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標題: 人類第四型胜肽精胺酸去亞胺酶第三, 四, 五號鈣離子結合位對於酵素活性以及結構穩定性的影響
Influences of Calcium Binding Sites 3, 4, 5 on the Structural Stability and Activity of Peptidylarginine Deiminase Type 4
作者: 蔡一成
Tsai, I-Chen
關鍵字: Peptidylarginine Deiminase Type 4
出版社: 生命科學系所
引用: 1. Zendman, A.J., et al., ABAP: antibody-based assay for peptidylarginine deiminase activity. Anal Biochem, 2007. 369(2): p. 232-40. 2. Girbal-Neuhauser, E., et al., The epitopes targeted by the rheumatoid arthritis-associated antifilaggrin autoantibodies are posttranslationally generated on various sites of (pro)filaggrin by deimination of arginine residues. J Immunol, 1999. 162(1): p. 585-94. 3. Nienhuis, R.L. and E. Mandema, A New Serum Factor in Patients with Rheumatoid Arthritis; the Antiperinuclear Factor. Ann Rheum Dis, 1964. 23: p. 302-5. 4. Vossenaar, E.R., et al., PAD, a growing family of citrullinating enzymes: genes, features and involvement in disease. Bioessays, 2003. 25(11): p. 1106-18. 5. Nakashima, K., T. Hagiwara, and M. Yamada, Nuclear localization of peptidylarginine deiminase V and histone deimination in granulocytes. J Biol Chem, 2002. 277(51): p. 49562-8. 6. Hagiwara, T., et al., Deimination of arginine residues in nucleophosmin/B23 and histones in HL-60 granulocytes. Biochem Biophys Res Commun, 2002. 290(3): p. 979-83. 7. Thompson, P.R. and W. Fast, Histone citrullination by protein arginine deiminase: is arginine methylation a green light or a roadblock? ACS Chem Biol, 2006. 1(7): p. 433-41. 8. Barton, A., et al., A functional haplotype of the PADI4 gene associated with rheumatoid arthritis in a Japanese population is not associated in a United Kingdom population. Arthritis Rheum, 2004. 50(4): p. 1117-21. 9. Kang, C.P., et al., A functional haplotype of the PADI4 gene associated with increased rheumatoid arthritis susceptibility in Koreans. Arthritis Rheum, 2006. 54(1): p. 90-6. 10. Caponi, L., et al., A family based study shows no association between rheumatoid arthritis and the PADI4 gene in a white French population. Ann Rheum Dis, 2005. 64(4): p. 587-93. 11. Suzuki, A., et al., Functional haplotypes of PADI4, encoding citrullinating enzyme peptidylarginine deiminase 4, are associated with rheumatoid arthritis. Nat Genet, 2003. 34(4): p. 395-402. 12. Cantaert, T., et al., Functional haplotypes of PADI4: relevance for rheumatoid arthritis specific synovial intracellular citrullinated proteins and anticitrullinated protein antibodies. Ann Rheum Dis, 2005. 64(9): p. 1316-20. 13. Ikari, K., et al., Association between PADI4 and rheumatoid arthritis: a replication study. Arthritis Rheum, 2005. 52(10): p. 3054-7. 14. Fan, L.Y., et al., A functional haplotype and expression of the PADI4 gene associated with increased rheumatoid arthritis susceptibility in Chinese. Tissue Antigens, 2008. 72(5): p. 469-73. 15. Gandjbakhch, F., et al., A Functional Haplotype of PADI4 Gene in Rheumatoid Arthritis: Positive Correlation in a French Population. J Rheumatol, 2009. 16. Raptopoulou, A., et al., Anti-citrulline antibodies in the diagnosis and prognosis of rheumatoid arthritis: evolving concepts. Crit Rev Clin Lab Sci, 2007. 44(4): p. 339-63. 17. Vossenaar, E.R., et al., Expression and activity of citrullinating peptidylarginine deiminase enzymes in monocytes and macrophages. Ann Rheum Dis, 2004. 63(4): p. 373-81. 18. Masson-Bessiere, C., et al., In the rheumatoid pannus, anti-filaggrin autoantibodies are produced by local plasma cells and constitute a higher proportion of IgG than in synovial fluid and serum. Clin Exp Immunol, 2000. 119(3): p. 544-52. 19. El-Gabalawy, H.S. and J.A. Wilkins, Anti-Sa antibodies: prognostic and pathogenetic significance to rheumatoid arthritis. Arthritis Res Ther, 2004. 6(2): p. 86-9. 20. Vossenaar, E.R., et al., Rheumatoid arthritis specific anti-Sa antibodies target citrullinated vimentin. Arthritis Res Ther, 2004. 6(2): p. R142-50. 21. Kearney, P.L., et al., Kinetic characterization of protein arginine deiminase 4: a transcriptional corepressor implicated in the onset and progression of rheumatoid arthritis. Biochemistry, 2005. 44(31): p. 10570-82. 22. Santoro, M.M. and D.W. Bolen, Unfolding free energy changes determined by the linear extrapolation method. 1. Unfolding of phenylmethanesulfonyl alpha-chymotrypsin using different denaturants. Biochemistry, 1988. 27(21): p. 8063-8. 23. Hung, H.C. and G.G. Chang, Multiple unfolding intermediates of human placental alkaline phosphatase in equilibrium urea denaturation. Biophys J, 2001. 81(6): p. 3456-71. 24. Arita, K., et al., Structural basis for Ca(2+)-induced activation of human PAD4. Nat Struct Mol Biol, 2004. 11(8): p. 777-83.
