利用荧光生物传感器跟踪肝癌细胞中 1,6-二磷酸果糖的动态变化

IF 4.6 2区综合性期刊 Q1 MULTIDISCIPLINARY SCIENCES

iScience Pub Date : 2024-11-06 DOI:10.1016/j.isci.2024.111336

Israel Pérez-Chávez , John N. Koberstein , Julia Malo Pueyo , Eduardo H. Gilglioni , Didier Vertommen , Nicolas Baeyens , Daria Ezeriņa , Esteban N. Gurzov , Joris Messens

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引用次数: 0

摘要

HYlight 是一种基因编码的荧光生物传感器，可按比例监测 1,6-二磷酸果糖 (FBP)，这是一种关键的糖酵解代谢物。鉴于葡萄糖在肝癌代谢中的作用，我们在人类肝癌细胞和原代小鼠肝细胞中表达了 HYlight。通过体外、硅学和细胞内实验，我们发现 HYlight 能够监测与糖酵解而非葡萄糖生成相关的 FBP 变化。HYlight 与 FBP 的亲和力为 1 μM，并且在生理 pH 值范围内稳定。HYlight 与磷酸二羟丙酮的结合力较弱，其比率反应受离子强度和磷酸盐的影响。因此，有必要在体外模拟细胞膜条件，以便在 HYlight 的细胞反应和 FBP 浓度之间建立可靠的相关性。研究发现，FBP 的浓度在较低的微摩尔范围内，远远低于之前的毫摩尔估计值。总之，这种生物传感器方法能以单细胞分辨率实时监测FBP浓度，是了解癌症代谢的宝贵工具。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

Tracking fructose 1,6-bisphosphate dynamics in liver cancer cells using a fluorescent biosensor

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Tracking fructose 1,6-bisphosphate dynamics in liver cancer cells using a fluorescent biosensor

HYlight is a genetically encoded fluorescent biosensor that ratiometrically monitors fructose 1,6-bisphosphate (FBP), a key glycolytic metabolite. Given the role of glucose in liver cancer metabolism, we expressed HYlight in human liver cancer cells and primary mouse hepatocytes. Through in vitro, in silico, and in cellulo experiments, we showed HYlight’s ability to monitor FBP changes linked to glycolysis, not gluconeogenesis. HYlight’s affinity for FBP was ∼1 μM and stable within physiological pH range. HYlight demonstrated weak binding to dihydroxyacetone phosphate, and its ratiometric response was influenced by both ionic strength and phosphate. Therefore, simulating cytosolic conditions in vitro was necessary to establish a reliable correlation between HYlight’s cellular responses and FBP concentrations. FBP concentrations were found to be in the lower micromolar range, far lower than previous millimolar estimates. Altogether, this biosensor approach offers real-time monitoring of FBP concentrations at single-cell resolution, making it an invaluable tool for the understanding of cancer metabolism.

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来源期刊

iScience Multidisciplinary-Multidisciplinary

CiteScore

7.20

自引率

1.70%

发文量

1972

审稿时长

6 weeks

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