( 您好!臺灣時間:2022/08/15 03:52
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::


研究生(外文):Yun-Ru Lai
論文名稱(外文):Molecular Cloning and Functional Analysis of Rat 86-kDa Heat Shock Protein
指導教授(外文):Margaret Dah-Tsyr Chang
外文關鍵詞:heat shock protein
  • 被引用被引用:0
  • 點閱點閱:159
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
熱休克蛋白九十 (HSP90) 為一種廣泛存在於真核生物細胞質中的保護子,可使與細胞生長相關之蛋白質折疊以調控其正常生長;而當哺乳動物細胞受到高溫、藥物或是病毒的刺激時會誘發出逆境蛋白的合成,使變性的蛋白質重新折疊,以維持正常的生理功能。由目前已知的HSP90研究當中,可知HSP90是由兩組基因來負責調控,分別為HSP86和HSP84。因此我們利用物種間的DNA序列比對來設計PCR的引子,由大鼠基因組中夾擊出小片段的hsp86之保留區段,並以 [-32P] dCTP標定為探針後從基因組庫中篩選出完整的 hsp86 cDNA。
目前已定序完成的大鼠hsp86 cDNA共有2,795 bp,而其編碼區 (coding region) 包含有2,202 bp。HSP90可分為三個區段,為了分析HSP90的功能,將全長的hsp90 cDNA、ATPase區段以及含有突變的ATPase 區段分別建構到pET23(a)載體內,以ITPG誘導於大腸桿菌BL21內進行蛋白質的大量表現。表現完後將細胞收下可以12 % SDS-PAGE分析蛋白質的產量、純度與溶解度,最後再以鎳親和性管柱將重組蛋白質純化。
將純化完成的蛋白質利用[-32P] ATP進行ATPase的功能分析,由實驗結果可知,全長HSP86之ATPase活性為ATPase區段的十五倍,而加入ADP競爭亦可抑制蛋白質的ATPase活性。我們也發現鎂離子的存在可增加HSP86對ATP的水解能力;但在突變的ATPase 區段並無觀察到明顯的ATPase活性差異。

Heat shock protein 90 (HSP90), an abundant molecular chaperone in the eukaryotic cytosol, is involved the folding of a set of cell regulatory proteins and in the re-folding of stress-denatured polypeptides. In vertebrates, hsp90 has two isoforms, hsp84 and hsp86. Here we report the cDNA cloning and functional analysis of the rat hsp86. Based on the homology among human hsp90, horse hsp90, porcine hsp90 and mouse hsp86, the conserved region in rat hsp86 was amplified by RT-PCR and used as the probe. The full-length rat hsp86 cDNA was obtained from cDNA library screening. The cDNA sequence data contains 2,795 bp and the length of coding region was 2,202 bp (GeneBank accession number AJ428213). Based on a crystal structure of the N-terminal domain of human HSP90 with bound ADP-Mg, several mutants were made in the rat HSP86 ATPase domain. Our results show that the full-length HSP86 has 15-fold higher ATPase activity than the N-termunal ATPase domain only, but the ATPase activity of HSP84 ATPase domain was similar to HSP86 ATPase domain. In addition, we found that the presence of magnesium can increase the ability of ATP hydrolysis of HSP86F Therefore, the results establish that magnesium binding, ATP binding and hydrolysis are required for HSP90 function in vitro.

