Abstract
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 -[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.
Highlights
The human ASCT2 transporter is one of the seven members of the SLC1 family (SLC1A5) and represents one of the most studied proteins among the SoLute Carrier (SLC) members being considered a hot spot topic for either biochemical interest and pharmacological applications (Kanai et al, 2013; Scalise et al, 2019)
Plus, Hybond Electro Chemi Luminescence (ECL) membranes were from GE Healthcare; L[3H]Glutamine was from Perkin Elmer; anti-ASCT2 was from Millipore; conjugated anti-His antibody, Octaethylene glycol monododecyl ether (C12E8), Cholesteryl hemisuccinate, Amberlite XAD-4, egg yolk phospholipids (3sn-phosphatidylcholine from egg yolk), Sephadex G-75, Lglutamine, methyl-β-cyclodextrin (MβCD) and all the other reagents were from Sigma-Aldrich
Maximal stimulation was observed at a cholesterol concentration of 75 μg/mg total lipids corresponding to 7.5% cholesterol which falls within the physiological cholesterol concentration in cells (Litvinov et al, 2018); at higher concentrations, the activity dramatically decreased
Summary
The human ASCT2 transporter is one of the seven members of the SLC1 family (SLC1A5) and represents one of the most studied proteins among the SLC members being considered a hot spot topic for either biochemical interest and pharmacological applications (Kanai et al, 2013; Scalise et al, 2019). The rat transporter (Oppedisano et al, 2004, 2007) was studied employing the proteoliposome technology that allows investigating a single protein inserted in an artificial membrane with the same orientation as in the native cell membrane (Scalise et al, 2017a) These studies confirmed most of the characteristics described in intact cells. Cysteine, i.e., one of the amino acids underlying the acronym ASC(Cysteine)T2, has been shown to be a modulator of the transporter but not a substrate (Scalise et al, 2015) explaining overlooked old data (Utsunomiya-Tate et al, 1996) This peculiar regulation mode, together with the discovered responsiveness to GSH, H2S, and NO suggested that ASCT2 could be a redox sensor in physiological and pathological conditions.
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