摘要
Ubiquitin是一个由76个氨基酸残基组成的广泛并且只存在于真核细胞的多肽分子。Ubiquitination最重要的功能是介导底物蛋白通过26S蛋白酶体快速降解。Ubiquitin通过修饰各种重要的功能蛋白，精确地调节着细胞周期、细胞增殖与分化、细胞凋亡等生理过程。在真核细胞内，还存在类似于Ubiquitin的小分子多肽，称为UBLs(Ubiquitin-like modifiers)。Sumo(Small ubiquitin-like modifier)是研究得最为深入的一种UBLs。Sumo具有与Ubiquitin相类似的三维空间结构，以Ubiquitination类似的方式对相应的底物蛋白进行修饰。Sumos修饰不介导蛋白质降解，而是通过改变细胞定位、蛋白活性、蛋白之间相互作用等不同的机制在细胞的各种生理活动中发挥重要的调节作用。在本文中，我们从组学水平对肝细胞蛋白质的Ubiquitination进行了分析，并且研究了HDAC5的Sumo修饰机制。
我们利用蛋白质组学的研究手段对肝细胞内Ubiquitin底物蛋白进行了纯化分离，并对分离出的Ubiquitin底物蛋白进行了质谱鉴定。S5a是26S蛋白酶体的Ubiquitin识别亚基，能够通过两个独立的UIMs(Ubiquitin-interacting motifs)特异性结合发生Polyubiquitination的蛋白。我们利用S5a亲和层析从人正常肝细胞(Chang’liver cell line)中纯化了发生Polyubqiquitination的蛋白，并且使用高通量质谱方法LC／LC-MS／MS (Multidimensional chromatography coupled with tandem mass spectrometry)进行了蛋白质成分鉴定。通过上述实验，我们一共确定了81个Polyubiquitination的底物蛋白，同时通过数据分析预测了分布于17个蛋白中的19个可能的Ubiquitin特异性修饰位点。
与此同时，我们利用生物化学的方法对Sumolation的修饰机制进行了研究。我们鉴定了一个新的Sumo底物蛋白——组蛋白去乙酰化酶5(HDAC5)，并深入研究了Sumolation的修饰机制。组蛋白去乙酰化酶(Histone deacetylases，HDACs)通过改变组蛋白的乙酰化状态改变着染色质的结构，是十分重要的基因表达调节因子。HDAC4是Sumo的重要底物之一，我们通过体内Sumoylation分析实验发现了与HDAC4同属Ⅱa家族的HDAC5也能够被Sumo修饰。含有SP-RING结构域的PIASs(Protein inhibitorsof activated STAT proteins)家族蛋白是目前发现的最主要的Sumo E3连接酶，在基因的转录调节中发挥重要作用。我们通过免疫沉淀实验证明TPIASs和HDAC5在细胞内相互作用。将不同的PIASs成员加入HDAC5的Sumoylation体内分析系统，进一步发现PIAS1特异性促进Sumo1对HDAC5的修饰，而PIASy特异性促进Sumo2对HDAC5的修饰。我们的工作也发现在HDAC5中符合ψKXE的赖氨酸(K606)为Sumo2和Sumo3的特异性修饰位点。根据文献报道并结合本研究所得实验结果，我们推测第600氨基酸附近的区域为Ⅱa类HDACs保守的Sumolation修饰位置。
Ubiquitin is a highly conserved 76-amino acid polypeptide expressed in all eukaryotes but absent in bacteria and archaea. Polyubiquitin through Lys48 linkage targets modified proteins for ATP-dependent degradation by the 26S proteasome. Ubiquitination plays an essential role in cellular functions such as cell cycle progressing, cellular proliferation and differentiation, apoptosis, etc. UBLs (ubiquitin-like modifers) that share similarities with ubiquitin as a post- translational protein modifier have been identified in eukaryotic cells. The most well studied member of this ubiquitin-like family is Sumo (Small Ubiquitin-like modifier). With an ubiquitin-like 3D structure, Sumo is conjugated to proteins via a similar mechanism as ubiquitin. Unlike ubiquitination, which usually targets proteins to degaradation, sumolation mediates a number of cellular processes by regulate the behaviors of substrate proteins such as subcellular localization, enzymatic activity, protein-protein interaction. Here, we studied the ubiquitination in liver cells by proteomic approach and the sumolation of HDAC5 respectively.
