CUEDC2的表达纯化与溶液结构初步探索
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摘要
CUEDC2 (CUE Domain Containing 2)是一个功能未知的蛋白质。最近研究发现CUEDC2能够和孕激素受体结合,促进孕激素诱导的受体的泛素化和降解,可能作为一个新的肿瘤治疗靶点,因而具有十分重要的应用价值。为了更好的研究其生物学功能,我们拟用核磁共振方法研究其三维结构。通过生物信息学的分析表明CUEDC2包含一个CUE结构域。CUE结构域是一个约40个氨基酸的广泛存在于真核蛋白质中的结构域。CUE结构域具有多种功能,其中包括内质网上所合成的折叠错误和序列错误的蛋白质的降解过程。CUE结构域有着与UBA结构域相似的结构,都有三个α-螺旋折叠而成。CUE结构域的α-螺旋1中的保守的MFP motif和α-螺旋3中的LL motif与泛素的疏水表面相互作用,同时据文献报道CUE结构域可以形成二聚体,可以附加的结合多个泛素,因而具有较高的泛素亲和能力。许多包含CUE结构域的蛋白质既能识别单泛素化又能识别多泛素化。实验证明,许多包含CUE结构域的蛋白质都能够以一种CUE结构域依赖性的方法发生泛素化。CUEDC2是一个分子量约为32 kDa包含287个氨基酸的酸性蛋白质。由于分子量较大,我们计划将其截成2个片段,分别解析其溶液结构。
本文以CUEDC2为研究对象,分别构建了多个CUEDC2的截断体,并通过优化各种表达与纯化条件,获得了高表达的,且符合核磁共振实验要求的蛋白质样品溶液,并利用多维核磁共振技术对CUEDC2 N端包含133个氨基酸残基的结构域的溶液结构进行了初步的研究与探索。
首先,我们表达、纯化了GST-CUEDC(1-133),但是在用凝血酶切除GST标签蛋白时,我们发现在用凝血酶酶切16小时后仍然无法将GST标签蛋白切下。于是我们考虑构建有较小的标签的克隆。我们将CUEDC2 N端的133个氨基酸构建到pET-28a载体上,并对其表达、纯化条件及蛋白质序列进行优化。最终获得了折叠状态较好的CUEDC(1-133)cut蛋白质样品。之后,我们优化了核磁共振的实验温度并确定了三共振实验条件。我们采集了一系列的三维核磁共振实验图谱,包括用于主链谱峰指认的HNCA、HNCOCA、CBCANH、CBCA(CO)NH和用于侧链谱峰指认的HBHA(CO)NH、CCCONH、15N-NOESY-HSQC、HCCH-COSY和HCCH-TOCSY谱。然后我们利用NMRPipe软件包对谱图进行处理,利用CARA软件归属了大部分主链原子的化学位移。
本文还对CUEDC2(133-287)蛋白进行了初步的研究。表达、纯化了GST-CUEDC(133-287)。并利用凝血酶对GST-CUEDC(133-287)酶切,除去了GST标签蛋白,获得了高浓度的CUEDC(133-287)蛋白样品。然后我们1H核磁共振谱对其结构进行初步的评价。结果表明可能由于CUEDC2(133-287)这个截断体中缺失了某个关键的氨基酸序列,导致其折叠构象改变,蛋白质折叠不完全。我们希望通过构建其它的CUEDC2截断体来得到C末端折叠较好的CUEDC2蛋白,进行结构研究。
CUE Domain Containing 2 (CUEDC2 ) is a new protein with unidentified function. Recently, CUEDC2 was found to interact with progesterone receptor (PR) and promotes progesterone-induced PR degradation by the ubiquitin-proteasome pathway. CUEDC2 may be a new target for cancer therapeutics. The solvation of the solution structure of CUEDC2 will facilitate the research of its function.
Based on bioinformatic analysis, CUEDC2 was considered as a CUE domain-containing protein. The CUE domain is moderately conserved consisting of 40 amino acids and is structurally related to the ubiquitin-binding UBA domain, which is found in a variety of eukaryotic proteins.
