Intracellular quantification of the activity of RAS signaling regulators by in-cell NMR with genome editing technique

2022 Fiscal Year Final Research Report

• Project/Area Number: 21K20624
• Research Category: Grant-in-Aid for Research Activity Start-up
• Allocation Type: Multi-year Fund
• Review Section: 0701:Biology at molecular to cellular levels, and related fields
• Research Institution: Chiba University
• Principal Investigator: Zhao Qingci 千葉大学, 大学院薬学研究院, 助教 (60907682)
• Project Period (FY): 2021-08-30 – 2023-03-31
• Keywords: In-cell NMR / 核磁気共鳴法 / 分子夾雑 / ゲノム編集 / 低分子量GTPase

Outline of Final Research Achievements

The RAS protein is an important molecular switch in the cell proliferation signaling pathway, and its activation state within the cell is regulated by regulatory proteins such as GTPase-activating proteins (GAPs). In this study, we utilized cells in which GAP was knocked out through genome editing and performed quantitative analysis of the intracellular contribution of specific GAPs using in-cell NMR analysis. By comparing the proportions of active RAS in multiple GAP knockout cells, we found that one type of GAP, NF1, significantly contributes to the inhibition of RAS activation. Furthermore, simulation using a mathematical model revealed that the enzymatic activity of intracellular NF1 is notably suppressed compared to its quantitative value in vitro.

Free Research Field

構造生物学

Academic Significance and Societal Importance of the Research Achievements

低分子量GTPase RAS は細胞増殖のシグナル伝達における重要な分子スイッチであり、その制御の破綻はがんをはじめとする様々な疾患の原因となることが報告されているが、各制御因子が細胞内のRASの活性化状態にどの程度影響しているかは定量的に明らかにされていない。本解析手法は、実際の細胞内における特定の制御因子の寄与を定量的に捉えるものであり、様々な分子スイッチとその制御因子に対して適用可能であると考える。さらに、解析手法は創薬標的となるタンパク質の探索や、阻害剤の効果の評価にも応用可能である。