Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22695
標題: 第三, 四, 五號鈣離子結合位在人類第四型胜肽精胺酸去亞胺酶中所扮演的角色
Functional Characterization of Calcium Binding Sites 3, 4, 5 in Peptidylarginine Deiminase type 4
作者: 張家維
Chang, Chia-Wei
關鍵字: Peptidylarginine Deiminase type 4;第四型胜肽精胺酸去亞胺酶
出版社: 生命科學系所
引用: 1. Ishigami A, Ohsawa T, Asaga H, Akiyama K, Kuramoto M, Maruyama N: Human peptidylarginine deiminase type II: molecular cloning, gene organization, and expression in human skin. Arch Biochem Biophys 2002, 407(1):25-31. 2. Guerrin M, Ishigami A, Mechin MC, Nachat R, Valmary S, Sebbag M, Simon M, Senshu T, Serre G: cDNA cloning, gene organization and expression analysis of human peptidylarginine deiminase type I. Biochem J 2003, 370(Pt 1):167-174. 3. Kanno T, Kawada A, Yamanouchi J, Yosida-Noro C, Yoshiki A, Shiraiwa M, Kusakabe M, Manabe M, Tezuka T, Takahara H: Human peptidylarginine deiminase type III: molecular cloning and nucleotide sequence of the cDNA, properties of the recombinant enzyme, and immunohistochemical localization in human skin. J Invest Dermatol 2000, 115(5):813-823. 4. Nakashima K, Hagiwara T, Ishigami A, Nagata S, Asaga H, Kuramoto M, Senshu T, Yamada M: Molecular characterization of peptidylarginine deiminase in HL-60 cells induced by retinoic acid and 1alpha,25-dihydroxyvitamin D(3). J Biol Chem 1999, 274(39):27786-27792. 5. Chavanas S, Mechin MC, Takahara H, Kawada A, Nachat R, Serre G, Simon M: Comparative analysis of the mouse and human peptidylarginine deiminase gene clusters reveals highly conserved non-coding segments and a new human gene, PADI6. Gene 2004, 330:19-27. 6. Vossenaar ER, Zendman AJ, van Venrooij WJ, Pruijn GJ: PAD, a growing family of citrullinating enzymes: genes, features and involvement in disease. Bioessays 2003, 25(11):1106-1118. 7. Vossenaar ER, Radstake TR, van der Heijden A, van Mansum MA, Dieteren C, de Rooij DJ, Barrera P, Zendman AJ, van Venrooij WJ: Expression and activity of citrullinating peptidylarginine deiminase enzymes in monocytes and macrophages. Ann Rheum Dis 2004, 63(4):373-381. 8. Nakashima K, Hagiwara T, Yamada M: Nuclear localization of peptidylarginine deiminase V and histone deimination in granulocytes. J Biol Chem 2002, 277(51):49562-49568. 9. Thompson PR, Fast W: Histone citrullination by protein arginine deiminase: is arginine methylation a green light or a roadblock? ACS Chem Biol 2006, 1(7):433-441. 10. Hagiwara T, Nakashima K, Hirano H, Senshu T, Yamada M: Deimination of arginine residues in nucleophosmin/B23 and histones in HL-60 granulocytes. Biochem Biophys Res Commun 2002, 290(3):979-983. 11. Firestein GS: Evolving concepts of rheumatoid arthritis. Nature 2003, 423(6937):356-361. 12. Reparon-Schuijt CC, van Esch WJ, van Kooten C, Schellekens GA, de Jong BA, van Venrooij WJ, Breedveld FC, Verweij CL: Secretion of anti-citrulline-containing peptide antibody by B lymphocytes in rheumatoid arthritis. Arthritis Rheum 2001, 44(1):41-47. 13. Masson-Bessiere C, Sebbag M, Girbal-Neuhauser E, Nogueira L, Vincent C, Senshu T, Serre G: The major synovial targets of the rheumatoid arthritis-specific antifilaggrin autoantibodies are deiminated forms of the alpha- and beta-chains of fibrin. J Immunol 2001, 166(6):4177-4184. 14. Mizoguchi M, Manabe M, Kawamura Y, Kondo Y, Ishidoh K, Kominami E, Watanabe K, Asaga H, Senshu T, Ogawa H: Deimination of 70-kD nuclear protein during epidermal apoptotic events in vitro. J Histochem Cytochem 1998, 46(11):1303-1309. 15. Asaga H, Yamada M, Senshu T: Selective deimination of vimentin in calcium ionophore-induced apoptosis of mouse peritoneal macrophages. Biochem Biophys Res Commun 1998, 243(3):641-646. 