The positively charged interface of TXNIP central arrestin domain is essential for GLUT1 interaction and glucose metabolism.

(A) (Top) Schematic figure demonstrates the group of positively charged residues located in the central arrestin domain of TXNIP. (PDB: 4LL1) (Bottom) Sequence alignment of alpha-arrestin family members shows conserved arrangement of positively charged residues in the central arrestin domain. (B) HeLa cells were transfected as indicated, and HA-IP was carried out to determine the interaction between GLUT1 and TXNIP-ARR mutants. (C) HeLa cells were transduced with lentiviruses carrying vector control, TXNIP, TXNIP-C247S, or TXNIP-3KA genes, and cell lysates were examined for endogenous GLUT1, HA, and ACTIN. (D) Confocal image of HeLa cells expressing GLUT1-GFP and TXNIP-WT-mCherry or TXNIP-3KA-mCherry. (E) (Left) HeLa cells stably expressing HA-GLUT1 were transduced with lentiviruses carrying vector control, TXNIP-WT, TXNIP-C247S, or TXNIP-3KA genes, and surface HA-GLUT1 was examined by FACS analysis. (Right) Quantification of surface HA-GLUT1 on the left panel. Data are represented as mean ± SEM. (F) HeLa cells were transduced with lentiviruses carrying vector control, TXNIP, TXNIP-C247S, or TXNIP-3KA genes, and the relative glucose uptake was examined by 3H-2DG tracing. Data are represented as mean ± SEM. (G) Kinects of 3H-2DG uptake in HeLa cells expressing vector control, HA-TXNIP-WT, HA-TXNIP-C247S, or HA-TXNIP-3KA. (3H-2DG uptake were examined at 5 min, 12 min and 33 min after adding 3H-2DG). Data are represented as mean ± SEM. (H) Principal Component Analysis (PCA) of LC-MS metabolomics in HEK293T cells expressing vector control, TXNIP-WT, TXNIP-C247S, or TXNIP-3KA.