跳到主要內容

臺灣博碩士論文加值系統

(44.212.99.208) 您好!臺灣時間:2024/04/17 17:18
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:陳佳儀
研究生(外文):Chia-Yi Chen
論文名稱:鉀離子通道結合蛋白在二價離子誘導下構型影響及聚合作用之研究
論文名稱(外文):Divalent cation-induced conformational changes and oligomerization of KChIP1
指導教授:張榮賢
指導教授(外文):Long-Sen Chang
學位類別:碩士
校院名稱:國立中山大學
系所名稱:生物醫學科學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:66
中文關鍵詞:聚合作用鉀離子通道構型變化
外文關鍵詞:KChIPconformational changeoligomerization
相關次數:
  • 被引用被引用:0
  • 點閱點閱:169
  • 評分評分:
  • 下載下載:6
  • 收藏至我的研究室書目清單書目收藏:0
摘要
KChIPs family為鉀離子通道結合蛋白,可與電位驅動型鉀離子通道(Kv4)��-subunit之N端產生分子間作用力,並調控其功能。 此蛋白質家族經alternative splicing作用產生許多isoforms,其胺基酸序列主要差異在N端,而C端含有保留性之4個EF hands。 而KChIP3/Calsenilin/DREAM已被證實為一具有multi-function之蛋白質,在細胞中不同位置可與不同目標分子結合,且受到鈣離子調控其聚合作用以及DNA結合性質。 本論文利用5’-RACE得到KChIPs cDNA之後,進一步轉殖入表現載體於E.coli系統成功表現重組蛋白。 藉1-anilinonaphthalene-8-sulfonate(ANS)可與蛋白質疏水性區域結合之特性,偵測KChIPs重組蛋白在二價金屬離子(Mg2+,Ca2+,Sr2+,Ba2+)誘導下之構形變化;並以螢光強度變化相對於離子濃度求得解離常數。 結果得到KChIP1及KChIP2具有兩種鈣離子親和性,而對於其他離子則只有一種。 去除掉第四個EF hand之後僅可測得一種鈣離子解離常數,且親和性與其他金屬離子相似,顯示第四個EF hand對於高親和性鈣離子結合部位扮演重要功能。 去除第三及第四個EF hands之後,仍具有鈣離子結合功能,但已無法結合ANS,且其CD spectra與wild type顯著不同,顯示第三及第四個EF hands在KChIP1構形上之重要性。 此外,in vitro蛋白質分子間聚合實驗結果得到KChIP1及KChIP2之聚合作用亦受到鈣離子調控,而pull down則證實兩者可形成hetero-oligomer,且N端並未參與此蛋白質分子間之聚合反應。
Abstract

