LUBAC) were co-transfected in 293 T cells and the linear ubiquitination chain binding ability of Aurora-A was identified by Western blot.(3) LUBAC was interfered in with siR NA and then synchronized in HeL a cells. The regulation of linear ubiquitination on the biological function of potential substrate Aurora A was observe by Western blotting. RESULTS(1) Western blotting results suggested that the UBAN probe could bind to the linear ubiquitin chain on NEMO, which confirmed the success of the system.(2) Including Aurora-A, a total of 403 proteins were identified as potential substrates of linear ubiquitination by mass spectrometry. The ability to bind to the linear ubiquitination chain was confirmed by immunoprecipitation between Aurora-A and the linear ubiquitination chains.(3) Western blotting results showed that compared with the control group, Aurora-A phosphorylation level was up-regulated, which meant the occurrence of self-activation after knockdown of LUBAC. CONCLUSION The probe constructed with the UBAN domain can efficiently capture and enrich linear ubiquitination substrates. And the self-activation of Aurora-A may be regulated by linear ubiquitination. Significantly, the strategy of identifying the linear ubiquitination substrates established in this paper provides a novel idea and method for further exploration of biological functions of linear ubiquitination.
OBJECTIVE In this research, the ubiquitin binding in ABIN and NEMO proteins(UBAN)domain of NF-κB essential modulator(NEMO) was used as a protein probe to screen potent linear ubiquitination substrates. Adopting the proteomics strategy, the putative substrates could be enriched and identified. This work will greatly expand our knowledge on the molecular mechanism of linear ubiquitination regulation in various biological events. METHODS(1) The glutathione S-transferase(GST)-UBAN protein was overexpressed by Escherichia Coli and connected with glutathione agarose beads by GST tag to be used as the UBAN probe. Then, the function of the probe was verified in linear ubiquitination chain binding ability test by immunoprecipitation and Western blotting in 293 T cells.(2) Using the UBAN probe,linear ubiquitination substrates were enriched and identified by mass spectrometry in 293 T cell lysate and analyzed by bioinformatics. Among them, the potential linear ubiquitination substrates Aurora kinase A(Aurora-A) and linear ubiquitin chain assembly complex(LUBAC) were co-transfected in 293 T cells and the linear ubiquitination chain binding ability of Aurora-A was identified by Western blot.(3) LUBAC was interfered in with siR NA and then synchronized in HeL a cells. The regulation of linear ubiquitination on the biological function of potential substrate Aurora A was observe by Western blotting. RESULTS(1) Western blotting results suggested that the UBAN probe could bind to the linear ubiquitin chain on NEMO, which confirmed the success of the system.(2) Including Aurora-A, a total of 403 proteins were identified as potential substrates of linear ubiquitination by mass spectrometry. The ability to bind to the linear ubiquitination chain was confirmed by immunoprecipitation between Aurora-A and the linear ubiquitination chains.(3) Western blotting results showed that compared with the control group, Aurora-A phosphorylation level was up-regulated, which meant the occurrence of self-activation after knockdown of LUBAC. CONCLUSION The probe constructed with the UBAN domain can efficiently capture and enrich linear ubiquitination substrates. And the self-activation of Aurora-A may be regulated by linear ubiquitination. Significantly, the strategy of identifying the linear ubiquitination substrates established in this paper provides a novel idea and method for further exploration of biological functions of linear ubiquitination.
引文
[1]Chen ZJ,Sun LJ.Nonproteolytic functions of ubiquitin in cell signaling[J].Mol Cell,2009,33(3):275-286.
[2]Jackson SP,Durocher D.Regulation of DNA damage responses by ubiquitin and SUMO[J].Mol Cell,2013,49(5):795-807.
[3]Kwon YT,Ciechanover A.The ubiquitin code in the ubiquitin-proteasome system and autophagy[J].Trends Biochem Sci,2017,42(11):873-886.
[4]Kirisako T,Kamei K,Murata S,Kato M,Fukumoto H,Kanie M,et al.A ubiquitin ligase complex assembles linear polyubiquitin chains[J].EMBO J,2006,25(20):4877-4887.
