EB病毒蛋白BNLF2a阻止抗原转运蛋白TAP的构象变化
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摘要
【背景】EB病毒是一个常见的病原,它能引起霍奇金淋巴瘤、伯基特淋巴瘤以及胃癌、鼻咽癌。该病毒编码的膜蛋白BNLF2a抑制抗原转运蛋白TAP (Transporter associated with antigen processing)从而逃逸T细胞的清除。TAP属于ABC(ATP-bindingcassette)转运蛋白超家族,是由TAP1和TAP2两个亚基构成的。TAP通过ATP提供能量,跨膜转运抗原多肽,这一过程伴随着构象变化。【目的】旨在揭示BNLF2a是否影响TAP的构象变化。【方法】TAP蛋白核酸结合结构域的二聚体界面的D-loop进行点突变,引入半胱氨酸。在表达和不表达BNLF2a情况下,采用氧化性的二价铜离子交联半胱氨酸,并通过Westernblot对比TAP的半胱氨酸形成二硫键的比例。【结果】BNLF2a表达使TAP被交联的比例增高。【结论】BNLF2a可能将TAP稳定在核苷酸结合结构域二聚化的构象,从而同时抑制ATP和抗原多肽结合到TAP上来。
[Background] Epstein-Barr virus(EBV) is a common pathogen causing Burkitt's lymphoma,Hodgkin's disease, gastric cancer and nasopharyngeal carcinoma. A membrane protein BNLF2 a encoded by EBV inhibits antigen transportation by transporter associated with antigen processing(TAP) and thereby evades the elimination by cytotoxic T cells. TAP is a member of the ATP-binding cassette(ABC) superfamily and composed of two subunits of TAP1 and TAP2. Using the energy of ATP hydrolysis, TAP transports antigenic peptides across the membrane with a conformational change. [Objective] The aim of this study was to study if BNLF2 a affects the conformational switch of TAP. [Methods] Cysteines were introduced into the D-loop at the interface of TAP's nucleotide binding domains. TAP were cross-linked by oxidizing agent Cu(II). The ratios of disulfide formed TAP were compared in the presence or absence of BNLF2 a by western blot. [Results] The expression of BNLF2 a increased the ratio of cross-linked TAP. [Conclusion] BNLF2 a appears to stabilize TAP in the nucleotide binding domains dimerized conformation and thereby inhibit both of ATP and antigenic peptide binding to TAP.
[Background] Epstein-Barr virus(EBV) is a common pathogen causing Burkitt's lymphoma,Hodgkin's disease, gastric cancer and nasopharyngeal carcinoma. A membrane protein BNLF2 a encoded by EBV inhibits antigen transportation by transporter associated with antigen processing(TAP) and thereby evades the elimination by cytotoxic T cells. TAP is a member of the ATP-binding cassette(ABC) superfamily and composed of two subunits of TAP1 and TAP2. Using the energy of ATP hydrolysis, TAP transports antigenic peptides across the membrane with a conformational change. [Objective] The aim of this study was to study if BNLF2 a affects the conformational switch of TAP. [Methods] Cysteines were introduced into the D-loop at the interface of TAP's nucleotide binding domains. TAP were cross-linked by oxidizing agent Cu(II). The ratios of disulfide formed TAP were compared in the presence or absence of BNLF2 a by western blot. [Results] The expression of BNLF2 a increased the ratio of cross-linked TAP. [Conclusion] BNLF2 a appears to stabilize TAP in the nucleotide binding domains dimerized conformation and thereby inhibit both of ATP and antigenic peptide binding to TAP.
引文
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[2]Horst D,Favaloro V,Vilardi F,et al.EBV protein BNLF2a exploits host tail-anchored protein integration machinery to inhibit TAP[J].The Journal of Immunology,2011,186(6):3594-3605
[3]Wycisk AI,Lin JC,Loch S,et al.Epstein-barr viral BNLF2a protein hijacks the tail-anchored protein insertion machinery to block antigen processing by the transport complex TAP[J].The Journal of Biological Chemistry,2011,286(48):41402-41412
[4]Ortmann B,Copeman J,Lehner PJ,et al.A critical role for tapasin in the assembly and function of multimeric MHC class I-TAP complexes[J].Science,1997,277(5330):1306-1309
[5]Groothuis T,Neefjes J.The ins and outs of intracellular peptides and antigen presentation by MHC class I molecules[A]//Wiertz E,Kikkert M.Dislocation and Degradation of Proteins from the Endoplasmic Reticulum[M].Berlin,Heidelberg:Springer,2005,300:127-148
[6]El Hage F,Durgeau A,Mami-Chouaib F.TAP expression level in tumor cells defines the nature and processing of MHC class Ipeptides for recognition by tumor-specific cytotoxic Tlymphocytes[J].Annals of the New York Academy of Sciences,2013,1283(1):75-80
[7]Hollenstein K,Dawson RJP,Locher KP.Structure and mechanism of ABC transporter proteins[J].Current Opinion in Structural Biology,2007,17(4):412-418
[8]Davidson AL,Dassa E,Orelle C,et al.Structure,function,and evolution of bacterial ATP-binding cassette systems[J].Microbiology and Molecular Biology Reviews,2008,72(2):317-364
[9]Smith PC,Karpowich N,Millen L,et al.ATP binding to the motor domain from an ABC transporter drives formation of a nucleotide sandwich dimer[J].Molecular Cell,2002,10(1):139-149
[10]Chen J,Lu G,Lin J,et al.A tweezers-like motion of the ATP-binding cassette dimer in an ABC transport cycle[J].Molecular Cell,2003,12(3):651-661
[11]Hollenstein K,Frei DC,Locher KP.Structure of an ABCtransporter in complex with its binding protein[J].Nature,2007,446(7132):213-216
[12]Aller SG,Yu J,Ward A,et al.Structure of P-glycoprotein reveals a molecular basis for poly-specific drug binding[J].Science,2009,323(5922):1718-1722
[13]O?Mara ML,Tieleman DP.P-glycoprotein models of the apo and ATP-bound states based on homology with Sav1866 and MalK[J].FEBS Letters,2007,581(22):4217-4222
[14]Oliveira AS,Baptista AM,Soares CM.Conformational changes induced by ATP-hydrolysis in an ABC transporter:a molecular dynamics study of the Sav1866 exporter[J].Proteins-Structure Function and Bioinformatics,2011,79(6):1977-1990
[15]Dawson RJ,Locher KP.Structure of a bacterial multidrug ABCtransporter[J].Nature,2006,443(7108):180-185
[16]Hislop AD,Taylor GS,Sauce D,et al.Cellular responses to viral infection in humans:lessons from Epstein-Barr virus[J].Annual Review of Immunology,2007,25:587-617