KChIPs are Kv channel-interacting proteins that bind to the cytoplasmic N-terminus of Kv4 ��-subunits and regulate the ion-current of Kv channel. Several isomeric KChIPs arise from alternative splicing have been identified. These KChIPs share a high degree of sequence homology at their C-terminal region which is constituted with four EF hands, but their N-terminus sequence is notably diversified. Among them, KChIP3/Calseniline/Dream has been found to be a multifunctional protein, and interacts with different targets according to its cellular sub-localization. Noticeably, KChIP3 shows a Ca2+-dependent DNA-binding and oligomerization. In the present study, using 5’-RACE PCR, the KChIP 1 and KChIP2 were successfully amplified from human brain and heart cDNA libraries, respectively. The recombinant KChIP proteins were prepared from E. coli expression system, and purified by His-bind resin or GST-resin according to their fusing proteins. The interaction between the KChIP proteins with divalent cations (Mg2+, Ca2+, Sr2+ and Ba2+) was explored by 8-anilinonaphthalene sulfonate (ANS) fluorescence. The results showed that KChIPs possessed both high affinity and low affinity Ca2+-binding sites. However, only one kind of binding-affinity for the other divalent cations was noted. Removal of EF hand 4 caused an abolishment of the high Ca2+-affinity, and the resulting mutated protein exhibited a similar binding affinity for Ca2+, Mg2+, Sr2+ and Ba2+. The mutant lacked EF hands 3 and 4 lost its ANS-binding ability, but it still could bind with Ca2+. Moreover, this mutated KChIP showed a noticeable change in its secondary structure as evidenced by CD spectra. The results of in vitro cross-linking indicated that KChIP1 and KChIP2 could form homo-oligomer in a Ca2+-dependent manner. Interestingly, hetero-oligomerization between KChIP1 and KChIP2 was noted as revealed by pull down assay. The N-terminal region was not involved in homo- or hetero-oligomerization of KChIPs. Taken together, our findings suggest that the integrity of EF hand 4 is essential for the high affinity for Ca2+, and both EF hands 3 and 4 are crucial for maintaining the conformation of KChIPs. However, the oligomerization reaction is highly dependent upon the presence of EF hands.
目錄
頁次
壹、中文摘要……….………...……………………………………….1
貳、英文摘要…………….….………………………………………...2
參、縮 寫……………..…….……………………………………..4
肆、緒 論.…………….….………………………………………..5
伍、材 料……….…….………………………………………….11
陸、實驗方法….………….…….……………………………………13
cDNA端點快速放大……………………………………………………..13
去氧核醣核酸序列分析……...…………………………………………..14
表現載體之建構……………...…………………………………………..14
基因轉型………………...………………………………………………..16
重組蛋白質的大量表現…………...……………………………………..17
蛋白質定量…………………...…………………………………………..18
SDS-PAGE…………………...…………………………………………...18
螢光測定……………………………...…………………………………..19
偏極光譜分析…………………...………………………………………..20
鈣離子結合能力之測定……………...…………………………………..20
蛋白質聚合反應……………...…………………………………………..21
Pull down assay……………………...…………………………………....21
西方墨點法….…………....………………………………………………22
DNA結合能力之分析…….…………….…………………………….…22
NATIVE-PAGE…………………...………………………………………23
柒、實驗結果……….……….……………………………………….24
KChIPs cDNA之選殖……………………………………………………24
重組蛋白質之表現與純化………...……………………………………..24
螢光測定………………….…..…………………………………………..26
鈣離子結合能力分析…………...………………………………………..27
二級結構圖譜分析……………………...………………………………..28
蛋白質分子間交互作用………………...………………………………..28
DRE結合能力分析……………….……………………………………..30
His-KChIP重組蛋白之抗原性偵測…………………………………….30
捌、討 論.…….……….…………………………………………31
玖、圖 表…………...……………………………………………37
拾、參考文獻…………………...……………………………………61
參考文獻
Ames, J. B., Tanaka, T., Stryer, L. and Ikura, M.(1996)Portrait of a myristoyl switch protein. Curr. Opin. Struct. Biol. 6, 432-438.

An, W. F., Bowlby, M. R., Betty, M., Cao, J., Ling, H. P., Mendoza, G., Hinson, J. W., Mattsson, K. I., Strassle, B. W., Trimmer, J. S. and Rhodes, K. J.(2000)Modulation of A-type potassium channels by a family of calcium sensors. Nature 403, 553-556.

Bahring, R., Dannenberg, J., Peters, H. C., Leicher, T., Pongs, O. and Isbrandt, D.(2001)Conserved Kv4 N-terminal domain critical for effects of Kv channel-interacting protein 2.2 on channel expression and gating. J. Biol. Chem. 276, 23888-23894.

Berg, J. M., Tymoczko, J. L. and Stryer, L.(2001)Signal-transduction pathways : an introduction to information metabolism., In : Biochemistry 5th ed. pp. 410, W. H. Freeman and Company, New York.

Braunewell, K. H. and Gundelfinger, E. D.(1999)Intracellular neuronal calcium sensor proteins:a family of EF-hand calcium-binding proteins in search of a function. Cell Tissue Res. 295, 1-12.

Buxbaum, J. D., Choi, E. K., Luo, Y., Lilliehook, C., Crowley, A. C., Merriam, D. E. and Wasco, W.(1998)Calsenilin:a calcium-binding protein that interacts with the presenilins and regulates the levels of a presenilin fragment. Nature Med. 4, 1177-1181.

Carrion, A. M., Mellstrom, B. and Naranjo, J. R.(1998)Protein kinase A-dependent derepression of the human prodynorphin gene via differential binding to an intragenic silencer element. Mol. Cell. Biol. 18, 6921-6929.

