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Structural characterisation reveals insights into substrate recognition by the glutamine transporter ASCT2/SLC1A5.

ABSTRACT: Cancer cells undergo a shift in metabolism where they become reliant on nutrients such as the amino-acid glutamine. Glutamine enters the cell via the alanine/serine/cysteine transporter 2 (ASCT2) that is upregulated in several cancers to maintain an increased supply of this nutrient and are therefore an attractive target in cancer therapeutic development. ASCT2 belongs to the glutamate transporter (SLC1A) family but is the only transporter in this family able to transport glutamine. The structural basis for glutamine selectivity of ASCT2 is unknown. Here, we identify two amino-acid residues in the substrate-binding site that are responsible for conferring glutamine selectivity. We introduce corresponding mutations into a prokaryotic homologue of ASCT2 and solve four crystal structures, which reveal the structural basis for neutral amino acid and inhibitor binding in this family. This structural model of ASCT2 may provide a basis for future development of selective ASCT2 inhibitors to treat glutamine-dependent cancers.

SUBMITTER: Scopelliti AJ

PROVIDER: S-EPMC5750217 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Structural characterisation reveals insights into substrate recognition by the glutamine transporter ASCT2/SLC1A5.

Scopelliti Amanda J AJ Font Josep J Vandenberg Robert J RJ Boudker Olga O Ryan Renae M RM

Nature communications 20180102 1

Cancer cells undergo a shift in metabolism where they become reliant on nutrients such as the amino-acid glutamine. Glutamine enters the cell via the alanine/serine/cysteine transporter 2 (ASCT2) that is upregulated in several cancers to maintain an increased supply of this nutrient and are therefore an attractive target in cancer therapeutic development. ASCT2 belongs to the glutamate transporter (SLC1A) family but is the only transporter in this family able to transport glutamine. The structur ...[more]

PMID: 29295993

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Project description: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 Naex+ -[3H]glutamineex/glutaminein 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 Vmax of transport without affecting the Km 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.

Project description:Alanine, serine, cysteine-preferring transporter 2 (ASCT2; SLC1A5) mediates uptake of glutamine, a conditionally essential amino acid in rapidly proliferating tumour cells. Uptake of glutamine and subsequent glutaminolysis is critical for activation of the mTORC1 nutrient-sensing pathway, which regulates cell growth and protein translation in cancer cells. This is of particular interest in breast cancer, as glutamine dependence is increased in high-risk breast cancer subtypes. Pharmacological inhibitors of ASCT2-mediated transport significantly reduced glutamine uptake in human breast cancer cell lines, leading to the suppression of mTORC1 signalling, cell growth and cell cycle progression. Notably, these effects were subtype-dependent, with ASCT2 transport critical only for triple-negative (TN) basal-like breast cancer cell growth compared with minimal effects in luminal breast cancer cells. Both stable and inducible shRNA-mediated ASCT2 knockdown confirmed that inhibiting ASCT2 function was sufficient to prevent cellular proliferation and induce rapid cell death in TN basal-like breast cancer cells, but not in luminal cells. Using a bioluminescent orthotopic xenograft mouse model, ASCT2 expression was then shown to be necessary for both successful engraftment and growth of HCC1806 TN breast cancer cells in vivo. Lower tumoral expression of ASCT2 conferred a significant survival advantage in xenografted mice. These responses remained intact in primary breast cancers, where gene expression analysis showed high expression of ASCT2 and glutamine metabolism-related genes, including GLUL and GLS, in a cohort of 90 TN breast cancer patients, as well as correlations with the transcriptional regulators, MYC and ATF4. This study provides preclinical evidence for the feasibility of novel therapies exploiting ASCT2 transporter activity in breast cancer, particularly in the high-risk basal-like subgroup of TN breast cancer where there is not only high expression of ASCT2, but also a marked reliance on its activity for sustained cellular proliferation.

Dataset Information

Structural characterisation reveals insights into substrate recognition by the glutamine transporter ASCT2/SLC1A5.

Publications

Structural characterisation reveals insights into substrate recognition by the glutamine transporter ASCT2/SLC1A5.

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