16. Suzuki A, Yamada R, Chang X, Tokuhiro S, Sawada T, Suzuki M, Nagasaki M, Nakayama-Hamada M, Kawaida R, Ono M et al: Functional haplotypes of PADI4, encoding citrullinating enzyme peptidylarginine deiminase 4, are associated with rheumatoid arthritis. Nat Genet 2003, 34(4):395-402. 17. Cantaert T, Coucke P, De Rycke L, Veys EM, De Keyser F, Baeten D: Functional haplotypes of PADI4: relevance for rheumatoid arthritis specific synovial intracellular citrullinated proteins and anticitrullinated protein antibodies. Ann Rheum Dis 2005, 64(9):1316-1320. 18. Barton A, Bowes J, Eyre S, Spreckley K, Hinks A, John S, Worthington J: 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):1117-1121. 19. Kang CP, Lee HS, Ju H, Cho H, Kang C, Bae SC: A functional haplotype of the PADI4 gene associated with increased rheumatoid arthritis susceptibility in Koreans. Arthritis Rheum 2006, 54(1):90-96. 20. Hung HC, Lin CY, Liao YF, Hsu PC, Tsay GJ, Liu GY: The functional haplotype of peptidylarginine deiminase IV (S55G, A82V and A112G) associated with susceptibility to rheumatoid arthritis dominates apoptosis of acute T leukemia Jurkat cells. Apoptosis 2007, 12(3):475-487. 21. Arita K, Hashimoto H, Shimizu T, Nakashima K, Yamada M, Sato M: Structural basis for Ca(2+)-induced activation of human PAD4. Nat Struct Mol Biol 2004, 11(8):777-783. 22. Arita K, Shimizu T, Hashimoto H, Hidaka Y, Yamada M, Sato M: Structural basis for histone N-terminal recognition by human peptidylarginine deiminase 4. Proc Natl Acad Sci U S A 2006, 103(14):5291-5296. 23. Kearney PL, Bhatia M, Jones NG, Yuan L, Glascock MC, Catchings KL, Yamada M, Thompson PR: Kinetic characterization of protein arginine deiminase 4: a transcriptional corepressor implicated in the onset and progression of rheumatoid arthritis. Biochemistry 2005, 44(31):10570-10582. 24. Liao YF, Hsieh HC, Liu GY, Hung HC: A continuous spectrophotometric assay method for peptidylarginine deiminase type 4 activity. Anal Biochem 2005, 347(2):176-181. 25. Calhoun DB, Vanderkooi JM, Englander SW: Penetration of small molecules into proteins studied by quenching of phosphorescence and fluorescence. Biochemistry 1983, 22(7):1533-1539. 26. Lehrer SS, Leavis PC: Solute quenching of protein fluorescence. Methods in enzymology 1978, 49:222-236. 27. Schuck P: On the analysis of protein self-association by sedimentation velocity analytical ultracentrifugation. Anal Biochem 2003, 320(1):104-124. 28. Schwede T, Kopp J, Guex N, Peitsch MC: SWISS-MODEL: An automated protein homology-modeling server. Nucleic acids research 2003, 31(13):3381-3385. 29. Nakayama-Hamada M, Suzuki A, Kubota K, Takazawa T, Ohsaka M, Kawaida R, Ono M, Kasuya A, Furukawa H, Yamada R et al: Comparison of enzymatic properties between hPADI2 and hPADI4. Biochem Biophys Res Commun 2005, 327(1):192-200.
摘要: 
胜肽精胺酸去亞胺酶 (Peptidylarginine deiminase)是一種後轉譯修飾的酵素,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),胜肽精胺酸去亞胺酶總共有五種異構型 (isoforms),其中第四型胜肽精胺酸去亞胺酶近年來被認為是偵測類風濕性關節炎的重要因子。到目前為止,只有第四型胜肽精胺酸去亞胺酶的結構被結晶出來,從蛋白結構中可以知道第四型胜肽精胺酸去亞胺酶有五個鈣離子的結合位,分別為將這些鈣離子簡稱為Ca1、Ca2、Ca3、Ca4、Ca5,酵素在鈣離子的結合之下會促使結構的變化,使催化中心區域可以跟受質結合。過去的研究已知,Ca1和Ca2的結合位置鄰近催化中心並且會協助催化中心的形成,然而Ca3、Ca4、Ca5的功能目前還不確定,所以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做研究,這些胺基酸分別為N153、D155、D157、D165、D168、E170、D176、D179、E252、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醯胺胺基酸 (amide 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,即使將Ca5結合位完全去除,酵素仍保有一半左右的活性,然而Ca3和Ca4結合位的破壞,會導致酵素活性大為減低;從蛋白質螢光 (fluorescence)的實驗中,我們也得知Ca3、Ca4、Ca5結合位的胺基酸對於酵素的三級結構有不同程度的影響性,這也間接解釋了Ca3、Ca4、Ca5結合位對於酵素結構的重要性。總結以上的實驗,我們可以知道雖然Ca3、Ca4、Ca5結合位不像Ca1、Ca2結合位這麼接近活性中心,但是Ca3、Ca4、Ca5結合位的存在,對於酵素的三級結構及活性都有一定程度上的影響。

