Ligand specificity in the CRAL-TRIO protein family
ABSTRACT Intracellular trafficking of hydrophobic ligands is often mediated by specific binding proteins. The CRAL-TRIO motif is common to several lipid binding proteins including the cellular retinaldehyde binding protein (CRALBP), the alpha-tocopherol transfer protein (alpha-TTP), yeast phosphatidylinositol transfer protein (Sec14p), and supernatant protein factor (SPF). To examine the ligand specificity of these proteins, we measured their affinity toward a variety of hydrophobic ligands using a competitive [(3)H]-RRR-alpha-tocopherol binding assay. Alpha-TTP preferentially bound RRR-alpha-tocopherol over all other tocols assayed, exhibiting a K(d) of 25 nM. Binding affinities of other tocols for alphaTTP closely paralleled their ability to inhibit in vitro intermembrane transfer and their potency in biological assays. All other homologous proteins studied bound alpha-tocopherol but with pronouncedly weaker (> 10-fold) affinities than alpha-TTP. Sec14p demonstrated a K(d) of 373 nM for alpha-tocopherol, similar to that for its native ligand, phosphatidylinositol (381 nM). Human SPF had the highest affinity for phosphatidylinositol (216 nM) and gamma-tocopherol (268 nM) and significantly weaker affinity for alpha-tocopherol (K(d) 615 nM). SPF bound [(3)H]-squalene more weakly (879 nM) than the other ligands. Our data suggest that of all known CRAL-TRIO proteins, only alphaTTP is likely to serve as the physiological mediator of alpha-tocopherol's biological activity. Further, ligand promiscuity observed within this family suggests that caution should be exercised when suggesting protein function(s) from measurements utilizing a single ligand.
- SourceAvailable from: Angelo Azzi
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- "The competition with these ligands for a common binding site and their exchange could affect phospholipid-dependent transport and signalling pathways. Accordingly, αT stimulates in vitro squalene epoxidase and phosphatidylinositol-3-kinase gamma (PI3Kγ) activity possibly by forcing the release of squalene or phosphatidylinositol, respectively, and/or facilitating their presentation to the enzymes , , , , . Thus, αT may act as competing heterotypic ligand to PI or squalene as proposed for PC in stimulating the phosphorylation of PI by PI kinases . "
ABSTRACT: The vitamin E derivative, alpha-tocopheryl phosphate (αTP), is detectable in cultured cells, plasma and tissues in small amounts, suggesting the existence of enzyme(s) with α-tocopherol (αT) kinase activity. Here, we characterize the production of αTP from αT and [γ-32P]-ATP in primary human coronary artery smooth muscle cells (HCA-SMC) using separation by thin layer chromatography (TLC) and subsequent analysis by Ultra Performance Liquid Chromatography (UPLC). In addition to αT, although to a lower amount, also γT is phosphorylated. In THP-1 monocytes, γTP inhibits cell proliferation and reduces CD36 scavenger receptor expression more potently than αTP. Both αTP and γTP activate the promoter of the human vascular endothelial growth factor (VEGF) gene with similar potency, whereas αT and γT had no significant effect. The recombinant human tocopherol associated protein 1 (hTAP1, hSEC14L2) binds both αT and αTP and stimulates phosphorylation of αT possibly by facilitating its transport and presentation to a putative αT kinase. Recombinant hTAP1 reduces the in vitro activity of the phosphatidylinositol-3-kinase gamma (PI3Kγ) indicating the formation of a stalled/inactive hTAP1/PI3Kγ heterodimer. The addition of αT, βT, γT, δT or αTP differentially stimulates PI3Kγ, suggesting facilitated egress of sequestered PI from hTAP1 to the enzyme. It is suggested that the continuous competitive exchange of different lipophilic ligands in hTAPs with cell enzymes and membranes may be a way to make these lipophiles more accessible as substrates for enzymes and as components of specific membrane domains.PLoS ONE 07/2014; 9(7):e101550. DOI:10.1371/journal.pone.0101550 · 3.23 Impact Factor