The CUE domain has been found in proteins with a variety of functions, including the degradation of misfolded proteins in the endoplasmic reticulum and protein sorting. The CUE domain is folded into a three-helix bundle. A conserved MFP motif inα-helix1 and an LL motif inα-helix 3 interact with the conserved hydrophobic patch of ubiquitin. It has been reported that the CUE domain exists as a domain-swapped dimmer to make additional contacts with ubiquitin, resulting in higher binding affinity with ubiquitin. Proteins containing CUE domains, such as Vps9 and Cue1, can bind ubiqutin in mono and polyubiquitin manners. Some CUE-containing proteins are ubiquitinylated in a CUE domain-dependent manner. CUEDC2 is an acidic protein consisting of 287 amino acids with a molecular weight of 32 kDa. The full length protein is too large for NMR study and was thus divided into separate N-terminal domain (1-133) and C-terminal domain (133-287) for solution structure determination.
In this thesis have been described the structural studies of CUEDC2.A few truncated forms of CUEDC2 were constructed, expressed in E. coli and purified with optimized conditions. High quality protein samples suitable for NMR experiments were obtained. Preliminary results of the solution structure of the N-terminal domain CUEDC2(1-133) have been obtained based on heteronuclear multidimensional NMR experiments.
First of all, the CUEDC2(1-133) was expressed as GST-fusion protein. But it turned out to be difficult to cut off GST protein tag with thrombin even after 16 h incubation. Therefore, a new construct of CUEDC2(1-133) was made from pET-28a plasmid, resulting in a His-tag fusion protein of CUEDC2(1-133). The conditions for the expression and purification of this fusion protein were optimized. Finally, a protein sample of CUEDC2(1-133) was obtained with good folding as checked by NMR experiments. The NMR experimental conditions were optimized and a set of 3D NMR experiments were carried out, which including HNCA, HN(CO)CA, CBCANH, CBCA(CO)NH for the assignments of backbone, and HBHA(CO)NH, CC(CO)NH, 15N-NOESY-HSQC, HCCH-COSY, HCCH-TOCSY for theassignments of sidechain atoms. All the spectra were processed with NMRPipe Software Package, and chemical shift assignments were carried out using CARA and NMRView. Most of the backbone signals have been assigned.
The C-terminal domain GST-CUEDC2(133-287) has also been expressed as GST fusion protein and purified with GST-tag removed. with the preliminary 1H NMR spectra showed that CUEDC2(133-287) was not well folded. It might be due to the lack of key amino acids in this truncated form. New constructs of this truncated domain have been carrying on to solve this problem.
本文以CUEDC2为研究对象,分别构建了多个CUEDC2的截断体,并通过优化各种表达与纯化条件,获得了高表达的,且符合核磁共振实验要求的蛋白质样品溶液,并利用多维核磁共振技术对CUEDC2 N端包含133个氨基酸残基的结构域的溶液结构进行了初步的研究与探索。
首先,我们表达、纯化了GST-CUEDC(1-133),但是在用凝血酶切除GST标签蛋白时,我们发现在用凝血酶酶切16小时后仍然无法将GST标签蛋白切下。于是我们考虑构建有较小的标签的克隆。我们将CUEDC2 N端的133个氨基酸构建到pET-28a载体上,并对其表达、纯化条件及蛋白质序列进行优化。最终获得了折叠状态较好的CUEDC(1-133)cut蛋白质样品。之后,我们优化了核磁共振的实验温度并确定了三共振实验条件。我们采集了一系列的三维核磁共振实验图谱,包括用于主链谱峰指认的HNCA、HNCOCA、CBCANH、CBCA(CO)NH和用于侧链谱峰指认的HBHA(CO)NH、CCCONH、15N-NOESY-HSQC、HCCH-COSY和HCCH-TOCSY谱。然后我们利用NMRPipe软件包对谱图进行处理,利用CARA软件归属了大部分主链原子的化学位移。
本文还对CUEDC2(133-287)蛋白进行了初步的研究。表达、纯化了GST-CUEDC(133-287)。并利用凝血酶对GST-CUEDC(133-287)酶切,除去了GST标签蛋白,获得了高浓度的CUEDC(133-287)蛋白样品。然后我们1H核磁共振谱对其结构进行初步的评价。结果表明可能由于CUEDC2(133-287)这个截断体中缺失了某个关键的氨基酸序列,导致其折叠构象改变,蛋白质折叠不完全。我们希望通过构建其它的CUEDC2截断体来得到C末端折叠较好的CUEDC2蛋白,进行结构研究。
CUE Domain Containing 2 (CUEDC2 ) is a new protein with unidentified function. Recently, CUEDC2 was found to interact with progesterone receptor (PR) and promotes progesterone-induced PR degradation by the ubiquitin-proteasome pathway. CUEDC2 may be a new target for cancer therapeutics. The solvation of the solution structure of CUEDC2 will facilitate the research of its function.
