Elucidation of induction mechanism of redox perturbed cell death by human tumor suppressor gene candidate 101F6

2018 Fiscal Year Final Research Report

• Project/Area Number: 16K07323
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Biophysics
• Research Institution: Kobe University
• Principal Investigator: Tsubaki Motonari 神戸大学, 理学研究科, 教授 (00145046)
• Co-Investigator(Kenkyū-buntansha): 木村 哲就 神戸大学, 理学研究科, 特命講師 (70506906) ; 武内 総子 神戸大学, 大学教育推進機構, 助教 (00448169)
• Project Period (FY): 2016-04-01 – 2019-03-31
• Keywords: 膜タンパク質 / 電子伝達 / ヘム / アポトーシス / ナノディスク / シトクロムb561 / アスコルビン酸 / 生体膜

Outline of Final Research Achievements

We have succeeded to perform large-scale expression and high purity purification of 101F6 protein using the 101F6 protein expression system by imploying the methanol-utilizing yeast Pichia pastoris. Cell extracts from human cancer tissue-derived A549 cells were prepared using surfactant DDM or β-OG. After thoroughly mixing the cell extract with the purified 101F6 protein, followed by by magnetic beads antibody method using antibodies that specifically recognize and bind the 101F6 protein, we obtained a group of proteins that seems to interact with the 101F6 protein in human cells by SDS-PAGE and silver staining analyses. Then we have succeeded in reconstituting the purified 101F6 protein into nanodiscs which are very good models of biological membranes, Further, we have succeeded in measuring the enzyme activity of purified 101F6 protein for the first time.

Free Research Field

生化学

Academic Significance and Societal Importance of the Research Achievements

本研究により、101F6タンパク質はferric reductase活性を持つ事が既に報告されているDcytbと同様にferric reductase活性を持つ事が初めて証明された。この結果は、101F6の持つcaspase非依存性アポトーシス誘導作用がferric reductase 活性と何らかの関連を持つ事を示すものと考えられる。また、本研究において初めて精製したb561タンパク質ホモログそのものを用いてferric reductase酵素活性を直接に測定できる事を示した。そういう意味で本研究の意義は非常に大きい