Materials and Methods.....5
Figure and Legend........21

1. Argon, Y. and B. B. Simen (1999). "GRP94, an ER chaperone with protein and peptide binding properties." Semin Cell Dev Biol 10(5): 495-505.
2. Csermely, P., Y. Miyata, et al. (1997). "Binding affinity of proteins to hsp90 correlates with both hydrophobicity and positive charges. A surface plasmon resonance study." Life Sci 61(4): 411-8.
3. Csermely, P., T. Schnaider, et al. (1998). "The 90-kDa molecular chaperone family: structure, function, and clinical applications. A comprehensive review." Pharmacol Ther 79(2): 129-68.
4. Dale, E. C., X. Yang, et al. (1996). "Cloning and characterization of the promoter for murine 84-kDa heat-shock protein." Gene 172(2): 279-84.
5. Dale, E. C., X. Yang, et al. (1997). "Murine 86-kDa heat shock protein gene and promoter." Cell Stress Chaperones 2(2): 87-93.
6. Felts, S. J., B. A. Owen, et al. (2000). "The hsp90-related protein TRAP1 is a mitochondrial protein with distinct functional properties." J Biol Chem 275(5): 3305-12
7. Garcia-Cardena, G., R. Fan, et al. (1998). "Dynamic activation of endothelial nitric oxide synthase by Hsp90." Nature 392(6678): 821-4.
8. Huang, H., W. C. Lee, et al. (1999). "Molecular cloning and characterization of porcine cDNA encoding a 90-kDa heat shock protein and its expression following hyperthermia." Gene 226(2): 307-15.
9. Jakob, U., H. Lilie, et al. (1995). "Transient interaction of Hsp90 with early unfolding intermediates of citrate synthase. Implications for heat shock in vivo." J Biol Chem 270(13): 7288-94.
10. Lindquist, S. (1986). "The heat-shock response." Annu Rev Biochem 55: 1151-91.
11. Lindquist, S. and E. A. Craig (1988). "The heat-shock proteins." Annu Rev Genet 22: 631-77.
12. Maruya, M., M. Sameshima, et al. (1999). "Monomer arrangement in HSP90 dimer as determined by decoration with N and C-terminal region specific antibodies." J Mol Biol 285(3): 903-7.
13. McGuire, J. A., L. Poellinger, et al. (1992). "Cloning and regulation by glucocorticoid receptor ligands of a rat hsp90." J Steroid Biochem Mol Biol 42(8): 813-22.
14. McLaughlin, S. H., H. W. Smith, et al. (2002). "Stimulation of the weak ATPase activity of human hsp90 by a client protein." J Mol Biol 315(4): 787-98.
15. Moore, S. K., C. Kozak, et al. (1989). "Murine 86- and 84-kDa heat shock proteins, cDNA sequences, chromosome assignments, and evolutionary origins." J Biol Chem 264(10): 5343-51.
16. Nadeau, K., A. Das, et al. (1993). "Hsp90 chaperonins possess ATPase activity and bind heat shock transcription factors and peptidyl prolyl isomerases." J Biol Chem 268(2): 1479-87.
17. Neckers, L. (2002). "Hsp90 inhibitors as novel cancer chemotherapeutic agents." Trends Mol Med 8(4): S55-61.
18. Obermann, W. M., H. Sondermann, et al. (1998). "In vivo function of Hsp90 is dependent on ATP binding and ATP hydrolysis." J Cell Biol 143(4): 901-10.
19. Panaretou, B., C. Prodromou, et al. (1998). "ATP binding and hydrolysis are essential to the function of the Hsp90 molecular chaperone in vivo." Embo J 17(16): 4829-36.
20. Parsell, D. A., J. Taulien, et al. (1993). "The role of heat-shock proteins in thermotolerance." Philos Trans R Soc Lond B Biol Sci 339(1289): 279-85; discussion 285-6.
21. Pearl, L. H. and C. Prodromou (2000). "Structure and in vivo function of Hsp90." Curr Opin Struct Biol 10(1): 46-51.
22. Pratt, W. B. and D. O. Toft (1997). "Steroid receptor interactions with heat shock protein and immunophilin chaperones." Endocr Rev 18(3): 306-60.
23. Prodromou, C., S. M. Roe, et al. (1997). "Identification and structural characterization of the ATP/ADP-binding site in the Hsp90 molecular chaperone." Cell 90(1): 65-75.