Based on bioinformatic analysis, CUEDC2 was considered as a CUE domain-containing protein. The CUE domain is moderately conserved consisting of 40 amino acids and is structurally related to the ubiquitin-binding UBA domain, which is found in a variety of eukaryotic proteins.
The CUE domain has been found in proteins with a variety of functions, including the degradation of misfolded proteins in the endoplasmic reticulum and protein sorting. The CUE domain is folded into a three-helix bundle. A conserved MFP motif inα-helix1 and an LL motif inα-helix 3 interact with the conserved hydrophobic patch of ubiquitin. It has been reported that the CUE domain exists as a domain-swapped dimmer to make additional contacts with ubiquitin, resulting in higher binding affinity with ubiquitin. Proteins containing CUE domains, such as Vps9 and Cue1, can bind ubiqutin in mono and polyubiquitin manners. Some CUE-containing proteins are ubiquitinylated in a CUE domain-dependent manner. CUEDC2 is an acidic protein consisting of 287 amino acids with a molecular weight of 32 kDa. The full length protein is too large for NMR study and was thus divided into separate N-terminal domain (1-133) and C-terminal domain (133-287) for solution structure determination.
In this thesis have been described the structural studies of CUEDC2.A few truncated forms of CUEDC2 were constructed, expressed in E. coli and purified with optimized conditions. High quality protein samples suitable for NMR experiments were obtained. Preliminary results of the solution structure of the N-terminal domain CUEDC2(1-133) have been obtained based on heteronuclear multidimensional NMR experiments.
First of all, the CUEDC2(1-133) was expressed as GST-fusion protein. But it turned out to be difficult to cut off GST protein tag with thrombin even after 16 h incubation. Therefore, a new construct of CUEDC2(1-133) was made from pET-28a plasmid, resulting in a His-tag fusion protein of CUEDC2(1-133). The conditions for the expression and purification of this fusion protein were optimized. Finally, a protein sample of CUEDC2(1-133) was obtained with good folding as checked by NMR experiments. The NMR experimental conditions were optimized and a set of 3D NMR experiments were carried out, which including HNCA, HN(CO)CA, CBCANH, CBCA(CO)NH for the assignments of backbone, and HBHA(CO)NH, CC(CO)NH, 15N-NOESY-HSQC, HCCH-COSY, HCCH-TOCSY for theassignments of sidechain atoms. All the spectra were processed with NMRPipe Software Package, and chemical shift assignments were carried out using CARA and NMRView. Most of the backbone signals have been assigned.
The C-terminal domain GST-CUEDC2(133-287) has also been expressed as GST fusion protein and purified with GST-tag removed. with the preliminary 1H NMR spectra showed that CUEDC2(133-287) was not well folded. It might be due to the lack of key amino acids in this truncated form. New constructs of this truncated domain have been carrying on to solve this problem.
引文
[1]阎隆飞,孙之荣.蛋白质分子结构.北京:清华大学出版社, (1997): 131-154
[2]夏斌,金长文生物大分子结构及动力学的核磁共振研究。现代仪器(2002) 8(4) 1-5
[3]施蕴渝,吴季辉核磁共振波谱应用于结构生物学的研究进展。ACTA BIOPHYSICA SINICA (2007)Vol.23 No.4 241–245
[4] Cavanagh J. Protein NMR spectroscopy: principles and practice.2nd ed. Academic Press, Amsterdam; Boston, (2007). pp.ⅩⅩⅤ, 885
[5] Downing AK. Protein NMR techniques. 2nd ed. Totowa, NJ:Humana Press (2004) 289-301
[6]余亦华.蛋白质三维空间结构研究的里程碑。科学,(2003),55(2): 57-58
[7] David G. Reid, Lesley K., Protein NMR Techniques, Humana Press (1997) 53-69
[8]施燕红,郭晨云,林东海蛋白质NMR样品制备技术CHEMISTRY OF LIFE (2006), 26(2) 166-169
[9] Masatsune Kainosho, Takuya Torizawa Optimal isotope labelling for NMR protein structure determinations (2006), 440, 10.1038
