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研究生:劉宏文
研究生(外文):Hong-Wen Liu
論文名稱:核醣體蛋白質L7的胺基酸重複序列在內質網結合與核醣體合成中之特性
論文名稱(外文):Characterization of the role of tandem repeated sequence of the ribosomal protein L7 in binding to the endoplasmic reticulum and in the ribosome assembly
指導教授:林茂榮林茂榮引用關係
指導教授(外文):Alan Lin
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:遺傳學研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2001
畢業學年度:89
語文別:中文
論文頁數:65
中文關鍵詞:核醣體蛋白質胺基酸重複序列內質網結合生物感測系統
外文關鍵詞:Ribosomal proteinTandem repeated sequenceER-bindingBiosensor
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根據本實驗室先前之研究發現核醣體上的核醣體蛋白L7,除了參與核醣體的合成外,還主導核醣體與內質網之間的結合作用。本篇論文主要是探討核醣體蛋白L7如何與內質網相互作用及其作用的結合區域。我們運用基因工程建構了一系列核醣體蛋白L7之變異蛋白質,然後利用內質網微粒漂浮實驗(microsome floating assay),証明當L7僅具氨基端前五十個胺基酸的重複序列時,便可與內質網微粒作用結合;反之,缺乏此重複序列之變異蛋白質則喪失了其結合內質網微粒的親和力。因此說明了此重複序列對於結合內質網是有其必要性並且具有專一性。我們也利用生物感測系統(biosensor: IAsys)來偵測其結合動力學上的分解平衡常數(KD),測得重複序列對內質網的結合親和力其數據與核醣體之KD值相類似。並且發現L7氨基端重複序列的個數之多寡對結合內質網微粒的親和力影響不大,但蛋白質至少須含一個或以上的重複序列才具有其結合內質網的能力。此推論也可經由比對多種L7同源蛋白質的初級結構以及此區域二級結構之親水性質的一致性得到佐證。另外,本篇論文也証明了此序列中帶有核集訊號(nuclear localization signal),並不能成功的進入核仁。同時應用生化免疫之技術測得此重複序列雖然屬於核醣體蛋白的一部分,卻不須參與核醣體的合成。此結果間接地說明此重複序列可能是暴露在核醣體的表面而參與核醣體與內質網之間的相互結合。
According to the previous finding of our laboratory, the ribosomal protein L7 is responsible for binding to the ER membrane. In this thesis, I examine the binding region of ribosomal protein L7 and determine the binding affinity to the ER. The results show that the NH2 terminal region of ribosomal protein L7 makes up a tandem repeated sequence and is essential for binding to the ER membrane. Using a biosensor assay, the dissociation equilibrium constant (KD) of the NH2 terminal tandem repeated sequence to bind to the ER is 2.89 x 10-8 M which is similar to the binding affinity of ribosome, suggesting that this sequence involves in the formation of rough ER. In the same assay, no significant difference in binding affinity to the ER is found for modified proteins regardless the number of repeated units, but protein without any repeated unit does not bind, suggesting that the repeated unit alone rather the number of copies is important to bind. This study suggests that the tandem repeated sequence of the ribosomal protein L7 does not itself intrinsically interact with the ribosomal components but externally engages in binding with the ER membrane assisting the ribosome in the formation of rough ER.
