磷酸化调控泛素单体与Rad23A/Ubiquilin-1中泛素结合域互作的检测
|
摘要
泛素(ubiquitin,Ub)是一种广泛存在、高度保守的信号蛋白质,它能够特异性识别成千上万种靶蛋白,以非共价方式行使不同的功能,其中包含蛋白质降解.Ubiquilin-1(Ubql-1)和Rad23A作为两种蛋白降解的转运因子,都包含有与泛素结合的结构域,被称为泛素结合域(ubiquitin-associated domain, UBA).2014年,泛素S65位磷酸化修饰的特异性激酶PINK1被发现,磷酸化使泛素在溶液中呈现舒展态与收缩态两种互相转换的构象.本文通过核磁共振(nuclear magnetic resonance, NMR)技术对UBA和磷酸化泛素之间的相互作用进行检测,观测磷酸化对UBA和泛素结合的影响.实验结果表明Rad23A-UBA2与Ubql-1 UBA都特异性的与磷酸化泛素的舒展态相互作用,但是磷酸化未改变泛素与UBA之间的亲和力.值得注意的是与Ubql-1 UBA相互作用时,磷酸化促进了泛素收缩态向舒展态的转换.
Ubiquitin is a conserved signaling protein. It is able to interact with thousands of target proteins specifically and perform its biological functions via non-covalent interactions. Ubiquilin-1(Ubql-1) and Rad23 A are two transport factors in the protein-degradation process, and both contain ubiquitin-associated domains(UBAs). S65 of the ubiquitin can be phosphorylated specifically by the kinase PINK1, and the phosphorylated ubiquitin has two interchangeable conformations in solution, the relaxed conformation and the retracted conformation. In this work, nuclear magnetic resonance(NMR) methods were used to characterize the regulation of interactions between the ubiquitin and the UBAs of Rad23 A/Ubql-1 by phosphorylation. The results indicated that the UBAs selectively interacted with the relaxed conformation of the phosphorylated ubiquitin without affecting the affinity. When interacting with the UBA of Ubql-1, inter-protein binding appeared to be able to promote the transferring from the retracted conformation to the relaxed conformation.
Ubiquitin is a conserved signaling protein. It is able to interact with thousands of target proteins specifically and perform its biological functions via non-covalent interactions. Ubiquilin-1(Ubql-1) and Rad23 A are two transport factors in the protein-degradation process, and both contain ubiquitin-associated domains(UBAs). S65 of the ubiquitin can be phosphorylated specifically by the kinase PINK1, and the phosphorylated ubiquitin has two interchangeable conformations in solution, the relaxed conformation and the retracted conformation. In this work, nuclear magnetic resonance(NMR) methods were used to characterize the regulation of interactions between the ubiquitin and the UBAs of Rad23 A/Ubql-1 by phosphorylation. The results indicated that the UBAs selectively interacted with the relaxed conformation of the phosphorylated ubiquitin without affecting the affinity. When interacting with the UBA of Ubql-1, inter-protein binding appeared to be able to promote the transferring from the retracted conformation to the relaxed conformation.
引文
[1]KOMANDER D,RAPE M.The ubiquitin code[J].Annu Rev Biochem,2012,81:203-229.
[2]DIKIC I,WAKATSUKI S,WALTERS K J.Ubiquitin-binding domains-from structures to functions[J].Nat Rev Mol Cell Biol,2009,10(10):659-671.
[3]FOROUD T,UNIACKE S K,LIU L,et al.Heterozygosity for a mutation in the parkin gene leads to later onset Parkinson disease[J].Neurology,2003,60(5):796-801.
[4]DENG H X,CHEN W,HONG S T,et al.Mutations in UBQLN2 cause dominant X-linked juvenile and adult-onset ALS and ALS/dementia[J].Nature,2011,477(7363):211-215.
[5]EL AYADI A,STIEREN E S,BARRAL J M,et al.Ubiquilin-1 regulates amyloid precursor protein maturation and degradation by stimulating K63-linked polyubiquitination of lysine 688[J].Proc Natl Acad Sci U S A,2012,109(33):13416-13421.
[6]ZHANG D N,RAASI S,FUSHMAN D.Affinity makes the difference:nonselective interaction of the UBA domain of Ubiquilin-1 with monomeric ubiquitin and polyubiquitin chains[J].J Mol Biol,2008,377(1):162-180.