[10] Pervushin et al. Attenuated T2 relaxation by mutual cancellation of dipole–dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological PNAS USA, (1997) 94, p12366
[11] Jocelyne Fiaux*, Eric B. Bertelsen, Arthur L. Horwich & Kurt Wu¨thrich NMR analysis of a 900K GroEL–GroES Nature,(2002),418:207-211
[12] Bodenhausen, G. and D.J. Ruben.. Natural abundance nitrogen-15 NMR by enhanced heteronuclear spectroscopy.Chem. Phys. Lett. (1980) 69:185–189
[13] Gerhard Wider , Structure Determination of Biological Macromolecules in Solution Using NMR spectroscopy Biotechniques. (2000) 29(6):1278-82, 1284-90
[14] Lewis E. Kay, Mitsuhiko Ikura, Rolf Tschudin and Ad Bax Three-dimensional triple-resonance NMR spectroscopy of isotopically enriched proteins. Journal of Magnetic Resonance (1990) 89, 3, 1, 496-514
[15] B.T. Farmer . R.A. Venters. L.D. Spicer. M.G. Wittekind and L.Muller A refocused and optimized HNCA: Increased sensitivity and resolution in large macromolecules. Journal of Biomolecular NMR. (1992) 2 195-202
[16] Stephan Grzesiek* and Ad Bax .Improved 3D triple-resonance NMR techniques applied to a 31 kDa protein. Journal of Magnetic Resonance (1992) 96, 2, 432-440
[17] Ad Bax and Mitsuhiko Ikura, An efficient 3D NMR technique for correlating the proton and15N backbone amide resonances with theα-carbon of the preceding residue in uniformly15N/13C enriched proteins. Journal of Biomolecular NMR (1991), 1, 1 99-104
[18] Stephan Grzesiek, Ad Bax Correlating backbone amide and side chain resonances in larger proteins by multiple relayed triple resonance NMR. J. Am. Chem. Soc., (1992) 114 (16), 6291–6293
[19] Stephan Grzesiek* and Ad Bax. An efficient experiment for sequential backbone assignment of medium-sized isotopically enriched proteins. Journal of Magnetic Resonance (1992) 99, 1, 201-207
[20] J. Cavanagh, W. Fairbrother,A.G. Palmer III and N.J. Skleton. Protein NMR Spectroscopy - Principles and Practice Academic Press 399-410.
[21] D. Marion, P.C. Driscoll, L.E. Kay, P.T. Wingfield, A. Bax, A.M. Gronenborn and G.M. Clore Overcoming the overlap problem in the assignment of proton NMR spectra of larger proteins by use of three-dimensional heteronuclear proton-nitrogen-15 Hartmann-Hahn-multiple quantum coherence and nuclear Overhauser-multiple quantum coherence spectroscopy: application to interleukin 1.beta. Biochemistry (1989)28 6150-6156.
[22] D. Marion, L.E. Kay, S.W. Sparks, D.A. Torchia and A. Bax Three-dimensional heteronuclear NMR of nitrogen-15 labeled proteins J. Am. Chem. Soc. (1989) 111 1515-1517.
[23] E.R.P. Zuiderweg and S.W. Fesik Heteronuclear three-dimensional NMR spectroscopy of the inflammatory protein C5a Biochemistry (1989) 28 2387-2391.
[24] A. Bax, G.M. Clore, P.C. Driscoll, A.M.Gronenborn, M. Ikura and L.E. Kay Practical aspects of proton-carbon-carbon-proton three-dimensional correlation spectroscopy of 13C-labeled proteinsJ. Magn. Reson. (1990) 87 620-627.
[25] L.E. Kay, M. Ikura and A. Bax Proton-proton correlation via carbon-carbon couplings: a three-dimensional NMR approach for the assignment of aliphatic resonances in proteins labeled with carbon-13J. Am. Chem. Soc. (1990) 112 888-889.
[26] M. Ikura, L.E. Kay and A. Bax Improved three-dimensional1H?13C?1H correlation spectroscopy of a13C-labeled protein using constant-time evolution J. Biomol. NMR (1991) 1 299-304.
[27] A. Bax, G.M. Clore and A.M. Gronenborn 1H---1H correlation via isotropic mixing of 13C magnetization, a new three-dimensional approach for assigning 1H and 13C spectra of 13C-enriched proteins J. Magn. Reson. (1990) 88 425-431.
[28] E.T. Olejniczak, R.X. Xu and S.W. Fesik A 4D HCCH-TOCSY experiment for assigning the side chain1H and13C resonances of proteins. J. Biomol. NMR (1992) 2 655-659.