Carrion, A. M., Link, W. A., Ledo, F., Mellstrom, B. and Naranjo, J. R.(1999)DREAM is a Ca2+-regulated transcriptional repressor. Nature 398, 80-84.

Cheng, H. Y., Pitcher, G. M., Laviolette, S. R., Whishaw, I. Q., Tong, K. I., Kockeritz, L. K., Wada, T., Joza, N. A., Crackower, M., Goncalves, J., Sarosi, I., Woodgett, J. R., Oliveira-dos-Santos, A. J., Ikura, M., van der Kooy, D., Salter, M. W. and Penninger, J. M.(2002)DREAM is a critical transcriptional repressor for pain modulation. Cell 108, 31-43.

Choe, S.(2002)Potassium channel structures. Nat. Rev. Neurosci. 3, 115-121.

Craig, T. A., Benson, L. M., Venyaminov, S. Y., Klimtchuk, E. S., Bajzer, Z., Prendergast, F. G., Naylor, S. and Kumar, R.(2002)The metal-binding properties of DREAM. J. Biol. Chem. 277, 10955-10966.

Decher, N., Uyguner, O., Scherer, C. R., Karaman, B., Yuksel-Apak, M., Busch,A.E., Steinmeyer,K. and Wollnik,B.(2001)hKChIP2 is a functional modifier of hKv4.3 potassium channels : cloning and expression of a short hKChIP2 splice variant. Cardiovasc. Res. 52, 255-264.

Deschenes, I., DiSilvestre, D., Juang, G. J., Wu, R. C., An, W. F. and Tomaselli, G. F.(2002)Regulation of Kv4.3 current by KChIP2 splice variants : a component of native cardiac I(to)? Circulation 106, 423-429.

Dixon, J. E., Shi, W., Wang, H. S., McDonald, C., Yu, H., Wymore, R. S., Cohen, I. S. and McKinnon, D.(1996)Role of the Kv4.3 K+ channel in ventricular muscle. A molecular correlate for the transient outward current. Circ. Res. 79, 659-668.

Falke, J. J., Synder, E. E., Thatcher, K. C. and Voertler, C. S.(1991)Quantitating and engineering the ion specificity of an EF-hand-like Ca2+ binding site. Biochemistry 30, 8690-8697.

Hoffman, D. A., Magee, J. C., Colbert, C. M., and Johnston, D.(1997)K+ channel regulation of signal propagation in dendrites of hippocampal pyramidal neurons. Nature 387, 869-875.

Holmes, T. C., Fadool, D. A., Ren, R. and Levitan, I. B.(1996)Association of Src tyrosine kinase with a human potassium channel mediated by SH3 domain. Science 274, 2089-2091.

Holmqvist, M. H., Cao, J., Knoppers, M. H., Jurman, M. E., Distefano, P. S., Rhodes, K. J., Xie, Y. and An, W. F.(2001)Kinetic modulation of Kv4-mediated A-current by arachidonic acid is dependent on potassium channel interacting proteins. J. Neurosci. 21, 4154-4161.

Holmqvist, M. H., Cao, J., Hernandez-Pineda, R., Jacobson, M. D., Carroll, K. I., Sung, M. A., Betty, M., Ge, P., Gilbride, K. J., Brown, M. E., Jurman, M. E., Lawson, D., Silos-Santiago, I., Xie, Y., Covarrubias, M., Rhodes, K. J., Distefano,P.S. and An,W.F.(2002)Elimination of fast inactivation in Kv4 A-type potassium channels by an auxiliary subunit domain. Proc. Natl. Acad. Sci. 99, 1035-1040.

Johnson, J. D., El-Bayoumi, M. A., Weber, L. D. and Tulinsky, A.(1979)Interaction of ��-chymotrypsin with the fluorescent probe 1-anilinonaphthalene-8-sulfonate in solution. Biochemistry 18, 1292-1296.

Ledo, F., Carrion, A. M., Link, W. A., Mellstrom, B. and Naranjo, J. R.(2000)DREAM-�哸REM interaction via leucine-charged domains derepresses downstream regulatory element-dependent transcription. Mol. Cell. Biol. 20, 9120-9126.

