Red wine and green tea flavonoids are cis-allosteric activators and competitive inhibitors of glucose transporter 1 (GLUT1)-mediated sugar uptake.

The antioxidant- and flavonoid-rich contents of red wine and green tea are reported to offer protection against cancer, cardiovascular disease, and diabetes. Some studies, however, show that flavonoids inhibit GLUT1-mediated, facilitative glucose transport, raising the possibility that their interaction with GLUT1 and subsequent downstream effects on carbohydrate metabolism may also impact health. The present study explores the structure-function relationships of flavonoid-GLUT1 interactions. We find that low concentrations of flavonoids act as cis-allosteric activators of sugar uptake, whereas higher concentrations competitively inhibit sugar uptake and noncompetitively inhibit sugar exit. Studies with heterologously expressed human GLUT1, -3, or -4 reveal that quercetin-GLUT1 and -GLUT4 interactions are stronger than quercetin-GLUT3 interactions, that epicatechin gallate (ECG) is more selective for GLUT1, and that epigallocatechin gallate (EGCG) is less GLUT isoform-selective. Docking studies suggest that only one flavonoid can bind to GLUT1 at any instant, but sugar transport and ligand-binding studies indicate that human erythrocyte GLUT1 can bind at least two flavonoid molecules simultaneously. Quercetin and EGCG are each characterized by positive, cooperative binding, whereas ECG shows negative cooperative binding. These findings support recent studies suggesting that GLUT1 forms an oligomeric complex of interacting, allosteric, alternating access transporters. We discuss how modulation of facilitative glucose transporters could contribute to the protective actions of the flavonoids against diabetes and Alzheimer's disease.

Kinetic Basis of Cis- and Trans-Allostery in GLUT1-Mediated Sugar Transport.

A growing body of evidence demonstrates that GLUT1-mediated erythrocyte sugar transport is more complex than widely assumed and that contemporary interpretations of emergent GLUT1 structural data are incompatible with the available transport and biochemical data. This study examines the kinetic basis of one such incompatibility-transport allostery-and in doing so suggests how the results of studies examining GLUT1 structure and function may be reconciled. Three types of allostery are observed in GLUT1-mediated, human erythrocyte sugar transport: (1) exofacial cis-allostery in which low concentrations of extracellular inhibitors stimulate sugar uptake while high concentrations inhibit transport; (2) endofacial cis-allostery in which low concentrations of intracellular inhibitors enhance cytochalasin B binding to GLUT1 while high concentrations inhibit binding, and (3) trans-allostery in which low concentrations of ligands acting at one cell surface stimulate ligand binding at or sugar transport from the other surface while high concentrations inhibit these processes. We consider several kinetic models to account for these phenomena. Our results show that an inhibitor can only stimulate then inhibit sugar uptake if (1) the transporter binds two or more molecules of inhibitor; (2) high-affinity binding to the first site stimulates transport, and (3) low-affinity binding to the second site inhibits transport. Reviewing the available structural, transport, and ligand binding data, we propose that exofacial cis-allostery results from cross-talk between multiple, co-existent ligand interaction sites present in the exofacial cavity of each GLUT1 protein, whereas trans-allostery and endofacial cis-allostery require ligand-induced subunit-subunit interactions.

Reconciling contradictory findings: Glucose transporter 1 (GLUT1) functions as an oligomer of allosteric, alternating access transporters.

