Activation mechanism of ubiquitin ligase parkin by PINK1 and phosphorylated ubiquitin

2018 Fiscal Year Final Research Report

• Project/Area Number: 15H04342
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Structural biochemistry
• Research Institution: Kyoto University
• Principal Investigator: Sugase Kenji 京都大学, 工学研究科, 准教授 (00300822)
• Research Collaborator: MIZUTANI Akihiro
• Project Period (FY): 2015-04-01 – 2019-03-31
• Keywords: parkin / リフォールディング / 大量発現系 / NMR

Outline of Final Research Achievements

Parkin, an E3 ubiquitin ligase, regulates mitophagy pathway by conjugation of ubiquitin chains on damaged mitochondria. Parkin is activated by phosphorylation and binding of phosphorylated ubiquitin. Although tertiary structures of parkin in the autoinhibited and activated states have been determined, its activation mechanism remains unclear, especially dynamic processes in activation are still elusive. In this study, we aim at elucidating structural dynamics of parkin by NMR to reveal the activation mechanism. For NMR measurements, a large amount of isotope-labeled protein is required, but parkin was obtained only in insoluble fractions in E. coli when we used M9 media. Here, we established a refolding method of parkin, in which insoluble parkin was solubilized by denaturant and the denaturant was removed gradually. The refolded parkin was monomeric and had ubiquitination activity.

Free Research Field

生物物理学

Academic Significance and Societal Importance of the Research Achievements

遺伝性パーキンソン病患者の多くにparkinの変異が報告されている。parkinの機能不全がパーキンソン病発症に直結するため、parkinの反応機構の解明はパーキンソン病の発症機構の解明や疾患の治療薬の創出につながると期待されている。そして、その実現にはNMRによるparkinの研究が不可欠である。これまでNMR研究用の安定同位体標識parkinの大量調製は極めて困難であったが、本研究ではその基盤となる安定同位体標識parkinの大量試料調製を可能とした。そのため、今後のNMR研究によりparkinの活性化機構の理解が深まることが期待される。