MTF-1之SIM結構對SUMO化MTF-1表達及純化的影響

Effect of SIM on the expression and purification of SUMOylated MTF-1

林昌儀(Chang-Yi Lin)

指導教授 ： 林立元

國立清華大學/生命科學院/分子與細胞生物研究所/碩士(2010年)

摘要

金屬感應轉錄因子 (MTF-1) 調控體內多個影響金屬平衡及氧化壓力的基因。實驗室研究指出金屬感應轉錄因子可以被類泛素蛋白 (SUMO) 所修飾，進而影響其轉錄活性。我們試圖表現並純化受SUMO所修飾的金屬感應轉錄因子，以進行SUMOylation後對其生理功能上影響之研究。在我們的實驗中，將MTF-1以及in vivo SUMOylation system共同轉型表現到大腸桿菌中。測試經過不同誘導條件下可獲得表現量最高的誘導條件，但純化的步驟中發現會受到未被SUMO修飾之MTF-1的汙染。分析MTF-1之胺基酸序列發現MTF-1之C端具有SUMO-interaction motif (SIM) 能與SUMO產生厭水性交互作用，並且證實SUMOylated MTF-1會透過SIM與MTF-1結合，因而難以純化SUMOylated MTF-1wt，同時SIM與SUMO間的交互作用也會抑制MTF-1之SUMOylation。我們以EMSA實驗比較MTF-1wt與SIM mutant MTF-1於DNA結合能力上之差異性，結果發現無明顯差異，並且SUMOylated SIM mutant MTF-1不藉由CHO-K1細胞萃取液協助即與metal-response element (MRE) 探針結合。顯示SUMOylated MTF-1轉錄活性下降並非由於MTF-1與DNA結合能力下降，而是透過別種方式改變MTF-1轉錄活性。結果顯示SUMOylated MTF-1與MTF-1間的交互作用可能是調控MTF-1的重要因素。

關鍵字

金屬感應轉錄因子 ； 類小泛素修飾化 ； 類小泛素 ； 共同轉型 ； 類小泛素交互作用區

並列摘要

Metal-responsive transcriptional factor 1 (MTF-1) regulates a variety of genes involved in metal homeostasis and oxidative stress. Recently, we have demonstrated that MTF-1 can be SUMOylated and resulted in an alternation of the transcriptional activity. We attempted to express and purify SUMOylated MTF-1 for functional study. In our experiment, MTF-1 and the in vivo SUMOylation system were co-expressed into E. coli. Various conditions were examined to obtain the optimal production of the protein. However, purification of the SUMOylated product was unsuccessful since native MTF-1 consistently presented in the eluted fractions with the modified protein. Analysis of the MTF-1 primary sequence reveals a consensus SUMO-interaction motif (SIM) located at the carboxyl terminal region, which may interact with SUMO by hydrophobic interaction. Mutation at the SIM region caused the loss of interaction and thus SUMOylated MTF-1 can be isolated. This result susggests a cross-interaction of MTF-1 and its SUMO-conjugated product in the cells. DNA-binding activity of the native MTF-1 and SIM mutant was analyzed by electrophoretic mobility shift assay. Native MTF-1 binds DNA only when cell extract is present. However, SUMOylated MTF-1 can form complex directly with DNA. The result implies a formation of special conformation after SUMO modification that allows the MTF-1 to react with DNA.

並列關鍵字

SUMOylation ； SUMO ； MTF-1 ； Co-transform ； SIM ； in vivo SUMOylation system ； SUMO-interacting motif

參考文獻

Auf der Maur A, Belser T, Wang Y, Gunes C, Lichtlen P, Georgiev O, Schaffner W (2000) Characterization of the mouse gene for the heavy metal-responsive transcription factor MTF-1. Cell Stress Chaperones 5: 196-206.

Bae SH, Jeong JW, Park JA, Kim SH, Bae MK, Choi SJ, Kim KW (2004) Sumoylation increases HIF-1alpha stability and its transcriptional activity. Biochem Biophys Res Commun 324: 394-400.

Bayer P, Arndt A, Metzger S, Mahajan R, Melchior F, Jaenicke R, Becker J (1998) Structure determination of the small ubiquitin-related modifier SUMO-1. J Mol Biol 280: 275-286.

Bittel DC, Smirnova IV, Andrews GK (2000) Functional heterogeneity in the zinc fingers of metalloregulatory protein metal response element-binding transcription factor-1. Journal of Biological Chemistry 275: 37194-37201.

Bohren KM, Nadkarni V, Song JH, Gabbay KH, Owerbach D (2004) A M55V polymorphism in a novel SUMO gene (SUMO-4) differentially activates heat shock transcription factors and is associated with susceptibility to type I diabetes mellitus. J Biol Chem 279: 27233-27238.