[29] Cornilescu G. Delaglio F and Bax A. Protein backbone angle restraints from searching a database for chemical shift and sequence homology. J.Biomol.NMR,(1999) 13:289-302.
[30] Monleon D.,Colson K.,Moseley H.N., Anklin C., Oswald R., Szyperski T. and Montelione G.T. Rapid analysis of protein backbone resonance assignments using cryogenic probes, a distributed Linux-based computing architecture, and an integrated set of spectral analysis tools. J.Struct.Funct.Genomics,(2002) 2:93-101
[31] Pei-Jing Zhang, Jie Zhao, Hui-Yan Li et al. CUE domain containing 2 regulates degradation of progesterone receptor by ubiquitin–proteasome. EMBO J. (2007) 26, 1831-1842
[32] Hui-Yan Li, Hui Liu, Chen-Hui Wang et al. Deactivation of the kinase IKK by CUEDC2 through recruitment of the phosphatase PP1. NATURE IMMUNOLOGY (2008) 9 (5): 533-541
[33]王艳,霍翔,沈洪兵,雌激素受体和孕激素受体与乳腺癌关系的研究进展.中国肿瘤(2007) 16: 12
[34]王颖,曹旭晨,乳腺癌雌激素受体研究进展。会议:第三届中-法乳腺癌高级学术论坛
[35] Kang, R.S et al. Solution Structure of a CUE-Ubiquitin Complex Reveals a Conserved Mode of Ubiquitin Binding(2003) Cell 113(5), 621–630
[2]夏斌,金长文生物大分子结构及动力学的核磁共振研究。现代仪器(2002) 8(4) 1-5
[3]施蕴渝,吴季辉核磁共振波谱应用于结构生物学的研究进展。ACTA BIOPHYSICA SINICA (2007)Vol.23 No.4 241–245
[4] Cavanagh J. Protein NMR spectroscopy: principles and practice.2nd ed. Academic Press, Amsterdam; Boston, (2007). pp.ⅩⅩⅤ, 885
[5] Downing AK. Protein NMR techniques. 2nd ed. Totowa, NJ:Humana Press (2004) 289-301
[6]余亦华.蛋白质三维空间结构研究的里程碑。科学,(2003),55(2): 57-58
[7] David G. Reid, Lesley K., Protein NMR Techniques, Humana Press (1997) 53-69
[8]施燕红,郭晨云,林东海蛋白质NMR样品制备技术CHEMISTRY OF LIFE (2006), 26(2) 166-169
[9] Masatsune Kainosho, Takuya Torizawa Optimal isotope labelling for NMR protein structure determinations (2006), 440, 10.1038
[10] Pervushin et al. Attenuated T2 relaxation by mutual cancellation of dipole–dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological PNAS USA, (1997) 94, p12366
[11] Jocelyne Fiaux*, Eric B. Bertelsen, Arthur L. Horwich & Kurt Wu¨thrich NMR analysis of a 900K GroEL–GroES Nature,(2002),418:207-211
[12] Bodenhausen, G. and D.J. Ruben.. Natural abundance nitrogen-15 NMR by enhanced heteronuclear spectroscopy.Chem. Phys. Lett. (1980) 69:185–189
[13] Gerhard Wider , Structure Determination of Biological Macromolecules in Solution Using NMR spectroscopy Biotechniques. (2000) 29(6):1278-82, 1284-90
[14] Lewis E. Kay, Mitsuhiko Ikura, Rolf Tschudin and Ad Bax Three-dimensional triple-resonance NMR spectroscopy of isotopically enriched proteins. Journal of Magnetic Resonance (1990) 89, 3, 1, 496-514
[15] B.T. Farmer . R.A. Venters. L.D. Spicer. M.G. Wittekind and L.Muller A refocused and optimized HNCA: Increased sensitivity and resolution in large macromolecules. Journal of Biomolecular NMR. (1992) 2 195-202
[16] Stephan Grzesiek* and Ad Bax .Improved 3D triple-resonance NMR techniques applied to a 31 kDa protein. Journal of Magnetic Resonance (1992) 96, 2, 432-440
[17] Ad Bax and Mitsuhiko Ikura, An efficient 3D NMR technique for correlating the proton and15N backbone amide resonances with theα-carbon of the preceding residue in uniformly15N/13C enriched proteins. Journal of Biomolecular NMR (1991), 1, 1 99-104
[18] Stephan Grzesiek, Ad Bax Correlating backbone amide and side chain resonances in larger proteins by multiple relayed triple resonance NMR. J. Am. Chem. Soc., (1992) 114 (16), 6291–6293
[19] Stephan Grzesiek* and Ad Bax. An efficient experiment for sequential backbone assignment of medium-sized isotopically enriched proteins. Journal of Magnetic Resonance (1992) 99, 1, 201-207
[20] J. Cavanagh, W. Fairbrother,A.G. Palmer III and N.J. Skleton. Protein NMR Spectroscopy - Principles and Practice Academic Press 399-410.