We purified and identified the ubiquitinated proteins by proteomics-based approaches. The S5a subunit of 26S proteasome was originally identified as a protein capable of binding poly ubiquitin chains through Lys48 linkage. 81 putative polyubiquitinated proteins in normal liver cells (Chang' liver cell) were identified in this study using S5a-affinity chromatography coupled LC/LC-MS/MS analysis. Moreover, 19 putative ubiquitination sites were identified by mass spectrometry.
Moreover, we show that HDAC5 is sumolated in vivo and this modification is regulated by PIASs. By deacetylating histone and modifying chramotin structure, HDACs (Histone Deacetylase) play essential roles in regulating gene expression. Previous research showed that HDAC4 is modified by sumo, we found that HDAC5 is a new substrate of sumo by in vivo sumoylation assays in this report. PIASs (Protein Inhibitors of Activated STAT Protein), which contain a conserved SP-RING domain, are considered as the common E3 ligase in sumolation pathway. After confirming the interaction between PIASs and HDAC5 by co-immunoprecipitation, we found that PIAS1 enhances the Sumo1-modification of HDAC5, while PIASy enhances the Sumo2-modification of HDAC5 specifically. Our data also showed that K606 on HDAC5, which matches a consensus sumoylation motif, is a specifical Sumo2/3 modification site. Bioinforrnatic analysis leads us to assume that the sumolation on conserved region (about the 600 amino acid from the N-terminus) is a common characteristic of ClassⅡa HDACs.
引文
[1] A. Hershko, and A. Ciechanover, The ubiquitin system, Annu Rev Biochem 67 (1998) 425-479.
[2] S. Fang, and A. M. Weissman, A field guide to ubiquitylation, Cell Mol Life Sci 61 (2004) 1546-1561.
[3] N. Shcherbik, and D. S. Haines, Ub on the move, J Cell Biochem 93 (2004) 11-19.
[4] C. M. Pickart, Mechanisms underlying ubiquitination, Annu Rev Biochem 70 (2001) 503-533.
[5] T. Jenuwein, and C. D. Allis, Translating the histone code, Science 293 (2001) 1074-1080.
[6] B. D. Strahl, and C. D. Allis, The language of covalent histone modifications, Nature 403 (2000) 41-45.
[7] E. Verdin, F. Dequiedt, and H. G. Kasler, Class II histone deacetylases: versatile regulators, Trends Genet 19 (2003) 286-293.
[8] N. Sengupta, and E. Seto, Regulation of histone deacetylase activities, J Cell Biochem 93 (2004) 57-67.
[9] D. C. Schwartz, and M. Hochstrasser, A superfamily of protein tags: ubiquitin, SUMO and related modifiers, Trends Biochem Sci 28 (2003) 321-328.
[10] K. M. Bohren, V. Nadkarni, J. H. Song, K. H. Gabbay, and D. Owerbach, A M55V polymorphism in a novel SUMO gene (SUMO-4) differentially activates heat shock transcription factors and is associated with susceptibility to type I diabetes mellitus, J Biol Chem 279 (2004) 27233-27238.
[11] N. Hattori, and Y. Mizuno, Pathogenetic mechanisms of parkin in Parkinson's disease, Lancet 364 (2004) 722-724.
[12] K. I. Kim, and S. H. Baek, SUMOylation code in cancer development and metastasis, Mol Cells 22 (2006) 247-253.
[13] M. Li, D. Guo, C. M. Isales, D. L. Eizirik, M. Atkinson, J. X. She, and C. Y. Wang, SUMO wrestling with type 1 diabetes, J Mol Med 83 (2005) 504-513.
[14] G. Gill, Post-translational modification by the small ubiquitin-related modifier SUMO has big effects on transcription factor activity, Curr Opin Genet Dev 13 (2003) 108-113.