[7]HANAOKA E,OZAKI T,OHIRA M,et al.Molecular cloning and expression analysis of the human DA41 gene and its mapping to chromosome9q21.2-q21.3[J].J Hum Genet,2000,45(3):188-191.
[8]WITHERS-WARD E S,MUELLER T D,CHEN I S,et al.Biochemical and structural analysis of the interaction between the UBA(2)domain of the DNA repair protein HHR23A and HIV-1 Vpr[J].Biochemistry,2000,39(46):14103-14112.
[9]HEESSEN S,MASUCCI M G,DANTUMA N P.The UBA2 domain functions as an intrinsic stabilization signal that protects Rad23 from proteasomal degradation[J].Mol Cell,2005,18(2):225-235.
[10]KOYANO F,OKATSU K,KOSAKO H,et al.Ubiquitin is phosphorylated by PINK1 to activate parkin[J].Nature,2014,510(7503):162-166.
[11]LEE H J,NA K,KWON M S,et al.Quantitative analysis of phosphopeptides in search of the disease biomarker from the hepatocellular carcinoma specimen[J].Proteomics,2009,9(12):3395-3408.
[12]ZHOU H J,DI PALMA S,PREISINGER C,et al.Toward a comprehensive characterization of a human cancer cell phosphoproteome[J].JProteome Res,2013,12(1):260-271.
[13]SCHWEPPE D K,RIGAS J R,GERBER S A.Quantitative phosphoproteomic profiling of human non-small cell lung cancer tumors[J].JProteomics,2013,91:286-296.
[14]TSAI C F,WANG Y T,YEN H Y,et al.Large-scale determination of absolute phosphorylation stoichiometries in human cells by motif-targeting quantitative proteomics[J].Nat Commun,2015,6:6622.
[15]WAUER T,SWATEK K N,WAGSTAFF J L,et al.Ubiquitin Ser65 phosphorylation affects ubiquitin structure,chain assembly and hydrolysis[J].EMBO J,2015,34(3):307-325.
[16]DONG X,GONG Z,LU Y B,et al.Ubiquitin S65 phosphorylation engenders a pH-sensitive conformational switch[J].Proc Natl Acad Sci U SA,2017,114(26):6770-6775.
[17]SWATEK K N,KOMANDER D.Ubiquitin modifications[J].Cell Res,2016,26(4):399-422.
[18]SWANEY D L,RODRíGUEZ-MIAS R A,VILLéN J.Phosphorylation of ubiquitin at Ser65 affects its polymerization,targets,and proteome-wide turnover[J].EMBO Rep,2015,16(9):1131-1144.
[19]ZHANG N X,WU J,ZHANG H Q,et al.Protein-protein interactions studied by NMR-A review using the ubiquitin-proteasome[J].Chinese JMagn Reson,2012,29(2):182-189.张乃霞,吴娟,张华群,等.NMR技术在蛋白质-蛋白质相互作用研究中的应用-泛素-蛋白水解酶体通路研究实例介绍[J].波谱学杂志,2012,29(2):182-189.
[20]ZHANG D,RAASI S,FUSHMAN D.Affinity makes the difference:nonselective interaction of the UBA domain of Ubiquilin-1 with monomeric ubiquitin and polyubiquitin chains[J].J Mol Biol,2008,377(1):162-180.
[21]LI S L,ZHU Q J,LIU M L,et al.Characteristics of protein NMR resonances and chemical shift assignments[J].Chinese J Magn Reson,2017,34(2):137-147.李双利,朱勤俊,刘买利,等.蛋白质分子核磁共振谱峰的特性及其化学位移归属[J].波谱学杂志,2017,34(2):137-147.
[2]DIKIC I,WAKATSUKI S,WALTERS K J.Ubiquitin-binding domains-from structures to functions[J].Nat Rev Mol Cell Biol,2009,10(10):659-671.
[3]FOROUD T,UNIACKE S K,LIU L,et al.Heterozygosity for a mutation in the parkin gene leads to later onset Parkinson disease[J].Neurology,2003,60(5):796-801.
[4]DENG H X,CHEN W,HONG S T,et al.Mutations in UBQLN2 cause dominant X-linked juvenile and adult-onset ALS and ALS/dementia[J].Nature,2011,477(7363):211-215.