[5]Tokunaga F,Sakata S,Saeki Y,Satomi Y,Kirisako T,Kamei K,et al.Involvement of linear polyubiquitylation of NEMO in NF-kappaB activation[J].Nat Cell Biol,2009,11(2):123-132.
[6]Inn KS,Gack MU,Tokunaga F,Shi M,Wong LY,Iwai K,et al.Linear ubiquitin assembly complex negatively regulates RIG-I-and TRIM25-mediated typeⅠinterferon induction[J].Mol Cell,2011,41(3):354-365.
[7]Fiil BK,Damgaard RB,Wagner SA,Keusekotten K,Fritsch M,Bekker-Jensen S,et al.OTULIN restricts Met1-linked ubiquitination to control innate immune signaling[J].Mol Cell,2013,50(6):818-830.
[8]Rodgers MA,Bowman JW,Fujita H,Orazio N,Shi M,Liang Q,et al.The linear ubiquitin assembly complex(LUBAC)is essential for NLRP3inflammasome activation[J].J Exp Med,2014,211(7):1333-1347.
[9]Goto E,Tokunaga F.Decreased linear ubiquitination of NEMO and FADD on apoptosis with caspasemediated cleavage of HOIP[J].Biochem Biophys Res Commun,2017,485(1):152-159.
[10]de Almagro MC,Goncharov T,Newton K,Vucic D.Cellular IAP proteins and LUBAC differential y regulate necrosome-associated RIP1 ubiquitination[J/OL].Cell Death Dis,2015,6:e1800(2015-06-25).https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4669837/
[11]Tang Y,Joo D,Liu G,Tu H,You J,Jin J,et al.Linear ubiquitination of cFLIP induced by LUBACcontributes to TNFα-induced apoptosis[J].J Biol Chem,2018,293(52):20062-20072.
[12]Newton K,Matsumoto ML,Wertz IE,Kirkpatrick DS,Lill JR,Tan J,et al.Ubiquitin chain editing revealed by polyubiquitin linkage-specific antibodies[J].Cel,2008,134(4):668-678.
[13]Franco M,Seyfried NT,Brand AH,Peng J,Mayor U.A novel strategy to isolate ubiquitin conjugates reveals wide role for ubiquitination during neural development[J/OL].Mol Cell Proteomics,2011,10(5):M110.002188(2011-09-22).https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3098581/
[14]Shi Y,Xu P,Qin J.Ubiquitinated proteome:ready for global?[J/OL].Mol Cell Proteomics,2011,10(5):R110.006882(2011-02-21).https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3098603/
[15]Kensche T,Uejima T,Bloor S,Komander D,Randow F,Wakatsuki S.et al.Specific recognition of linear ubiquitin chains by NEMO is important for NF-kappa B activation[J].Cel,2009,136(6):1098-1109.
[16]Wu CJ,Conze DB,Li T,Srinivasula SM,Ashwel JD.Sensing of Lys 63-linked polyubiquitination by NEMOis a key event in NF-kappaB activation[corrected][J].Nat Cell Biol,2006,8(4):398-406.
[17]Fennell LM,Rahighi S,Ikeda F.Linear ubiquitin chain-binding domains[J].FEBS J,2018,285(15):2746-2761.
[18]Bloor S,Ryzhakov G,Wagner S,Butler PJ,Smith DL,Krumbach R,et al.Signal processing by its coil zipper domain activates IKK gamma[J].Proc Natl Acad Sci USA,2008,105(4):1279-1284.
[19]Lo YC,Lin SC,Rospigliosi CC,Conze DB,Wu CJ,Ashwell JD,et al.Structural basis for recognition of diubiquitins by NEMO[J].Mol Cell,2009,33(5):602-615.
[20]Park JW,Chae YC,Kim JY,Oh H,Seo SB.Methylation of Aurora kinase A by MMSET reduces p53stability and regulates cell proliferation and apoptosis[J].Oncogene,2018,37(48):6212-6224.
[2]Jackson SP,Durocher D.Regulation of DNA damage responses by ubiquitin and SUMO[J].Mol Cell,2013,49(5):795-807.
[3]Kwon YT,Ciechanover A.The ubiquitin code in the ubiquitin-proteasome system and autophagy[J].Trends Biochem Sci,2017,42(11):873-886.