24. Roe, S. M., C. Prodromou, et al. (1999). "Structural basis for inhibition of the Hsp90 molecular chaperone by the antitumor antibiotics radicicol and geldanamycin." J Med Chem 42(2): 260-6.
25. Scheibel, T., H. I. Siegmund, et al. (1999). "The charged region of Hsp90 modulates the function of the N-terminal domain." Proc Natl Acad Sci U S A 96(4): 1297-302.
26. Scheibel, T., T. Weikl, et al. (1998). "Two chaperone sites in Hsp90 differing in substrate specificity and ATP dependence." Proc Natl Acad Sci U S A 95(4): 1495-9.
27. Schneider, C., L. Sepp-Lorenzino, et al. (1996). "Pharmacologic shifting of a balance between protein refolding and degradation mediated by Hsp90." Proc Natl Acad Sci U S A 93(25): 14536-41.
28. Schulte, T. W., M. V. Blagosklonny, et al. (1995). "Disruption of the Raf-1-Hsp90 molecular complex results in destabilization of Raf-1 and loss of Raf-1-Ras association." J Biol Chem 270(41): 24585-8.
29. Segnitz, B. and U. Gehring (1997). "The function of steroid hormone receptors is inhibited by the hsp90-specific compound geldanamycin." J Biol Chem 272(30): 18694-701.
30. Sharma, S. V., T. Agatsuma, et al. (1998). "Targeting of the protein chaperone, HSP90, by the transformation suppressing agent, radicicol." Oncogene 16(20): 2639-45.
31. Soga, S., T. Kozawa, et al. (1998). "Radicicol leads to selective depletion of Raf kinase and disrupts K-Ras-activated aberrant signaling pathway." J Biol Chem 273(2): 822-8.
32. Song, H. Y., J. D. Dunbar, et al. (1995). "Identification of a protein with homology to hsp90 that binds the type 1 tumor necrosis factor receptor." J Biol Chem 270(8): 3574-81.
33. Stebbins, C. E., A. A. Russo, et al. (1997). "Crystal structure of an Hsp90-geldanamycin complex: targeting of a protein chaperone by an antitumor agent." Cell 89(2): 239-50.
34. Sweitzer, T. D. and J. A. Hanover (1996). "Calmodulin activates nuclear protein import: a link between signal transduction and nuclear transport." Proc Natl Acad Sci U S A 93(25): 14574-9.
35. Thomas, J. G. and F. Baneyx (2000). "ClpB and HtpG facilitate de novo protein folding in stressed Escherichia coli cells." Mol Microbiol 36(6): 1360-70.
36. Vidair, C. A., S. J. Doxsey, et al. (1993). "Heat shock alters centrosome organization leading to mitotic dysfunction and cell death." J Cell Physiol 154(3): 443-55.
37. Whitesell, L., E. G. Mimnaugh, et al. (1994). "Inhibition of heat shock protein HSP90-pp60v-src heteroprotein complex formation by benzoquinone ansamycins: essential role for stress proteins in oncogenic transformation." Proc Natl Acad Sci U S A 91(18): 8324-8.
38. Whitesell, L., P. D. Sutphin, et al. (1998). "The physical association of multiple molecular chaperone proteins with mutant p53 is altered by geldanamycin, an hsp90-binding agent." Mol Cell Biol 18(3): 1517-24.
39. Williams, N. E. and E. M. Nelsen (1997). "HSP70 and HSP90 homologs are associated with tubulin in hetero-oligomeric complexes, cilia and the cortex of Tetrahymena." J Cell Sci 110 ( Pt 14): 1665-72.
40. Yamamoto, M., Y. Takahashi, et al. (1991). "Characterization of the hydrophobic region of heat shock protein 90." J Biochem (Tokyo) 110(1): 141-5.
41. Young, J. C., I. Moarefi, et al. (2001). "Hsp90: a specialized but essential protein-folding tool." J Cell Biol 154(2): 267-73.
42. Young, J. C., C. Schneider, et al. (1997). "In vitro evidence that hsp90 contains two independent chaperone sites." FEBS Lett 418(1-2): 139-43.
43. Yufu, Y., J. Nishimura, et al. (1992). "High constitutive expression of heat shock protein 90 alpha in human acute leukemia cells." Leuk Res 16(6-7): 597-605.
44. Zhang, S. L., J. Yu, et al. (1999). "Regulation of human hsp90alpha gene expression." FEBS Lett 444(1): 130-5.

第一頁 上一頁 下一頁 最後一頁 top