[15] G. Gill, Something about SUMO inhibits transcription, Curr Opin Genet Dev 15 (2005) 536-541.
[16] O. Kirsh, J. S. Seeler, A. Pichler, A. Gast, S. Muller, E. Miska, M. Mathieu, A. Harel-Bellan, T. Kouzarides, F. Melchior, and A. Dejean, The SUMO E3 ligase RanBP2 promotes modification of the HDAC4 deacetylase, Embo J 21 (2002) 2682-2691.
[17] Q. Deveraux, C. Jensen, and M. Rechsteiner, Molecular cloning and expression of a 26 S protease subunit enriched in dileucine repeats, J Biol Chem 270 (1995) 23726-23729.
[18] Q. Deveraux, V. Ustrell, C. Pickart, and M. Rechsteiner, A 26 S protease subunit that binds ubiquitin conjugates, J Biol Chem 269 (1994) 7059-7061.
[19] M. Mann, and O. N. Jensen, Proteomic analysis of post-translational modifications, Nat Biotechnol 21(2003)255-261.
[20] J. Peng, D. Schwartz, J. E. Elias, C. C. Thoreen, D. Cheng, G. Marsischky, J. Roelofs, D. Finley, and S. P. Gygi, A proteomics approach to understanding protein ubiquitination, Nat Biotechnol 21 (2003) 921-926.
[21] A. M. Weissman, Themes and variations on ubiquitylation, Nat Rev Mol Cell Biol 2 (2001) 169-178.
[22] J. S. Thrower, L. Hoffman, M. Rechsteiner, and C. M. Pickart, Recognition of the polyubiquitin proteolytic signal, Embo J 19 (2000) 94-102.
[23] H. Kawadler, and X. Yang, Lys63-linked polyubiquitin chains: linking more than just ubiquitin, Cancer Biol Ther 5 (2006) 1273-1274.
[24] C. M. Pickart, and D. Fushman, Polyubiquitin chains: polymeric protein signals, Curr Opin Chem Biol 8 (2004) 610-616.
[25] T. A. McKinsey, C. L. Zhang, J. Lu, and E. N. Olson, Signal-dependent nuclear export of a histone deacetylase regulates muscle differentiation, Nature 408 (2000) 106-111.
[26] C. L. Zhang, T. A. McKinsey, S. Chang, C. L. Antos, J. A. Hill, and E. N. Olson, Class II histone deacetylases act as signal-responsive repressors of cardiac hypertrophy, Cell 110 (2002) 479-488.
[27] S. Chang, T. A. McKinsey, C. L. Zhang, J. A. Richardson, J. A. Hill, and E. N. Olson, Histone deacetylases 5 and 9 govern responsiveness of the heart to a subset of stress signals and play redundant roles in heart development, Mol Cell Biol 24 (2004) 8467-8476.
[28] D. Nowis, E. McConnell, and C. Wojcik, Destabilization of the VCP-Ufdl-Npl4 complex is associated with decreased levels of ERAD substrates, Exp Cell Res 312 (2006) 2921-2932.
[29] S. Gregoire, and X. J. Yang, Association with class IIa histone deacetylases upregulates the sumoylation of MEF2 transcription factors, Mol Cell Biol 25 (2005) 2273-2287.
[30] X. Zhao, T. Sternsdorf, T. A. Bolger, R. M. Evans, and T. P. Yao, Regulation of MEF2 by histone deacetylase 4- and SIRT1 deacetylase-mediated lysine modifications, Mol Cell Biol 25 (2005) 8456-8464.
[31] D. Schmidt, and S. Muller, PIAS/SUMO: new partners in transcriptional regulation, Cell Mol Life Sci 60 (2003) 2561-2574.
[32] D. Schmidt, and S. Muller, Members of the PIAS family act as SUMO ligases for c-Jun and p53 and repress p53 activity, Proc Natl Acad Sci U S A 99 (2002) 2872-2877.
[33] V. Yurchenko, Z. Xue, and M. J. Sadofsky, SUMO modification of human XRCC4 regulates its localization and function in DNA double-strand break repair, Mol Cell Biol 26 (2006) 1786-1794.