[21] D. Marion, P.C. Driscoll, L.E. Kay, P.T. Wingfield, A. Bax, A.M. Gronenborn and G.M. Clore Overcoming the overlap problem in the assignment of proton NMR spectra of larger proteins by use of three-dimensional heteronuclear proton-nitrogen-15 Hartmann-Hahn-multiple quantum coherence and nuclear Overhauser-multiple quantum coherence spectroscopy: application to interleukin 1.beta. Biochemistry (1989)28 6150-6156.
[22] D. Marion, L.E. Kay, S.W. Sparks, D.A. Torchia and A. Bax Three-dimensional heteronuclear NMR of nitrogen-15 labeled proteins J. Am. Chem. Soc. (1989) 111 1515-1517.
[23] E.R.P. Zuiderweg and S.W. Fesik Heteronuclear three-dimensional NMR spectroscopy of the inflammatory protein C5a Biochemistry (1989) 28 2387-2391.
[24] A. Bax, G.M. Clore, P.C. Driscoll, A.M.Gronenborn, M. Ikura and L.E. Kay Practical aspects of proton-carbon-carbon-proton three-dimensional correlation spectroscopy of 13C-labeled proteinsJ. Magn. Reson. (1990) 87 620-627.
[25] L.E. Kay, M. Ikura and A. Bax Proton-proton correlation via carbon-carbon couplings: a three-dimensional NMR approach for the assignment of aliphatic resonances in proteins labeled with carbon-13J. Am. Chem. Soc. (1990) 112 888-889.
[26] M. Ikura, L.E. Kay and A. Bax Improved three-dimensional1H?13C?1H correlation spectroscopy of a13C-labeled protein using constant-time evolution J. Biomol. NMR (1991) 1 299-304.
[27] A. Bax, G.M. Clore and A.M. Gronenborn 1H---1H correlation via isotropic mixing of 13C magnetization, a new three-dimensional approach for assigning 1H and 13C spectra of 13C-enriched proteins J. Magn. Reson. (1990) 88 425-431.
[28] E.T. Olejniczak, R.X. Xu and S.W. Fesik A 4D HCCH-TOCSY experiment for assigning the side chain1H and13C resonances of proteins. J. Biomol. NMR (1992) 2 655-659.
[29] Cornilescu G. Delaglio F and Bax A. Protein backbone angle restraints from searching a database for chemical shift and sequence homology. J.Biomol.NMR,(1999) 13:289-302.
[30] Monleon D.,Colson K.,Moseley H.N., Anklin C., Oswald R., Szyperski T. and Montelione G.T. Rapid analysis of protein backbone resonance assignments using cryogenic probes, a distributed Linux-based computing architecture, and an integrated set of spectral analysis tools. J.Struct.Funct.Genomics,(2002) 2:93-101
[31] Pei-Jing Zhang, Jie Zhao, Hui-Yan Li et al. CUE domain containing 2 regulates degradation of progesterone receptor by ubiquitin–proteasome. EMBO J. (2007) 26, 1831-1842
[32] Hui-Yan Li, Hui Liu, Chen-Hui Wang et al. Deactivation of the kinase IKK by CUEDC2 through recruitment of the phosphatase PP1. NATURE IMMUNOLOGY (2008) 9 (5): 533-541
[33]王艳,霍翔,沈洪兵,雌激素受体和孕激素受体与乳腺癌关系的研究进展.中国肿瘤(2007) 16: 12
[34]王颖,曹旭晨,乳腺癌雌激素受体研究进展。会议:第三届中-法乳腺癌高级学术论坛
[35] Kang, R.S et al. Solution Structure of a CUE-Ubiquitin Complex Reveals a Conserved Mode of Ubiquitin Binding(2003) Cell 113(5), 621–630