Scalise Mariafrancesca, Pochini Lorena, Cosco Jessica, Aloe Emma, Mazza Tiziano, Console Lara, Esposito Antonella, Indiveri Cesare
Unit of Biochemistry and Molecular Biotechnology, Department DiBEST (Biologia, Ecologia, Scienze della Terra), University of Calabria, Cosenza, Italy.
Front Mol Biosci. 2019 Oct 23;6:110. doi: 10.3389/fmolb.2019.00110. eCollection 2019.
The human SLC1A5 commonly known as ASCT2 is a sodium-dependent neutral amino acid antiporter involved in transmembrane traffic of glutamine that is exchanged through the cell membrane with smaller amino acids such as serine or threonine. Due to the strong overexpression in human cancers, ASCT2 is widely studied for its relevance to human health. Of special interest are the aspects related to the regulation of its function. The role of cholesterol as a modulator of the transport activity has been studied using a combined strategy of computational and experimental approaches. The effect of cholesterol on the -[H]glutamine/glutamine antiport in proteoliposomes has been evaluated by adding cholesteryl hemisuccinate. A strong stimulation of transport activity was observed in the presence of 75 μg cholesteryl hemisuccinate per mg total lipids. The presence of cholesterol did not influence the proteoliposome volume, in a wide range of tested concentration, excluding that the stimulation could be due to effects on the vesicles. cholesteryl hemisuccinate, indeed, improved the incorporation of the protein into the phospholipid bilayer to some extent and increased about three times the V of transport without affecting the K for glutamine. Docking of cholesterol into the hASCT2 trimer was performed. Six poses were obtained some of which overlapped the hypothetical cholesterol molecules observed in the available 3D structures. Additional poses were docked close to CARC/CRAC motifs (Cholesterol Recognition/interaction Amino acid Consensus sequence). To test the direct binding of cholesterol to the protein, a strategy based on the specific targeting of tryptophan and cysteine residues located in the neighborhood of cholesterol poses was employed. On the one hand, cholesterol binding was impaired by modification of tryptophan residues by the Koshland's reagent. On the other hand, the presence of cholesterol impaired the interaction of thiol reagents with the protein. Altogether, these results confirmed that cholesterol molecules interacted with the protein in correspondence of the poses predicted by the docking analysis.
人类的SLC1A5通常被称为ASCT2，是一种钠依赖性中性氨基酸反向转运体，参与谷氨酰胺的跨膜运输，谷氨酰胺通过细胞膜与丝氨酸或苏氨酸等较小的氨基酸进行交换。由于在人类癌症中强烈过表达，ASCT2因其与人类健康的相关性而受到广泛研究。特别令人感兴趣的是与其功能调节相关的方面。已经使用计算和实验方法相结合的策略研究了胆固醇作为转运活性调节剂的作用。通过添加胆固醇半琥珀酸酯评估了胆固醇对蛋白脂质体中-[H]谷氨酰胺/谷氨酰胺反向转运的影响。在每毫克总脂质存在75μg胆固醇半琥珀酸酯的情况下，观察到转运活性受到强烈刺激。在广泛的测试浓度范围内，胆固醇的存在不影响蛋白脂质体的体积，排除了刺激可能是由于对囊泡的影响。实际上，胆固醇半琥珀酸酯在一定程度上改善了蛋白质掺入磷脂双层的情况，并使转运的V增加了约三倍，而不影响谷氨酰胺的K。进行了胆固醇与hASCT2三聚体的对接。获得了六个构象，其中一些与在可用的3D结构中观察到的假设胆固醇分子重叠。另外的构象对接在靠近CARC/CRAC基序（胆固醇识别/相互作用氨基酸共有序列）的位置。为了测试胆固醇与蛋白质的直接结合，采用了一种基于特异性靶向位于胆固醇构象附近的色氨酸和半胱氨酸残基的策略。一方面，通过科什兰德试剂修饰色氨酸残基会损害胆固醇结合。另一方面，胆固醇的存在会损害硫醇试剂与蛋白质的相互作用。总之，这些结果证实了胆固醇分子与对接分析预测的构象处的蛋白质相互作用。