[34] P. Gomez-del Arco, J. Koipally, and K. Georgopoulos, Ikaros SUMOylation: switching out of repression, Mol Cell Biol 25 (2005) 2688-2697.
[35] M. Hochstrasser, SP-RING for SUMO: new functions bloom for a ubiquitin-like protein, Cell 107 (2001) 5-8.
[36] P. K. Jackson, A new RING for SUMO: wrestling transcriptional responses into nuclear bodies with PIAS family E3 SUMO ligases, Genes Dev 15 (2001) 3053-3058.
[37] T. Kahyo, T. Nishida, and H. Yasuda, Involvement of PIAS1 in the sumoylation of tumor suppressor p53, Mol Cell 8 (2001) 713-718.
[38] Y. Takahashi, T. Kahyo, E. A. Toh, H. Yasuda, and Y. Kikuchi, Yeast Ull1/Siz1 is a novel SUMO1/Smt3 ligase for septin components and functions as an adaptor between conjugating enzyme and substrates, J Biol Chem 276 (2001) 48973-48977.
[39] M. S. Rodriguez, C. Dargemont, and R. T. Hay, SUMO-1 conjugation in vivo requires both a consensus modification motif and nuclear targeting, J Biol Chem 276 (2001) 12654-12659.
[40] V. Hietakangas, J. Anckar, H. A. Blomster, M. Fujimoto, J. J. Palvimo, A. Nakai, and L. Sistonen, PDSM, a motif for phosphorylation-dependent SUMO modification, Proc Natl Acad Sci U S A 103 (2006) 45-50.
[41] R. Hartmann-Petersen, M. Seeger, and C. Gordon, Transferring substrates to the 26S proteasome, Trends Biochem Sci 28 (2003) 26-31.
[42] R. Layfield, D. Tooth, M. Landon, S. Dawson, J. Mayer, and A. Alban, Purification of poly-ubiquitinated proteins by S5a-affinity chromatography, Proteomics 1 (2001) 773-777.
[43] D. A. Wolters, M. P. Washburn, and J. R. Yates, 3rd, An automated multidimensional protein identification technology for shotgun proteomics, Anal Chem 73 (2001) 5683-5690.
[44] B. Bukau, J. Weissman, and A. Horwich, Molecular chaperones and protein quality control, Cell 125(2006)443-451.
[45] S. C. Kaul, K. Taira, O. M. Pereira-Smith, and R. Wadhwa, Mortalin: present and prospective, Exp Gerontol 37 (2002) 1157-1164.
[46] P. J. Muchowski, and J. L. Wacker, Modulation of neurodegeneration by molecular chaperones, Nat Rev Neurosci 6 (2005) 11-22.
[47] S. Murata, Y. Minami, M. Minami, T. Chiba, and K. Tanaka, CHIP is a chaperone-dependent E3 ligase that ubiquitylates unfolded protein, EMBO Rep 2 (2001) 1133-1138.
[48] H. McDonough, and C. Patterson, CHIP: a link between the chaperone and proteasome systems, Cell Stress Chaperones 8 (2003) 303-308.
[49] S. Murata, T. Chiba, and K. Tanaka, CHIP: a quality-control E3 ligase collaborating with molecular chaperones, Int J Biochem Cell Biol 35 (2003) 572-578.
[50] J. Jiang, C. A. Ballinger, Y. Wu, Q. Dai, D. M. Cyr, J. Hohfeld, and C. Patterson, CHIP is a U-box-dependent E3 ubiquitin ligase: identification of Hsc70 as a target for ubiquitylation, J Biol Chem 276(2001)42938-42944.
[51] S. Hatakeyama, M. Yada, M. Matsumoto, N. Ishida, and K. I. Nakayama, U box proteins as a new family of ubiquitin-protein ligases, J Biol Chem 276 (2001) 33111-33120.
[52] A. Devoy, T. Soane, R. Welchman, and R. J. Mayer, The ubiquitin-proteasome system and cancer, Essays Biochem 41 (2005) 187-203.
