Spontaneous expression of heat shock protein 70 (HSP 70) in developing zebrafish (Danio rerio) and protein-protein interactions

• Main Authors: Chen Tun Yuan, 鄭惇遠
• Other Authors: 劉秀美
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/82598141503069816756

碩士 === 國立海洋大學 === 海洋生物研究所 === 91 === Heat shock protein 70 (HSP 70) is known to act as an ATP-dependent molecular chaperone that interacts with a cochaperone to assist the refolding of denatured proteins (Walter and Bunchner, 2002). A spontaneous high expression of HSP 70 was previously detected in 72 to 84-hr-old developing zebrafish (Danio rerio) (Yeh and Hsu, 2000) and no coordinate increase in HSP 70 mRNA synthesis could be found in these zebrafish, suggesting that the spontaneous HSP 70 productions was controlled at the translation level (Yeh and Hsu, 2002). Heat shock cognate 70 (HSC 70) generally present in unstressed conditions appeared to be the predominant form of the highly expressed HSP 70 in developing zebrafish after immunoblot analysis of 36 to 108-hr-old zebrafish extract proteins separated by 2-D gel electrophoresis. A low level of spontaneous expression of stress-inducible HSP 70 that had a more basic pI than HSC 70 was also observed. RT-PCR using different set of primers targeting various positions of zebrafish HSC 70 cDNA gave no difference in band intensity. Immunoblot analysis detected the presence of a 42-kDa molecule structurally similar to the human cochaperone dj-2 in zebrafish extracts and the expression of this molecule was found to increase parallely with HSC 70 in developing zebrafish. Although incubation of 36, 80, and 84-hr-old zebrafish extracts with the protein crosslinking agent EGS all generated protein complexes with similar eletrophoretic mobility, HSC 70 and the dj-2 like molecule were present only in complexes produced from 80 an 84-hr-old zebrafish extracts. The covalent protein complexes containing HSC 70 and dj-2 could be partially purified by anion exchange chromatography eluting with 0.3M NaCl. Two polypeptides estimated to be 40 and 25 kDa in molecular mass were specifically released from EGS-crosslinked 72 and 84-hr-old zebrafish protein complexes, as shown by hydroxylamine cleavage and SDS-PAGE. It is very likely that the two polypeptides were proteins chaperoned by HSC 70 and dj-2 highly expressed in 72 to 84-hr-old zebrafish. The nature of the possibly chaperoned proteins remains to be determined.