Peptidylarginine deiminase (PAD) is a post-translational modifying enzyme which catalyzes the conversion of protein arginine to citrulline in the presence of calcium. The PAD family has five isoforms and one of them, PAD4, is considered as the important factor of rheumatoid arthritis. The structures of PAD4 without or with calcium ions have been resolved by X-ray crystallography. There are five calcium ions occupying on the enzyme that are designated Ca1, Ca2, Ca3, Ca4, and Ca5, respectively. Calcium-binding induces PAD4 conformational change to form the active-site cleft. The Ca1 and Ca2, which are near to the catalytic site, are defined as critical calcium ions that assist in recognition of the substrate. The other three calcium ions are in the N-terminal domain away from the catalytic site. The functional roles of Ca3, Ca4, and Ca5 are still undefined. Here we use site-directed mutagenesis and enzyme kinetic analysis to interpret the role of Ca3, Ca4, and Ca5 binding sites. The ten residues, N153, D155, D157, D165, D168, E170, D176, D179, E252, and D388, are replaced by alanine or amide amino acid to interrupt the calcium binding on this region. These mutants at the Ca3 and Ca4 binding sites show higher Km and lower kcat than WT, suggesting that Ca3 and Ca4 are essential for PAD4 catalysis. Our fluorescence studies further demonstrate that these mutants display different conformations as compared with WT. The mutants of D168, E170, and E252, which participate the Ca5 binding, have weaker effects on Km and lower kcat than Ca3 and Ca4. Our findings show that Ca3 and Ca4, although far away from the active site, still have significant influence on substrate binding and calcium cooperativity.
URI: http://hdl.handle.net/11455/22695
其他識別: U0005-1807200818295500
Appears in Collections:生命科學系所

Show full item record
 

Google ScholarTM

Check


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.