Department of DiBEST (Biologia, Ecologia, Scienze Della Terra) Unit of Biochemistry and Molecular Biotechnology, University of Calabria, via Bucci 4C, 87036, Arcavacata di Rende, Italy.
Department of Pharmaceutical Chemistry, University of Vienna, Althanstrasse 14, 1090, Wien, Austria.
Sci Rep. 2020 Oct 7;10(1):16738. doi: 10.1038/s41598-020-73757-y.
The plasma membrane transporter hLAT1 is responsible for providing cells with essential amino acids. hLAT1 is over-expressed in virtually all human cancers making the protein a hot-spot in the fields of cancer and pharmacology research. However, regulatory aspects of hLAT1 biology are still poorly understood. A remarkable stimulation of transport activity was observed in the presence of physiological levels of cholesterol together with a selective increase of the affinity for the substrate on the internal site, suggesting a stabilization of the inward open conformation of hLAT1. A synergistic effect by ATP was also observed only in the presence of cholesterol. The same phenomenon was detected with the native protein. Altogether, the biochemical assays suggested that cholesterol and ATP binding sites are close to each other. The computational analysis identified two neighboring regions, one hydrophobic and one hydrophilic, to which cholesterol and ATP were docked, respectively. The computational data predicted interaction of the ϒ-phosphate of ATP with Lys 204, which was confirmed by site-directed mutagenesis. The hLAT1-K204Q mutant showed an impaired function and response to ATP. Interestingly, this residue is conserved in several members of the SLC7 family.
质膜转运蛋白 hLAT1 负责为细胞提供必需氨基酸。hLAT1 在几乎所有人类癌症中过度表达，使该蛋白成为癌症和药理学研究领域的热点。然而，hLAT1 生物学的调节方面仍知之甚少。在存在生理水平胆固醇的情况下，观察到转运活性的显著刺激，同时对内位点对底物的亲和力选择性增加，表明 hLAT1 的内向开放构象稳定。只有在胆固醇存在的情况下才观察到 ATP 的协同作用。同样的现象也在天然蛋白中检测到。总之，生化测定表明胆固醇和 ATP 结合位点彼此靠近。计算分析确定了两个相邻的区域，一个疏水，一个亲水，分别与胆固醇和 ATP 对接。计算数据预测了 ATP 的 γ-磷酸与 Lys 204 的相互作用，这通过定点突变得到了证实。hLAT1-K204Q 突变体显示功能受损，对 ATP 的反应减弱。有趣的是，该残基在 SLC7 家族的几个成员中保守。
