Molecular Imaging Biosensor Monitors p53 Sumoylation in Cells and Living Mice

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• 作者：Thillai V. Sekar ; Kira Foygel ; Rammohan Devulapally ; Vineet Kumar ; Sanjay Malhotra ; Tarik F. Massoud ; Ramasamy Paulmurugan
• 刊名：Analytical Chemistry
• 出版年：2016
• 出版时间：December 6, 2016
• 年：2016
• 卷：88
• 期：23
• 页码：11420-11428
• 全文大小：622K
• ISSN：1520-6882

文摘

Small molecule mediated stabilization of p53 tumor suppressor protein through sumoylation is a promising new strategy for improving cancer chemotherapy. A molecular tool that monitors p53 sumoylation status and expedites screening for drugs that enhance p53 sumoylation would be beneficial. We report a molecularly engineered reporter fragment complementation biosensor based on optical imaging of Firefly luciferase (FLuc), to quantitatively image p53 sumoylation and desumoylation in cells and living mice. We initially characterized this biosensor by successfully imaging sumoylation of several target proteins, achieving significant FLuc complementation for ERα (p < 0.01), p53 (p < 0.005), FKBP12 (p < 0.03), ID (p < 0.03), and HDAC1 (p < 0.002). We then rigorously tested the sensitivity and specificity of the biosensor using several variants of p53 and SUMO1, including deletion mutants, and those with modified sequences containing the SUMO-acceptor site of target proteins. Next we evaluated the performance of the biosensor in HepG2 cells by treatment with ginkgolic acid, a drug that reduces p53 sumoylation, as well as trichostatin A, a potential inducer of p53 sumoylation by enhancement of its nuclear export. Lastly, we demonstrated the in vivo utility of this biosensor in monitoring and quantifying the effects of these drugs on p53 sumoylation in living mice using bioluminescence imaging. Adoption of this biosensor in future high throughput drug screening has the important potential to help identify new and repurposed small molecules that alter p53 sumoylation, and to preclinically evaluate candidate anticancer drugs in living animals.