[4]Kirisako T,Kamei K,Murata S,Kato M,Fukumoto H,Kanie M,et al.A ubiquitin ligase complex assembles linear polyubiquitin chains[J].EMBO J,2006,25(20):4877-4887.
[5]Tokunaga F,Sakata S,Saeki Y,Satomi Y,Kirisako T,Kamei K,et al.Involvement of linear polyubiquitylation of NEMO in NF-kappaB activation[J].Nat Cell Biol,2009,11(2):123-132.
[6]Inn KS,Gack MU,Tokunaga F,Shi M,Wong LY,Iwai K,et al.Linear ubiquitin assembly complex negatively regulates RIG-I-and TRIM25-mediated typeⅠinterferon induction[J].Mol Cell,2011,41(3):354-365.
[7]Fiil BK,Damgaard RB,Wagner SA,Keusekotten K,Fritsch M,Bekker-Jensen S,et al.OTULIN restricts Met1-linked ubiquitination to control innate immune signaling[J].Mol Cell,2013,50(6):818-830.
[8]Rodgers MA,Bowman JW,Fujita H,Orazio N,Shi M,Liang Q,et al.The linear ubiquitin assembly complex(LUBAC)is essential for NLRP3inflammasome activation[J].J Exp Med,2014,211(7):1333-1347.
[9]Goto E,Tokunaga F.Decreased linear ubiquitination of NEMO and FADD on apoptosis with caspasemediated cleavage of HOIP[J].Biochem Biophys Res Commun,2017,485(1):152-159.
[10]de Almagro MC,Goncharov T,Newton K,Vucic D.Cellular IAP proteins and LUBAC differential y regulate necrosome-associated RIP1 ubiquitination[J/OL].Cell Death Dis,2015,6:e1800(2015-06-25).https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4669837/
[11]Tang Y,Joo D,Liu G,Tu H,You J,Jin J,et al.Linear ubiquitination of cFLIP induced by LUBACcontributes to TNFα-induced apoptosis[J].J Biol Chem,2018,293(52):20062-20072.
[12]Newton K,Matsumoto ML,Wertz IE,Kirkpatrick DS,Lill JR,Tan J,et al.Ubiquitin chain editing revealed by polyubiquitin linkage-specific antibodies[J].Cel,2008,134(4):668-678.
[13]Franco M,Seyfried NT,Brand AH,Peng J,Mayor U.A novel strategy to isolate ubiquitin conjugates reveals wide role for ubiquitination during neural development[J/OL].Mol Cell Proteomics,2011,10(5):M110.002188(2011-09-22).https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3098581/
[14]Shi Y,Xu P,Qin J.Ubiquitinated proteome:ready for global?[J/OL].Mol Cell Proteomics,2011,10(5):R110.006882(2011-02-21).https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3098603/
[15]Kensche T,Uejima T,Bloor S,Komander D,Randow F,Wakatsuki S.et al.Specific recognition of linear ubiquitin chains by NEMO is important for NF-kappa B activation[J].Cel,2009,136(6):1098-1109.
[16]Wu CJ,Conze DB,Li T,Srinivasula SM,Ashwel JD.Sensing of Lys 63-linked polyubiquitination by NEMOis a key event in NF-kappaB activation[corrected][J].Nat Cell Biol,2006,8(4):398-406.
[17]Fennell LM,Rahighi S,Ikeda F.Linear ubiquitin chain-binding domains[J].FEBS J,2018,285(15):2746-2761.
[18]Bloor S,Ryzhakov G,Wagner S,Butler PJ,Smith DL,Krumbach R,et al.Signal processing by its coil zipper domain activates IKK gamma[J].Proc Natl Acad Sci USA,2008,105(4):1279-1284.
[19]Lo YC,Lin SC,Rospigliosi CC,Conze DB,Wu CJ,Ashwell JD,et al.Structural basis for recognition of diubiquitins by NEMO[J].Mol Cell,2009,33(5):602-615.
[20]Park JW,Chae YC,Kim JY,Oh H,Seo SB.Methylation of Aurora kinase A by MMSET reduces p53stability and regulates cell proliferation and apoptosis[J].Oncogene,2018,37(48):6212-6224.
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