PDF(Pubmed)
Abstract：
Facilitative carbohydrate transporters (GLUTs, SLC2 gene family) are transmembrane proteins transporting hexoses and other sugars based on cellular metabolic demands. While a direct link between GLUTs and metabolic disorders has framed them as important biological and medicinal targets, targeting disease-relevant GLUTs remains challenging. In this study, we aimed to identify substrate-GLUT interactions that would discriminate between major fructose transporters. We examined the uptake distribution for conformational and configurational isomers of fructose using the corresponding conformationally locked fluorescently labeled mimetics as probes for assessing GLUT preferences in real time. Through comparative analysis of the uptake of the probes in the yeast-based single GLUT expression systems and the multi-GLUT mammalian cell environment, we established the ability of fructose transporters to discriminate between fructose conformers and epimers. We demonstrated that recreating the conformational and configurational mixture of fructose with molecular probes allows for the specific probe distribution, with fructofuranose mimetic being taken up preferentially through GLUT5 and β-d-fructopyranose mimetic passing through GLUT2. The uptake of α-d-fructopyranose mimetic was found to be independent of GLUT5 or GLUT2. The results of this study provide a new approach to analyzing GLUT5 and GLUT2 activity in live cells, and the findings can be used as a proof-of-concept for multi-GLUT activity screening in live cells. The research also provides new knowledge on substrate-GLUT interactions and new tools for monitoring alterations in GLUT activities.
摘要：
便利的碳水化合物转运蛋白(GLUTs，SLC2基因家族）是基于细胞代谢需求运输己糖和其他糖的跨膜蛋白。虽然GLUTs和代谢紊乱之间的直接联系已将它们视为重要的生物学和医学靶标，靶向疾病相关GLUTs仍然具有挑战性.在这项研究中,我们的目的是确定底物-GLUT相互作用，可以区分主要的果糖转运蛋白。我们使用相应的构象锁定的荧光标记的模拟物作为探针实时评估GLUT偏好，检查了果糖的构象和构型异构体的摄取分布。通过比较分析基于酵母的单GLUT表达系统和多GLUT哺乳动物细胞环境中探针的摄取，我们建立了果糖转运蛋白区分果糖构象异构体和差向异构体的能力。我们证明，用分子探针重建果糖的构象和构型混合物允许特定的探针分布，呋喃果糖模拟物优先通过GLUT5摄取，β-d-吡喃果糖模拟物通过GLUT2。发现α-d-吡喃果糖模拟物的摄取与GLUT5或GLUT2无关。这项研究的结果提供了一种分析活细胞中GLUT5和GLUT2活性的新方法，研究结果可作为活细胞多GLUT活性筛选的概念验证。该研究还提供了有关底物与GLUT相互作用的新知识，以及用于监测GLUT活动变化的新工具。