[5]EL AYADI A,STIEREN E S,BARRAL J M,et al.Ubiquilin-1 regulates amyloid precursor protein maturation and degradation by stimulating K63-linked polyubiquitination of lysine 688[J].Proc Natl Acad Sci U S A,2012,109(33):13416-13421.
[6]ZHANG D N,RAASI S,FUSHMAN D.Affinity makes the difference:nonselective interaction of the UBA domain of Ubiquilin-1 with monomeric ubiquitin and polyubiquitin chains[J].J Mol Biol,2008,377(1):162-180.
[7]HANAOKA E,OZAKI T,OHIRA M,et al.Molecular cloning and expression analysis of the human DA41 gene and its mapping to chromosome9q21.2-q21.3[J].J Hum Genet,2000,45(3):188-191.
[8]WITHERS-WARD E S,MUELLER T D,CHEN I S,et al.Biochemical and structural analysis of the interaction between the UBA(2)domain of the DNA repair protein HHR23A and HIV-1 Vpr[J].Biochemistry,2000,39(46):14103-14112.
[9]HEESSEN S,MASUCCI M G,DANTUMA N P.The UBA2 domain functions as an intrinsic stabilization signal that protects Rad23 from proteasomal degradation[J].Mol Cell,2005,18(2):225-235.
[10]KOYANO F,OKATSU K,KOSAKO H,et al.Ubiquitin is phosphorylated by PINK1 to activate parkin[J].Nature,2014,510(7503):162-166.
[11]LEE H J,NA K,KWON M S,et al.Quantitative analysis of phosphopeptides in search of the disease biomarker from the hepatocellular carcinoma specimen[J].Proteomics,2009,9(12):3395-3408.
[12]ZHOU H J,DI PALMA S,PREISINGER C,et al.Toward a comprehensive characterization of a human cancer cell phosphoproteome[J].JProteome Res,2013,12(1):260-271.
[13]SCHWEPPE D K,RIGAS J R,GERBER S A.Quantitative phosphoproteomic profiling of human non-small cell lung cancer tumors[J].JProteomics,2013,91:286-296.
[14]TSAI C F,WANG Y T,YEN H Y,et al.Large-scale determination of absolute phosphorylation stoichiometries in human cells by motif-targeting quantitative proteomics[J].Nat Commun,2015,6:6622.
[15]WAUER T,SWATEK K N,WAGSTAFF J L,et al.Ubiquitin Ser65 phosphorylation affects ubiquitin structure,chain assembly and hydrolysis[J].EMBO J,2015,34(3):307-325.
[16]DONG X,GONG Z,LU Y B,et al.Ubiquitin S65 phosphorylation engenders a pH-sensitive conformational switch[J].Proc Natl Acad Sci U SA,2017,114(26):6770-6775.
[17]SWATEK K N,KOMANDER D.Ubiquitin modifications[J].Cell Res,2016,26(4):399-422.
[18]SWANEY D L,RODRíGUEZ-MIAS R A,VILLéN J.Phosphorylation of ubiquitin at Ser65 affects its polymerization,targets,and proteome-wide turnover[J].EMBO Rep,2015,16(9):1131-1144.
[19]ZHANG N X,WU J,ZHANG H Q,et al.Protein-protein interactions studied by NMR-A review using the ubiquitin-proteasome[J].Chinese JMagn Reson,2012,29(2):182-189.张乃霞,吴娟,张华群,等.NMR技术在蛋白质-蛋白质相互作用研究中的应用-泛素-蛋白水解酶体通路研究实例介绍[J].波谱学杂志,2012,29(2):182-189.
[20]ZHANG D,RAASI S,FUSHMAN D.Affinity makes the difference:nonselective interaction of the UBA domain of Ubiquilin-1 with monomeric ubiquitin and polyubiquitin chains[J].J Mol Biol,2008,377(1):162-180.
[21]LI S L,ZHU Q J,LIU M L,et al.Characteristics of protein NMR resonances and chemical shift assignments[J].Chinese J Magn Reson,2017,34(2):137-147.李双利,朱勤俊,刘买利,等.蛋白质分子核磁共振谱峰的特性及其化学位移归属[J].波谱学杂志,2017,34(2):137-147.