英 文 摘 要 …………………………………………………………... 1
中 文 摘 要 …………………………………………………………... 2
緒 論 …………………………………………………………………... 3
材 料 與 方 法 ……………………………………………………... 7
結 果
一、人類核醣體蛋白質L7及其變異蛋白質基因之建構…………… 28
二、人類核醣體蛋白質L7及其變異蛋白質之表現及純化………… 28
三、人類核醣體蛋白質L7及其變異蛋白質之試管內結合內質網
微粒的能力 …………………………………………………… 29
四、人類核醣體蛋白質L7氨基端重複序列與內質網微粒結合之
動力學分析 …………………………………………………... 31
五、核醣體蛋白質L7的氨基端重複序列在真核細胞內與核醣體
的合成之關係 ………………………………………………... 32
六、核醣體蛋白質L7的氨基端重複序列在真核細胞內表現之情
形 ………………………………………………………………… 33
圖 表 ………………………………………………………………… . 36
討 論
一、人類核醣體蛋白質L7基因序列比對之差異 ………………….. 50
二、人類核醣體蛋白質L7在細胞內已知之生化功能 …………….. 50
三、人類核醣體蛋白質L7的氨基端重複序列參與核醣體與內質
網微粒之結合…………………………………………………... 51
四、從同源核醣體蛋白質L7的序列比對證實此氨基端重複序列
對於結合內質網之重要………………………………………... 52
五、人類核醣體蛋白質L7氨基端重複序列之細胞表現 ………….. 53
六、細胞內核醣體與內質網結合之機制…………………………... 54
附 圖 …………………………………………………………………. 56
參 考 文 獻 …………………………………………………………. 59
1. Adelman, M.R., Sabatini, D.D., and Blobel, G. (1973). Ribosome- membrane interaction. Nondestructive disassembly of rat liver rough microsomes into ribosomal and membranous components. J. Cell Biol. 56, 206-229.
2. Beckmann R., Bubeck D., Grassucci R., Penczek P., Verschoor A.,Blobel G., and Frank J. (1997). Alignment of conduits for the nascent polypeptide chain in the ribosome-Sec61 complex. Science 278, 2123-2126.
3. Berghofer-Hochheimer, Y., Zurek, C., Wolfl, S., Hemmerich, P., and Munder, T. (1998). L7 protein is a coregulator of vitamin D receptor-retinoid X receptor-mediated transactivation. J. Cell Biochem. 69, 1-12.
4. Borgese, N., Mok, W., Kreibich, G., and Sabatini, D.D. (1974). Ribosomal-membrane interaction: in vitro binding of ribosomes to microsomal membranes. J. Mol. Biol. 88, 559-580.
5. Brodsky, J.L. (1998). Translocation of protein across the endoplasmic reticulum membrane. Int. Rev. Cytol. 178, 277-328.
6. Connolly, T., and Gilmore, R. (1989). The signal recognition particle receptor mediates the GTP-dependent displacement of SRP from the signal sequence of the nascent polypeptide. Cell 57, 599-610.
7. Connolly, T., Rapiejko, P.J., and Gilmore, R. (1991). Requirement of GTP hydrolysis for dissociation of the signal recognition particle from its receptor. Science 252, 1171-1173.
8. Crowley, K.S., Reinhart, G.D., and Johnson, A.E. (1993). The signal sequence moves through a ribosomal tunnel into a noncytoplasmic aqueous environment at the ER membrane early in translocation. Cell 73, 1101-1115.
9. Crowley, K.S., Liao, S., Worrell, V.E., Reinhart, G.D., and Johnson, A.E. (1994). Secretory proteins move through the endoplasmic reticulum membrane via an aqueous, gated pore. Cell 78, 461-471.
10. Edwards, P.R., and Leatherbarrow, R.J. (1997). Determination of association rate constants by an optical biosensor using initial rate analysis. Anal. Biochem. 246, 1-6.
11. Gierasch, L.M. (1989). Signal sequences. Biochemistry 28, 923-930.
12. Gilmore, R., Walter, P., and Blobel, G. (1982). Protein translocation across the endoplasmic reticulum. II. Isolation and characterization of the signal recognition particle receptor. J. Cell Biol. 95, 470-477.
13. Gorlich, D., and Rapoport, T.A. (1993). Protein translocation into proteoliposomes reconstituted from purified components of the endoplasmic reticulum membrane. Cell 75, 615-630.
14. Hamman, B.D., Chen, J.C., Johnson, E.E., and Johnson, A.E. (1997). The aqueous pore through the translocon has a diameter of 40-60 A during cotranslational protein translocation at the ER membrane. Cell 89, 535-544.
15. Hamman, B.D., Hendershot, L.M., and Johnson, A.E. (1998). BiP maintains the permeability barrier of the ER membrane by sealing the lumenal end of the translocon pore before and early in translocation. Cell 92, 747-758.
16. Hanein D., Matlack K.E., Jungnickel B., Plath K., Kalies K.U., Miller K.R., Rapoport T.A., and Akey C.W. (1996). Oligomeric rings of the Sec61p complex induced by ligands required for protein translocation. Cell 87, 721-732.
