研究生物化學特性和結構特性來探討黑腹果蠅蛋白質酪氨酸亞硫酸化酵素

Abstract

蛋白質酪氨酸亞硫酸化酵素位於細胞內反式高爾基氏網，催化蛋白質中酪氨酸亞硫酸化反應，為一調控細胞外蛋白質交互影響力之重要因子，且調控許多生理上重要功能，例如：發炎作用，人類免疫系統缺乏病毒的入侵及甲狀腺機能減退侏儒症。但因缺乏同質性蛋白質酪氨酸亞硫酸化酵素來瞭解其生化上特性，使其在分子層級上之資訊所知甚少。在我的論文研究中，利用一凝血蛋白脢去除融合蛋白- 轉錄延長因子，首次能夠得到同質性黑腹果蠅蛋白質酪氨酸亞硫酸化酵素。藉由此瞭解黑腹果蠅蛋白質酪氨酸亞硫酸化酵素之酵素動力學，蛋白質四級結構，酵素穩定度和受質調控的特性。經分子篩層析法指出於溶液下其具有兩種結構,且在鹽與甘油的存在下得以穩定，目前已可以將其分離用於日後的研究。在人類與黑腹果蠅蛋白質酪氨酸亞硫酸化酵素上之點突變H269Q，H267Q並不會影響其比活性，但在大腸桿菌內表現量大幅降低導致其總活性隨之大幅減少。而相同的點突變被報導在家鼠上會造成侏儒症，推測可能的原因是此點突變會影響酵素的穩定度或表現量。

Protein tyrosine sulfation, mediated by tyrosylprotein sulfotransferase (TPST) that resides in trans-Golgi network, is a key modulator of extracellular protein-protein interactions and consequently regulates various physiological functions including inflammation, HIV infection, and hypothyroidism related dwarfism. Limited information at molecular level is available due to the lack of homogenous TPST for detailed biochemical characterization. In this study, a truncated Drosophila melanogaster TPST (DmTPST) was first prepared following thrombin proteolysis to remove NusA fusion protein. The kinetics, structure, stability, and substrate regulation of DmTPST were characterized. The result of gel filtration indicated that there were two configurations of DmTPST were simultaneously presented in the solution and could be isolated for future studies. DmTPST can be stabilized with salt and glycerol. A hypothyroidism-related mutation in DmTPST and hTPST2 did not cause any loss of specific activity. However, the total TPST activity was significantly decreased following its expression in E. coli. Similar mutation has been reported to cause dwarfism in mouse. It is proposed that such mutation may affect the stability or expression of TPST.