摘要: 胜肽精胺酸 (Peptidylarginine deiminase) 是一種後轉譯修飾酵素,在有鈣離子的情況下可以將蛋白質上的精胺酸 (arginine)轉變為瓜胺酸 (citrulline),胜肽精胺酸總共有五種異構型 (isoforms) ,其中,第四型胜肽精胺酸近年來被認為是研究類風濕性關節炎的重要對象。第四型胜肽精胺酸和鈣離子結合前後的結構,都已經被結晶分析出來。從蛋白結構中可以知道,第四型胜肽精胺酸有五個鈣離子的結合位,分別將這些鈣離子簡稱為 Ca1、Ca2、Ca3、Ca4、Ca5,酵素在鈣離子的結合下會促使結構的變化,使催化中心區域可以跟受質結合。由過去的研究已知,Ca1和Ca2的結合位置和催化中心相近,並且會協助催化中心的形成。然而,Ca3、Ca4、Ca5的位置離活性中心較遠,且其功能也尚未確定;在此,我們利用胺基酸點突變技術 (site-directed mutagenesis) 配合酵素動力學實驗,針對Ca3、Ca4、Ca5 結合位附近的胺基酸作討論,這些胺基酸分別為N153、D155、D157、D165、D176、D179、D388,將這些胺基酸分別置換為丙胺酸 (alanine)或是結構相近但不帶電的胺基化合物胺基酸 (amide amino acid),或是攜帶相同負電荷的胺基酸 (negatively charged amino acid)。從結果可以發現,Ca5結合位的重要性沒有Ca3、Ca4結合位來的明顯。從蛋白圓二色光譜 (Circular dichrois)以及螢光 (fluorescence)的實驗中,我們也得知Ca3、Ca4結合位的胺基酸對於酵素的二級以及三級結構的重要性。總結以上的實驗,我們知道雖然Ca3、Ca4、Ca5結合位雖然不像Ca1、Ca2結合位這麼接近活性中心,但是Ca3、Ca4、Ca5結合位的存在,對於酵素的結構以及活性上都有一定程度的影響。
As a post-translational modifying protein, peptidylarginine deiminase (PAD) catalyzes the conversion of protein arginine to citrulline in the presence of calcium. PAD4, one of the PAD isoform family, which is currently considered as the important factor of rheumatoid arthritis. The crystal structure of PAD4 with or without calcium ions have been resolved by X-ray crystallography. The inactive conformation of Ca2+-free PAD4 was changed to an active conformation after calcium binding. There are five calcium binding sites on PAD4, which are designated Ca1, Ca2, Ca3, Ca4, and Ca5, respectively. Ca1 and Ca2, near the enzyme catalytic site, are confirmed as critical calcium ions binding sites which assist enzyme with recognizing the substrate. There are additional three calcium ion binding sites in the N-terminal domain, the binding sties which are away from the catalytic site and the functional roles are still undefined. To interpret the role of Ca3, Ca4, and Ca5 binding sites, site-directed mutagenesis and enzyme kinetic analysis was used. Residues N153, D155, D157, D165, D176, D179, and D388, are replaced by alanine, amide, or negative charge amino acid to interrupt the calcium binding on this region. These alanine mutants at the Ca3 and Ca4 binding sites show higher Km and lower kcat than WT, suggesting that Ca3 and Ca4 are ennential for PAD4 catalysis. Circular dichroism and fluorescence studies further demonstrate that these mutants display different conformations as compared with WT. Our findings show that Ca3 and Ca4, although far away from the active site, still have significant influence on substrate binding, structural coordination, and calcium cooperativity.
其他識別: U0005-2107200910485000
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