Recent structural studies suggest that GLUT1 (glucose transporter 1)-mediated sugar transport is mediated by an alternating access transporter that successively presents exofacial (e2) and endofacial (e1) substrate-binding sites. Transport studies, however, indicate multiple, interacting (allosteric), and co-existent, exo- and endofacial GLUT1 ligand-binding sites. The present study asks whether these contradictory conclusions result from systematic analytical error or reveal a more fundamental relationship between transporter structure and function. Here, homology modeling supported the alternating access transporter model for sugar transport by confirming at least four GLUT1 conformations, the so-called outward, outward-occluded, inward-occluded, and inward GLUT1 conformations. Results from docking analysis suggested that outward and outward-occluded conformations present multiple ß-d-glucose and maltose interaction sites, whereas inward-occluded and inward conformations present only a single ß-d-glucose interaction site. Gln-282 contributed to sugar binding in all GLUT1 conformations via hydrogen bonding. Mutating Gln-282 to alanine (Q282A) doubled the Km(app) for 2-deoxy-d-glucose uptake and eliminated cis-allostery (stimulation of sugar uptake by subsaturating extracellular maltose) but not trans-allostery (uptake stimulation by subsaturating cytochalasin B). cis-Allostery persisted, but trans-allostery was lost in an oligomerization-deficient GLUT1 variant in which we substituted membrane helix 9 with the equivalent GLUT3 sequence. Moreover, Q282A eliminated cis-allostery in the oligomerization variant. These findings reconcile contradictory conclusions from structural and transport studies by suggesting that GLUT1 is an oligomer of allosteric, alternating access transporters in which 1) cis-allostery is mediated by intrasubunit interactions and 2) trans-allostery requires intersubunit interactions.

WZB117 (2-Fluoro-6-(m-hydroxybenzoyloxy) Phenyl m-Hydroxybenzoate) Inhibits GLUT1-mediated Sugar Transport by Binding Reversibly at the Exofacial Sugar Binding Site.

WZB117 (2-fluoro-6-(m-hydroxybenzoyloxy) phenyl m-hydroxybenzoate) inhibits passive sugar transport in human erythrocytes and cancer cell lines and, by limiting glycolysis, inhibits tumor growth in mice. This study explores how WZB117 inhibits the erythrocyte sugar transporter glucose transport protein 1 (GLUT1) and examines the transporter isoform specificity of inhibition. WZB117 reversibly and competitively inhibits erythrocyte 3-O-methylglucose (3MG) uptake with Ki(app) = 6 µm but is a noncompetitive inhibitor of sugar exit. Cytochalasin B (CB) is a reversible, noncompetitive inhibitor of 3MG uptake with Ki(app) = 0.3 µm but is a competitive inhibitor of sugar exit indicating that WZB117 and CB bind at exofacial and endofacial sugar binding sites, respectively. WZB117 inhibition of GLUTs expressed in HEK293 cells follows the order of potency: insulin-regulated GLUT4 ≫ GLUT1 ≈ neuronal GLUT3. This may explain WZB117-induced murine lipodystrophy. Molecular docking suggests the following. 1) The WZB117 binding envelopes of exofacial GLUT1 and GLUT4 conformers differ significantly. 2) GLUT1 and GLUT4 exofacial conformers present multiple, adjacent glucose binding sites that overlap with WZB117 binding envelopes. 3) The GLUT1 exofacial conformer lacks a CB binding site. 4) The inward GLUT1 conformer presents overlapping endofacial WZB117, d-glucose, and CB binding envelopes. Interrogating the GLUT1 mechanism using WZB117 reveals that subsaturating WZB117 and CB stimulate erythrocyte 3MG uptake. Extracellular WZB117 does not affect CB binding to GLUT1, but intracellular WZB117 inhibits CB binding. These findings are incompatible with the alternating conformer carrier for glucose transport but are consistent with either a multisubunit, allosteric transporter, or a transporter in which each subunit presents multiple, interacting ligand binding sites.

UPF1 is crucial for the infectivity of human immunodeficiency virus type 1 progeny virions.

The SF1 helicase MOV10 is an antiviral factor that is incorporated into human immunodeficiency virus type 1 (HIV-1) virions. We now report that HIV-1 virions also incorporate UPF1, which belongs to the same SF1 helicase subfamily as MOV10 and functions in the nonsense-mediated decay (NMD) pathway. Unlike ectopic MOV10, the overexpression of UPF1 does not impair the infectivity of HIV-1 progeny virions. However, UPF1 becomes a potent inhibitor of HIV-1 progeny virion infectivity when residues required for its helicase activity are mutated. In contrast, equivalent mutations abolish the antiviral activity of MOV10. Importantly, cells depleted of endogenous UPF1, but not of another NMD core component, produce HIV-1 virions of substantially lower specific infectivity. The defect is at the level of reverse transcription, the same stage of the HIV-1 life cycle inhibited by ectopic MOV10. Thus, whereas ectopic MOV10 restricts HIV-1 replication, the related UPF1 helicase functions as a cofactor at an early postentry step.