[53] S. Y. Fuchs, De-regulation of ubiquitin-dependent proteolysis and the pathogenesis of malignant melanoma, Cancer Metastasis Rev 24 (2005) 329-338.
[54] F. X. Bosch, J. Ribes, M. Diaz, and R. Cleries, Primary liver cancer: worldwide incidence and trends, Gastroenterology 127 (2004) S5-S16.
[55] T. Osada, M. Sakamoto, H. Nishibori, K. Iwaya, Y. Matsuno, T. Muto, and S. Hirohashi, Increased ubiquitin immunoreactivity in hepatocellular carcinomas and precancerous lesions of the liver, J Hepatol 26 (1997) 1266-1273.
[56] T. Osada, M. Sakamoto, H. Nagawa, J. Yamamoto, Y. Matsuno, A. Iwamatsu, T. Muto, and S. Hirohashi, Acquisition of glutamine synthetase expression in human hepatocarcinogenesis: relation to disease recurrence and possible regulation by ubiquitin-dependent proteolysis, Cancer 85 (1999) 819-831.
[57] S. J. Ding, Y. Li, X. X. Shao, H. Zhou, R. Zeng, Z. Y. Tang, and Q. C. Xia, Proteome analysis of hepatocellular carcinoma cell strains, MHCC97-H and MHCC97-L, with different metastasis potentials, Proteomics 4 (2004) 982-994.
[58] E. S. Johnson, Protein modification by SUMO, Annu Rev Biochem 73 (2004) 355-382.
[59] S. Sachdev, L. Bruhn, H. Sieber, A. Pichler, F. Melchior, and R. Grosschedl, PIASy, a nuclear matrix-associated SUMO E3 ligase, represses LEF1 activity by sequestration into nuclear bodies, Genes Dev 15 (2001) 3088-3103.
[60] T. H. Chun, H. Itoh, L. Subramanian, J. A. Iniguez-Lluhi, and K. Nakao, Modification of GATA-2 transcriptional activity in endothelial cells by the SUMO E3 ligase PIASy, Circ Res 92 (2003) 1201-1208.
[61] K. Shuai, and B. Liu, Regulation of gene-activation pathways by PIAS proteins in the immune system, Nat Rev Immunol 5 (2005) 593-605.
[62] J. K. Choe, M. Y. Dawood, W. A. Bardawil, and A. H. Andrews, Clinical and histologic evaluation of laser reanastomosis of the uterine tube, Fertil Steril 41 (1984) 754-760.
[63] J. Long, I. Matsuura, D. He, G. Wang, K. Shuai, and F. Liu, Repression of Smad transcriptional activity by PIASy, an inhibitor of activated STAT, Proc Natl Acad Sci U S A 100 (2003) 9791-9796.
[64] K. Petrie, F. Guidez, L. Howell, L. Healy, S. Waxman, M. Greaves, and A. Zelent, The histone deacetylase 9 gene encodes multiple protein isoforms, J Biol Chem 278 (2003) 16059-16072.
[1] Hershko A, Ciechanover A: The ubiquitin system. Annu Rev Biochem 1998, 67: 425-479.
[2] Pickart C. M. Mechanisms underlying ubiquitination. Annu. Rev. Biochem. 2001, 70: 503-533
[3] Natalia Shcherbik, Dale S. Haines : Ub on the Move. Journal of Cellular Biochemistry. 2004, 93:11-19
[4] S. Fanga, A. M.Weissman : A field guide to ubiquitylation. Cell. Mol. Life Sci. 2004, 61:1546-1561
[5] Schwartz DC, Hochstrasser M: A superfamily of protein tags: ubiquitin, SUMO and related modifiers. Trends Biochem Sci 2003,28:321-328.
