Table 1.
Conditions for detection of αTP by UPLC.
Figure 1.
Primary human coronary artery smooth muscle cells (HCA-SMC) contain αT phosphorylation activity.
(A) HCA-SMC cells were treated with 0.1% ethanol control (c) or αT, and the in situ phosphorylation reaction, lipid extraction, and thin layer chromatography performed as indicated in materials and methods. The TLC plate was subsequently exposed to film, and the labeled sample spots and control spots separated in parallel were scraped, extracted and the presence of αTP confirmed by UPLC (lower part). (B) Specificity of the αT phosphorylation reaction, concentration dependency and substrate specificity of αT phosphorylation. HCA-SMC cells were treated with 0.1% ethanol control (c) or αT, αTP, αTQ at the indicated concentrations, and the phosphorylation reaction, lipid extraction, and thin layer chromatography (TLC) were performed as indicated in materials and methods. (C) Comparison of αT and γT phosphorylation (mean±SEM, n = 2, *P<0.05 relative to control (c)).
Figure 2.
Comparison of cellular activities of αTP and γTP.
(A) Inhibition of THP-1 cell proliferation by αTP or γTP (both at 0, 10, 20, 30, 40 µM) after 4 h, 28 h and 52 h treatment (mean±SEM, n = 4, relative to untreated control at 0 h set to 100%). (B) Inhibition of CD36 cell surface exposition as analyzed by FACS after treatment with αTP (10 µM) or γTP (10 µM) for 24 h (mean±SEM, n = 4, *P<0.05 relative to control (c)).
Figure 3.
Binding of αT and αTP to recombinant hTAP1 and lipid exchange.
(A) Isoelectric Point Mobility Shift assay (IPMS) shows competition of αT but not of ritonavir (negative control) with phosphatidylinositol (PI). Recombinant hTAP1 (30 µg) was incubated with αT, ritonavir, and PI at the indicated concentrations and the IPMS assay performed as described in Materials and Methods. Gels were stained using a mixture of Coomassie Blue and Crocein Scarlet. (B) Isoelectric mobility shift assay shows competition of αTP with PI. The arrow indicates a supershift probably resulting from detergent-like denaturing effects of αTP at high concentrations. The experiments have been repeated twice with similar results. (C) Stimulation of αT phosphorylation reaction with recombinant hTAP1. HCA-SMC cells were treated with 0.1% ethanol control (c) or αT (50 µM) and two different amounts of recombinant hTAP1 (3 and 15 µg/2.5 ml ICB). The phosphorylation reaction, lipid extraction, and thin layer chromatography were performed as indicated in materials and methods, the control set to 100% and the mean±SEM of two experiments plotted (*P<0.05 relative to control (c)).
Figure 4.
Stimulation of phosphatidylinositol-3-phosphate kinase gamma (PI3Kγ) activity with different tocopherol analogues.
(A) In vitro PI3Kγ activity is modulated by recombinant hTAP1 (4 µg) in a tocopherol analogue specific manner. PI3Kγ activity was assessed as described in materials and methods and the mean±SEM results plotted (n = 3, *P<0.05 relative to untreated control (c) without hTAP1; #P<0.05 relative to αT in the presence of hTAP1). αT, βT, γT, δT: α-, β-, γ-, δ-tocopherols, respectively. W: wortmannin. (B) In vitro PI3Kγ activity is inhibited by wortmannin (W) (1 µM), and stimulated by αT (50 µM) and more by αTP (50 µM). Recombinant hTAP1 (4 µg) inhibits PI3Kγ activity possibly by forming a stalled/inactive complex; addition of αT or αTP reverts the inhibition by hTAP1, possibly by promoting dissociation of the inactive complex and/or competing with bound phosphatidylinositol allowing its egress from the hTAP1 binding site and the transfer to the enzyme. PI3Kγ activity was assessed as described in materials and methods, the control set to 100% and the mean±SEM plotted (n = 3, *P<0.05 relative to control (c)).
Figure 5.
PI3Kγ is involved in stimulating Akt(Ser473) phosphorylation by αTP in THP-1 monocytes.
(A) THP-1 monocytes were incubated with or without αT or αTP (both 40 µM) or the specific PI3Kγ inhibitor AS-605240 (1 µM) for 24 h and western blots performed as described in materials and methods (n = 3, *P<0.05 relative to untreated control (c)). (B) Differential regulation of VEGF promoter activity by tocopherol analogues (all 20 µM) in THP-1 monocytes. αTP and γTP significantly induce the VEGF promoter activity in THP-1 monocytes, whereas αT and γT had no effect (n = 4, *P<0.05 relative to untreated control (c)).
Figure 6.
Hypothetical molecular model for hTAPs in lipid transport and enzyme regulation.
(A) hTAPs transfer lipids from/to cellular import/export sites or between different membranes and membrane domains such as lipid rafts, e.g. between membranes of the Golgi, endoplasmic reticulum, mitochondria, vesicles or membranes of cilia in airway epithelia [28], [51]; in secretory cells lipid transfer may be polarized. (A and B) hTAPs mediated lipid transport may change lipid composition and membrane curvature and in this way influence signal transduction and secretion. (B) hTAPs bring lipid substrates (S) to specific enzymes (E), present them in the correct orientation and timing, and/or remove the lipid products (P) from the enzyme, thus enhancing lipid turnover at the catalytic center (CC). Lipid exchange may occur preferentially upon interaction of hTAPs with membranes, thus confining lipid presentation by hTAPs and subsequent lipid modification to enzymes located to membranes. Moreover, the affinity of different ligands to the ligand binding pocket can influence lipid exchange rate thus influence lipid-specificity to stimulate enzyme activity. The carboxy-terminal GOLD (G) domain in hTAPs may confine the exchange activity to certain sites and thus further increase the reaction specificity.