Leissring, M. A., Yamasaki, T. R., Wasco, W., Buxbaum, J. D., Parker, I. and LaFerla, F. M.(2000)Calsenilin reverses presenilin-mediated enhancement of calcium signaling. Proc. Natl. Acad. Sci. 97, 8590-8593.

Lilliehook, C., Chan, S., Choi, E. K., Zaidi, N. F., Wasco, W., Wattson, M. P. and Buxbaum, J. D.(2002)Calsenilin enhances apoptosis by altering endoplasmic reticulum calcium signaling. Mol. Cell. Neurosci. 19, 552-559.

Mattson, M. P., Guo, Q., Furukawa, K. and Pcdcrscn, W. A.(1998)Presenilins, the endoplasmic reticulum, and neuronal apoptosis in Alzheimer''s Disease. J. Neurochem. 70, 1-14.

Morohashi, Y., Hatano, N., Ohya, S., Takikawa, R., Watabiki, T., Takasugi, N., Imauzumi, Y., Tomita, T. and Iwatsubo, T.(2002)Molecular cloning and characterization of CALP/KChIP4, a novel EF-hand protein interacting with presenilin 2 and voltage-gated potassium channel subunit Kv4. J. Biol. Chem. 277, 14965-14975.

Ohya, S., Morohashi, Y., Muraki, K., Tomita, T., Watanabe, M., Iwatsubo, T. and Imaizumi, Y.(2001)Molecular cloning and expression of the novel splice variants of K(+) channel-interacting protein 2. Biochem. Biophys. Res. Commun. 282, 96-102.

Osawa, M., Tong, K. I., Lilliehook, C., Wasco, W., Buxbaum, J. D., Cheng, H. Y. M., Penninger, J. M., Ikura, M. and Ames, J. B.(2001)Calcium-regulated DNA binding and oligomerization of the neuronal calcium-sensing protein, calsenilin/DREAM/KChIP3. J. Biol. Chem. 276, 41005-41013.

Rosati, B., Pan, Z., Lypen, S., Wang, H. S., Cohen, I., Dixon, J. E. and McKinnon, D.(2001)Regulation of KChIP2 potassium channel b subunit gene expression underlies the gradient of transient outward current in canine and human ventricle. J. Physiol. 533, 119-125.

Sanz, C., Mellstrom, B., Link, W. A., Naranjo, J. R. and Fernandez-Luna, J. L.(2001)Interleukin 3-dependent activation of DREAM is involved in transcriptional silencing of the apoptotic hrk gene in hematopoietic progenitor cells. EMBO. 20, 2286-2292.

Serodio, P., Kentros, C. and Rudy, B.(1994)Identification of molecular components of A-type channels activating at subthreshold potentials. J. Neurophys. 72, 1516-1529.

Sherrington, R., Rogaev, E. I., Liang, Y., Rogaeva, E. A., Levesque, G., Ikeda, M., Chi, H., Lin, C., Li, G. and Holman, K.(1995)Cloning of a gene bearing missense mutations in early-onset familial Alzheimer''s disease. Nature 375, 754-760.

Silva, A. C. R., Kendrick, J. and Reinach, F. C.(1995)Determination of ion specificity on EF-hands sites. J. Biol. Chem. 270, 6773-6778.

Snyder, E. E., Buoscio, B. W. and Falke, J. J.(1990)Calcium(II) site specificity: effect of size and charge on metal ion binding to an EF-hand-like site? Biochemistry 29, 3937-3943.

Takimoto, K., Yang, E. K., and Conforti, L.(2002)Palmitoylation of KChIP splicing variants is required for efficient cell surface expression of Kv4.3 channels. J. Biol. Chem. 277, 26904-26911.

Wang, S., Patel, S. P., Qu, Y., Hua, P., Strauss, H. C. and Mora;es, M. J.(2002)Kinetic properties of Kv4.3 and their modulation by KChIP2b. Biochem. Biophys. Res. Commun. 295, 223-229.

Xia, W.(2001)Amyloid metabolism and secretases in Alzheimer’s disease. Curr. Neurol. Neurosci Rep. 5, 422-427.

Yuste, R.(1997)Potassium channels : Dendritic shock absorbers. Nature 387, 851-853.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top