17. Hemmerich, P., von Mikecz, A., Neumann, F., Sozeri, O., Wolff- Vorbeck, G., Zoebelein, R., and Krawinkel, U. (1993). Structural and functional properties of ribosomal protein L7 from humans and rodents. Nucleic Acids Res. 21, 223-231.
18. Hemmerich, P., Bosbach, S., von Mikecz, A., and Krawinkel, U. (1997). Human ribosomal protein L7 binds RNA with an alpha- helical arginine-rich and lysine-rich domain. Eur. J. Biochem. 245, 549-556.
19. Hofmann, K., Bucher, P., Falquet, L., and Bairoch, A. (1999). The PROSITE database, its status in 1999. Nucleic Acids Res. 27, 215-219.
20. Hortsch, M., Avossa, D., and Meyer, D.I. (1986). Characterization of secretory protein translocation: ribosome-membrane interaction in endoplasmic reticulum, J. Cell Biol. 103, 241-253.
21. Izard, J.W., and Kendall, D.A. (1994). Signal peptides: exquisitely designed transport promoters. Mol. Microbiol. 13, 765-773.
22. Jackson, T.A., Richer, J.K., Bain, D.L., Takimoto, G.S., Tung, L., and Horwitz, K.B. (1997). The partial agonist activity of antagonist- occupied steroid receptors is controlled by a novel hinge domain- binding coactivator L7/SPA and the corepressors N-CoR or SMRT. Mol. Endocrinol. 11, 693-705.
23. Johnson, A.E., and van Waes, M.A. (1999). The translocon: a dynamic gateway at the ER membrane. Ann. Rev. Cell Dev. Biol. 15, 799-842.
24. Kalies, K.U., Gorlich, D., and Rapoport, T.A. (1994). Binding of ribosomes to the rough endoplasmic reticulum mediated by the Sec61p-complex. J. Cell Biol. 126, 925-934.
25. Kalies, K.U., and Hartmann, E. (1998). Protein translocation into the endoplasmic reticulum (ER)--two similar routes with different modes. Eur. J. Biochem. 254, 1-5.
26. Landschulz, W.H., Johnson, P.F., and McKnight, S.L. (1988). The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science 240, 1759-1764.
27. Lauring, B., Sakai, H., Kreibich, G., and Wiedmann, M. (1995). Nascent polypeptide-associated complex protein prevents mistargeting of nascent chains to the endoplasmic reticulum. Proc. Natl. Acad. Sci. USA 92, 5411-5415.
28. Liao, S., Lin, J., Do, H., and Johnson, A.E. (1997). Both lumenal and cytosolic gating of the aqueous ER translocon pore are regulated from inside the ribosome during membrane protein integration. Cell 90, 31-41.
29. Lin, A., Chan, Y.L., McNally, J., Peleg, D., Meyuhas, O., and Wool, I.G. (1987). The primary structure of rat ribosomal protein L7. The presence near the amino terminus of L7 of five tandem repeats of a sequence of 12 amino acids. J. Biol. Chem. 262, 12665-12671.
30. Lin, A. (1991). Localization of surface peptide from ribosomal protein L7 on 80S ribosome by biotinylation. FEBS Lett. 287, 121-124
31. Martin, T.E., Rolleston, F.S., Low, R.B., and Wool, I.G. (1969). Dissociation and reassociation of skeletal muscle ribosomes. J. Mol. Biol. 26, 135-149.
32. McArthur, A.G., Morrison, H.G., Nixon, J.E., Passamaneck, N.Q., Kim, U., Hinkle, G., Crocker, M.K., Holder, M.E., Farr, R., Reich, C.I., Olsen, G.E., Aley, S.B., Adam, R.D., Gillin, F.D., and Sogin, M.L. (2000). The Giardia genome project database. FEMS Microbiol. Lett. 15, 271-273.
33. Meyer T.H., Menetret J.F., Breitling R., Miller K.R., Akey C.W., and Rapoport T.A. (1999). The bacterial SecY/E translocation complex forms channel-like structures similar to those of the eukaryotic Sec61p complex. J. Mol. Biol. 285, 1789-1800.
