【作者】 张波；

【导师】 叶升；

【作者基本信息】 浙江大学 ， 生物化学与分子生物学， 2020， 博士

【摘要】 单羧酸在细胞代谢中起到了非常重要的作用,它们参与到细胞的能量代谢以及生物合成途径中。它们在细胞内的浓度需要被细胞精确地调控。在细胞中起到调控单羧酸的是单羧酸转运体。在糖酵解活跃的细胞中,质子偶联的单羧酸转运体能够有效地将单羧酸转运出细胞以维持细胞的正常代谢。而在糖酵解不活跃的细胞中,质子偶联的单羧酸转运体的转运活性必须降低或者失去活性以维持单羧酸浓度在生理范围内。为了研究细胞是如何使用单羧酸转运体来实现细胞内单羧酸的稳态,我们研究分析了人源的单羧酸转运体2的转运活性。基于我们的功能实验,人源的单羧酸转运体2的转运活性依赖于细胞内底物的浓度。单羧酸转运体2的转运特点说明单羧酸转运体2转运底物时可能存在协同性。我们通过单颗粒冷冻电镜技术解析了人源的单羧酸转运体2的3.8?电镜结构。通过对电镜结构的分析,我们发现人源的单羧酸转运体2有着特殊的二聚体形式。单羧酸转运体2的亚基在形成二聚体时,氮端结构域和碳端结构域均参与二聚体的形成。这种特殊的二聚体结合方式可能解释为什么单羧酸转运体2转运底物时存在协同性。基于结构的突变体实验以及丙酮酸转运实验,我们明确了单羧酸转运体2中与协同转运有关的关键区域。通过对接模型的模拟以及功能实验,我们还确认了单羧酸转运体2的底物以及可能的质子的结合位点。基于与细菌同源结构的比较,我们最终提出了人源的单羧酸转运体2协同转运底物的模型。更多还原

【Abstract】 Monocarboxylates play important roles in metabolism in living cells.They participate in the energy metabolism and biosynthesis.The concentrations of monocarboxylates should be strictly controlled through monocarboxylate transporters by cells.In glycolytically active cells,proton-linked monocarboxylate transporters(MCTs)can efficiently transport monocarboxylates to maintain the proper metabolism for cells.However,in glycolytically inactive cells,the transport activity of MCTs should be reduced or even inactive to maintain a physiological monocarboxylate concentration.To demonstrate how MCTs maintain the monocarboxylate homeostasis,we studied and analyzed the transport activity of human monocarboxylate transporter 2(MCT2).Based on our functional study,the transport activity of MCT2 dependents on the concentration of intracellular substrate concentration.This property means MCT2 may transport substrate cooperatively.We solved the structure of human MCT2 at 3.8 ? with cryo-electron microscopy.By analyzing the cryo-EM structure,we found that human MCT2 has an unusual dimerization.The N-and C-terminal domains of MCT2 subunits are involved in dimerization.This unusual form of dimerization may explain why MCT2 transports substrate cooperatively.Structure-based mutagenesis in combination with pyruvate transport assay provides insights into the key region responsible for the cooperative transport of MCT2.With molecular docking and functional study,we also determined the key residues responsible for substrate recognition and proton coupling of MCT2.Based on the structure comparison between MCT2 and SfMCT,we finally proposed a model of cooperative transport in human MCT2.更多还原