Elucidating the mechanism of NLRP3 inflammasome activation using fluorescence and light emission protein-interaction sensors

2018 Fiscal Year Final Research Report

• Project/Area Number: 16K15527
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Pediatrics
• Research Institution: Kyoto University
• Principal Investigator: Heike Toshio 京都大学, 医学研究科, 名誉教授 (90190173)
• Co-Investigator(Kenkyū-buntansha): 八角 高裕 京都大学, 医学研究科, 講師 (00511891)
• Research Collaborator: OHARA Osamu
• Project Period (FY): 2016-04-01 – 2019-03-31
• Keywords: インフラマソーム / 自己炎症性疾患 / 蛋白質会合蛍光センサー

Outline of Final Research Achievements

For the purpose of evaluating the mechanism of NLRP3 inflammasome assembly and activation, we prepared constructs in which the luminescent substance is fused to the N- and C-terminus of each of the three component proteins of the NLRP3 inflammasome (NLRP3/ASC/CASP1). Constructs fused with HaloTag were also prepared. We then developed a screening system using an in vitro cell-free　transcription/translation system that enables selection of combinations of these proteins that are optimal for detection of final protein association. Currently, this system is being used to select the combination that produces the optimal light emission.

Free Research Field

免疫疾患

Academic Significance and Societal Importance of the Research Achievements

まだ研究の途中ではあるが、本研究により個々の細胞レベルでインフラマソーム構成分子の会合の視覚的な評価が可能となり、細胞による反応の強さや進行速度の違い、細胞内での複合体形成の位置や拡がり、細胞毎の刺激に対する反応閾値の違いなど、多角的な視点からの評価が可能となる。又、インフラマソーム複合体の形成と、最終的なcaspase 1の活性化を個別に捉える事が可能とあり、それぞれの律速段階や制御機構の解明へと繋がる可能性を秘めている。