34. Meyuhas, O., and Klein, A. (1990). The mouse ribosomal protein L7 gene. Its primary structure and functional analysis of the promoter region. J. Biol. Chem. 265, 11465-11473.
35. Neu, E., von Mikecz, A.H., Hemmerich, P.H., Peter, H.H., Fricke, M., Deicher, H., Genth, E., and Krawinkel, U. (1995). Autoantibodies against eukaryotic protein L7 in patients suffering from systemic lupus erythematosus and progressive systemic sclerosis: frequency and correlation with clinical, serological and genetic parameters. The SLE Study Group. Clin. Exp. Immunol. 100, 198-204.
36. Neumann, F., Hemmerich, P., von Mikecz, A., Peter, H.H., and Krawinkel, U. (1995). Human ribosomal protein L7 inhibits cell-free translation in reticulocyte lysates and affects the expression of nuclear proteins upon stable transfection into Jurkat T-lymphoma cells. Nucleic Acids Res. 23, 195-202.
37. Nice, E.C., and Catimel, B. (1999). Instrumental biosensors: new perspectives for the analysis of biomolecular interaction. Bioessays 21, 339-352.
38. Nomura, M. (1973). Assembly of bacterial ribosomes. Science 179, 864-873.
39. Pace, N.R., Olsen, G.J., and Woese, C. (1986). Ribosomal RNA phylogeny and the primary lines of evolutionary descent. Cell 45, 325-326.
40. Prinz, A., Behrens, C., Rapoport, T.A., Hartmann, E., and Kalies K.U. (2000). Evolutionarily conserved binding of ribosomes to the translocation channel via the large ribosomal RNA. EMBO J. 19, 1900-1906.
41. Rapoport, T.A., Rolls, M.M., and Jungnickel, B. (1996). Approaching the mechanism of protein transport across the ER membrane. Curr. Opin. Cell Biol. 8, 499-504.
42. Sommerville, J. (1986). Nucleolar structure and ribosome biogenesis. Trends Biochem. Sci. 11, 438-442.
43. von Heijne, G. (1990). The signal peptide. J. Membr. Biol. 115, 195-201.
44. von Mikecz, A.H., Hemmerich, P.H., Peter, H.H., and Krawinkel, U. (1995). Autoantigenic epitopes on eukaryotic L7. Clin. Exp. Immunol. 100, 205-213.
45. Walter, P., and Blobel, G. (1980). Purification of a membrane- associated protein complex required for protein translocation across the endoplasmic reticulum. Proc. Natl. Acad. Sci. USA 77, 7112- 7116.
46. Walter, P., and Blobel, G. (1982). Signal recognition particle contains a 7S RNA essential for protein translocation across the endoplasmic reticulum. Nature 299, 691-698.
47. Walter, P., and Lingappa, V.R. (1986). Mechanism of protein translocation across the endoplasmic reticulum membrane. Annu. Rev. Cell Biol. 2, 499-516.
48. Wang, L., Fast, D.G., and Attie, A.D. (1997). The enzymatic and non-enzymatic roles of protein-disulfide isomerase in apolipoprotein B secretion. J. Biol. Chem. 272, 27644-27651.
49. Witte, S., and Krawinkel, U. (1997). Specific interactions of the autoantigen L7 with multi-zinc finger protein ZNF7 and ribosomal protein S7. J. Biol. Chem. 272, 22243-22247.
50. Wool, I.G., Chan, Y.L., and Gluck, A. (1995). Structure and evolution of mammalian ribosomal proteins. Biochem. Cell Biol. 73, 933-947.
51. Wool, I.G. (1996). Extraribosomal functions of ribosomal proteins. Trends Biochem. Sci. 21, 164-165.
52. Wu, V-C., and Lin, A. (2001). The anchoring role of eukaryotic ribosomal protein L7 in making rough endoplasmic reticulum. Submitted to EMBO J.
53. 林達聰(1998). 核醣蛋白與內質網的結合作用. 國立陽明大學遺傳所碩士論文。
54. 吳偉正(2000). 真核核醣蛋白L7扮演一個"錨"的角色使核醣體能停靠在內質網上形成顆粒性內質網. 國立陽明大學遺傳所碩士論文。
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