不完整人類與大腸桿菌Thioredoxin形成Dimer及Dimer以上Polymer型態之探討

Abstract

Thioredoxin是一種12 KD小分子量的氧化還原酵素，本身可以被thioredoxin reductase利用輔酶NADPH進行還原反應循環，並可進行蛋白質間的雙硫鍵還原作用。其Thioredoxin的還原作用功能提供著生物體內諸多生物反應的調節，包括幫助蛋白質在高氧化環境的穩定，減少其高能量的自由基破壞、調節轉錄因子的活性、對於細胞凋零的抑制等等。

人類Thioredoxin催化著細胞內雙硫鍵的還原作用，同時也是與interleukin共同作用的co-cytokine，可以促使免疫細胞增長。而C端擷取片段thioredoxin蛋白質，在人類血液中被發現存在，其序列保留了N端的80個殘基，命名為Trx 80，其本身具有對人類週邊免疫單核球細胞(Human peripheral blood mononuclear cells)刺激增生的cytokine活性。且Trx 80在溶液內，被發現與wild type 之monomer不同的dimer型式存在，以CD光譜確認後，其二級結構相同。本研究表現與純化不同片段擷取的truncated人類以及大腸桿菌Thioredoxin重組蛋白，針對不同的tuncated突變片段(Human Trx 91、80、63以及E.coli Trx 96、83)，作其溶液中結構狀態的比較，觀察到各片段都有不同程度強烈的dimer(或polymer)型態產生的趨向。本研究比較片段與片段之間、以及human 與 E.coli的結果與程度之異同，針對其結果比較與探討。

Thioredoxin is a family of small redox proteins that undergo NADPH-dependent reduction by thioredoxin reductase. This results in a supply of reducing equivalents that cells use in a wide variety of biological reactions, which include maintaining reduced forms of the enzymes important for protection against damage from high-energy oxygen radicals, the regulation of transcription factor activity, and the inhibition of apoptosis.

Human thioredoxin catalyzes intracellular disulfide reductions but has also co-cytokine activity with interleukins after leaderless secretion. A recombinant truncated form of thioredoxin with the 80 N-terminal residues (Trx80) was discovered that Trx80 by itself is a potent mitogenic cytokine stimulating growth of resting human peripheral blood mononuclear cells (PBMC).Moreover, Trx80 is a dimer in solution ,with a secondary structure that resembles Trx measured by circular dichroic spectroscopy. In our research, we expressed and purified different truncated protein (Human Trx 91, Trx 80 ,Trx 63 and E.coli Trx 96, Trx 83), and found all of each has different level of inclination to form dimer (or polymer). In this study,we discuss the results between different forms of truncated human and E.coli thioredoxin.