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- "The presence of a CRALBP-like protein in Ciona (Ci-CRALBP) suggests that trapping and transport of 11-cis retinal derived from Ci-opsin3 could occur in both the larva and adult stages. Though Ci-CRALBP clusters with vertebrate CRALBP (unlike Branchiostoma CRALBP-like homolog , which clusters with α-tocopherol transfer protein (aTTP; data not shown), another member of the CRAL-TRIO family ), it has not yet been shown experimentally to actually bind 11-cis retinal. The absence of a robust LRAT ortholog (other than a SULT1-ST7-like homolog) in Ciona further weakens the case for a patent visual cycle in ascidians. "
ABSTRACT: In order to maintain visual sensitivity at all light levels, the vertebrate eye possesses a mechanism to regenerate the visual pigment chromophore 11-cis retinal in the dark enzymatically, unlike in all other taxa, which rely on photoisomerization. This mechanism is termed the visual cycle and is localized to the retinal pigment epithelium (RPE), a support layer of the neural retina. Speculation has long revolved around whether more primitive chordates, such as tunicates and cephalochordates, anticipated this feature. The two key enzymes of the visual cycle are RPE65, the visual cycle all-trans retinyl ester isomerohydrolase, and lecithin:retinol acyltransferase (LRAT), which generates RPE65's substrate. We hypothesized that the origin of the vertebrate visual cycle is directly connected to an ancestral carotenoid oxygenase acquiring a new retinyl ester isomerohydrolase function. Our phylogenetic analyses of the RPE65/BCMO and N1pC/P60 (LRAT) superfamilies show that neither RPE65 nor LRAT orthologs occur in tunicates (Ciona) or cephalochordates (Branchiostoma), but occur in Petromyzon marinus (Sea Lamprey), a jawless vertebrate. The closest homologs to RPE65 in Ciona and Branchiostoma lacked predicted functionally diverged residues found in all authentic RPE65s, but lamprey RPE65 contained all of them. We cloned RPE65 and LRATb cDNAs from lamprey RPE and demonstrated appropriate enzymatic activities. We show that Ciona ß-carotene monooxygenase a (BCMOa) (previously annotated as an RPE65) has carotenoid oxygenase cleavage activity but not RPE65 activity. We verified the presence of RPE65 in lamprey RPE by immunofluorescence microscopy, immunoblot and mass spectrometry. On the basis of these data we conclude that the crucial transition from the typical carotenoid double bond cleavage functionality (BCMO) to the isomerohydrolase functionality (RPE65), coupled with the origin of LRAT, occurred subsequent to divergence of the more primitive chordates (tunicates, etc.) in the last common ancestor of the jawless and jawed vertebrates.PLoS ONE 11/2012; 7(11):e49975. DOI:10.1371/journal.pone.0049975 · 3.23 Impact Factor
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- "However, each member binds diverse small hydrophobic ligands. For examples, the CRAL_TRIO domain of Sec14L group binds phosphotidylinositol , aTTP has affinity for tocopherol and CRALBP binds retinaldehyde . The ligand specificities of the RhoGEFs and Ras- GAP groups (represented by NF1 proteins) is currently unknown. "
ABSTRACT: The BNIP-2 and Cdc42GAP Homology (BCH) domains constitute a new and expanding family of highly conserved scaffold protein domains that regulate Rho, Ras and MAPK signaling, leading to cell growth, apoptosis, morphogenesis, migration and differentiation. Such versatility is achieved via their ability to target small GTPases and their immediate regulators such as GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs), their ability to form intra-molecular or inter-molecular interaction with itself or with other BCH domains, and also by their ability to bind diverse cellular proteins such as membrane receptors, isomerase, caspases and metabolic enzymes such as glutaminase. The presence of BCH and BCH-like domains in various proteins and their divergence from the ancestral lipid-binding CRAL-TRIO domain warrant the need to examine closely their structural, functional and regulatory plasticity in isolation or in concert with other protein modules present in the same proteins.FEBS letters 04/2012; 586(17):2674-91. DOI:10.1016/j.febslet.2012.04.023 · 3.17 Impact Factor