[6] Wolters, D. A., Washburn, M. P., Yates, J. R. An automated multidimensional protein identification technology for shotgun proteomics. Anal. Chem. 2001: 73, 5683-5690
[ 7] Peng, J. et al. A proteomics approach to understanding protein ubiquitination. Nature Biotech. 2003, 21: 921-926
[ 8] Hitchcock, A. L., Auld, K., Gygi, S. P., Silver, P. A. A subset of membrane-associated proteins is ubiquitinated in response to mutations in the endoplasmic reticulum degradation machinery. Proc. Natl Acad. Sci. USA 2003, 100: 12735-12740
[ 9] Donald S. Kirkpatrickl. et al Proteomic identification of ubiquitinated proteins from human cells expressing His-tagged ubiquitin Proteomics 2005, 5:2104-2111
[10] Tsirigotis, M. et al. Analysis of ubiquitination in vivo using a transgenic mouse model. iotechniques 2001, 31:120-130
[11] Carilee Denison, Steven P. Gygi A Proteomic Strategy for Gaining Insights into Protein Sumoylation in Yeast. Molecular & Cellular Proteomics 2005, 4:246-254
[12] Zhou W, Ryan JJ, Zhou H: Global analyses of sumoylated proteins in Saccharomyces cerevisiae. Induction of protein sumoylation by cellular stresses. J Biol Chem 2004, 279:32262-32268.
[13] Wohlschlegel JA, Johnson ES, Reed SI, Yates JR III: Global analysis of protein sumoylation in Saccharomyces cerevisiae. J Biol Chem 2004. 44:45662-45668
[14] Panse VG, Hardeland U, Werner T, Kuster B, Hurt E: A proteome wide approach identifies sumoylated substrate proteins in yeast. J Biol Chem 2004. 40: 41346-41351
[15] Zhao Y, Kwon SW, Anselmo A, Kaur K, White MA: Broad spectrum identification of cellular small ubiquitin-related modifier (SUMO) substrate proteins. J Biol Chem 2004, 279:20999-21002.
[16] Vertegaal AC, Ogg SC, Jaffray E, Rodriguez MS, Hay RT, Andersen JS, Mann M, Lamond AI: A Proteomic study of SUMO-2 target proteins. J Biol Chem 2004, 279:33791-33798.
[17] Germa' n Rosas-Acosta. et al. A Universal Strategy for Proteomic Studies of SUMO and Other Ubiquitin-like Modifiers. Molecular & Cellular Proteomics 2005, 4:56-72
[18] Gururaja T, Li W, Noble WS, Payan DG, Anderson DC: Multiple functional categories of proteins identified in an in vitro cellular ubiquitin affinity extract using shotgun peptide sequencing. J Proteome Res 2003, 2:394-404.
[19] Sato K, Hayami R, Wu W, Nishikawa T, Nishikawa H, Okuda Y, Ogata H, Fukuda M, Ohta.T. Nucleophosmin/B23 is a candidatesubstrate for the BRCA1-BARD1 ubiquitin ligase. J Biol Chem 2004, 279:30919-30922.
[20] Starita LM, Machida Y, Sankaran S, Elias JE, Griffin K, Schlegel BP, Gygi SP, Parvin JD: BRCA1-dependent ubiquitination of gamma-tubulin regulates centrosome number. Mol Cell Biol 2004, 24:8457-8466.
[21 ] Castro A, Bernis C, Vigneron S,Labbe JC,Lorca T. The anaphase-promoting complex: a key factor in the regulation of cell cycle. 2005 24(3):314-325
[22] Masaki Matsumoto, Shigetsugu Hatakeyama, Koji Oyamada, Yoshiya Oda,Toshihide Nishimura, and Keiichi I. Nakayamal, Large-scale analysis of the human ubiquitin-related proteome Proteomics. 2005, 5(16):4145-4151
[23] John Weekesl, Karen Morrison 1. Anthony Mullen, Robin Wait, Paul Barton, Michael J. Dunn. Hyperubiquitination of proteins in dilated cardiomyopathy. Proteomics. 2003, 3: 208-216
[24] Ficarro SB, McCleland ML, Stukenberg PT, Burke DJ, Ross MM, Shabanowitz J, Hunt DF, White FM. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nature Biotechnol. 2002, 20:301-305
[25] Coulombe, P., Rodier, G, Bonneil, E., Thibault, P. & Meloche, S. N-Terminal ubiquitination of extracellular signal-regulated kinase 3 and p21 directs their degradation by the proteasome. Mol. Cell. Biol. 2004,24:6140-6150.
