Cysteine accessibility in the hydrophilic cleft of human organic cation transporter 2

Organic cation transporters (OCTs) are involved in the renal elimination of many cationic drugs and toxins. A hypothetical three-dimensional structure of OCT2 based on a homology model that used the Escherichia coli glycerol 3-phosphate transporter as a template has been described (Zhang, X., Shirahatti, N. V., Mahadevan, D., and Wright, S. H. (2005) J. Biol. Chem. 280, 34813-34822). To further define OCT structure, the accessibility to hydrophilic thiol-reactive reagents of the 13 cysteine residues contained in the human ortholog of OCT2 was examined. Maleimide-PEO2-biotin precipitated (surface biotinylation followed by Western blotting) and reduced tetraethylammonium transport by OCT2 expressed in Chinese hamster ovary cells, effects that were largely reversed by co-exposure to substrates and transport inhibitors, suggesting interaction with cysteines that are near to or part of a substrate-binding surface. Cysteines at amino acid position 437, 451, 470, and 474 were identified from the model as being located in transmembrane helices that participate in forming the hydrophilic cleft, the proposed region of substrate-protein interaction. To determine which residues are exposed to the solvent, a mutant with all four of these cysteines converted to alanine, along with four variants of this mutant each with an individual cysteine restored, were created. Maleimide-PEO2-biotin was only effective at precipitating and reducing transport by wild-type OCT2 and the mutant with cysteine 474 restored. Additionally, the smaller thiol-reactive reagent, methanethiosulfonate ethylsulfonate, reduced transport by wild-type OCT2 and the mutant with cysteine 474 restored. These data demonstrate that cysteine 474 of OCT2 is exposed to the aqueous milieu of the cleft and contributes to forming a pathway for organic cation transport.     

Functional significance of conserved cysteines in the human organic cation transporter 2

The significance of conserved cysteines in the human organic cation transporter 2 (hOCT2), namely the six cysteines in the long extracellular loop (loop cysteines) and C474 in transmembrane helix 11, was examined. Uptake of tetraethylammonium (TEA) and 1-methyl-4-phenypyridinium (MPP) into Chinese hamster ovary cells was stimulated >20-fold by hOCT2 expression. Both cell surface expression and transport activity were reduced considerably following mutation of individual loop cysteines (C51, C63, C89, C103, and C143), and the C89 and C103 mutants had reduced Michaelis constants (K(t)) for MPP. The loop cysteines were refractory to interaction with thiol-reactive biotinylation reagents, except after pretreatment of intact cells with dithiothreitol or following cell membrane solubilization. Reduction of disulfide bridge(s) did not affect transport, but labeling the resulting free thiols with maleimide-PEO(2)-biotin did. Mutation of C474 to an alanine or phenylalanine did not affect the K(t) value for MPP. In contrast, the K(t) value associated with TEA transport was reduced sevenfold in the C474A mutant, and the C474F mutant failed to transport TEA. This study shows that some but not all of the six extracellular loop cysteines exist within disulfide bridge(s). Each loop cysteine is important for plasma membrane targeting, and their mutation can influence substrate binding. The effect of C474 mutation on TEA transport suggests that it contributes to a TEA binding surface. Given that TEA and MPP are competitive inhibitors, the differential effects of C474 modification on TEA and MPP binding suggest that the binding surfaces for each are distinct, but overlapping in area.     

Phosphoproteome Profiling of Isogenic Cancer Cell‐Derived Exosome Reveals HSP90 as a Potential Marker for Human Cholangiocarcinoma

The northeastern region of Thailand is well known to have a high incidence and mortality of cholangiocarcinoma (CCA). Protein phosphorylation status has been reported to reflect a key determinant of cellular physiology, but identification of phosphoproteins can be a problem due to the presence of phosphatase. Exosomes are stable toward circulating proteases and other enzymes in human blood and can be recognized before the onset of cancer progression. Here an in vitro metastatic model of isogenic CCA cells is used to provide insight into the phosphorylation levels of exosomal proteins derived from highly invasive cells. Gel‐based and gel‐free proteomics approaches are used to reveal the proteins differentially phosphorylated in relation to tumor cell phenotypes. Forty‐three phosphoproteins are identified with a significant change in phosphorylation level. Phos‐tag western blotting and immunohistochemistry staining are then employed to validate the candidate phosphoproteins. Heat shock protein 90 is successfully confirmed as being differentially phosphorylated in relation to tumor malignancy. Importantly, the aberrant phosphorylation of exosomal proteins might serve as a promising tool for the development of a biomarker for metastatic CCA.     

Interaction of H+ with the extracellular and intracellular aspects of hMATE1

Human multidrug and toxin extrusion 1 (hMATE1, SLC47A1) is a major candidate for being the molecular identity of organic cation/proton (OC/H(+)) exchange activity in the luminal membrane of renal proximal tubules. Although physiological function of hMATE1 supports luminal OC efflux, the kinetics of hMATE1-mediated OC transport have typically been characterized through measurement of uptake, i.e., the interaction between outward-facing hMATE1 and OCs. To examine kinetics of hMATE1-mediated transport in a more physiologically relevant direction, i.e., an interaction between inward-facing hMATE1 and cytoplasmic substrates, we measured the time course of hMATE1-mediated efflux of the prototypic MATE1 substrate, [(3)H]1-methyl-4-phenylpyridinium, under a variety of intra- and extracellular pH conditions, from Chinese hamster ovary cells that stably expressed the transporter. In this study, we showed that an IC(50)/K(i) for interaction between extracellular H(+) and outward-facing hMATE1 determined from conventional uptake experiments [12.9 ± 1.23 nM (pH 7.89); n = 9] and from the efflux protocol [14.7 ± 3.45 nM (pH 7.83); n = 3] was not significantly different (P = 0.6). Furthermore, kinetics of interaction between intracellular H(+) and inward-facing hMATE1 determined using the efflux protocol revealed an IC(50) for H(+) of 11.5 nM (pH 7.91), consistent with symmetrical interactions of H(+) with the inward-facing and outward-facing aspects of hMATE1.     

Possibility to Apply Strontium Aluminate to Produce Light-Emitting Plants: Efficiency and Safety

Light-emitting plants (LEPs) provides light in areas without electricity. The phosphorescent compound was used as a lighting material for LEP development. However, using the phosphorescent compound for LEPs development required optimization and phytotoxicity evaluation. Strontium aluminate (SrAl₂O₄) is a phosphorescent compound that can glow for a long time and is easily recharged by visible light. In this study, using SrAl₂O₄ to develop LEPs was evaluated. Additionally, plant stress under SrAl₂O₄ was investigated. Metabolomic analysis can explain the possible mechanism of plants’ stress under SrAl₂O₄. After, injecting 3 mL of 5 % (w/v) SrAl₂O₄ products 1, 2, and 3 into the stem of Ipomoea aquatica, the result showed that SrAl₂O₄ products 2 and 3 caused oxidative stress. The metabolomic analysis also indicated that I. aquatica responded to SrAl₂O₄ product 1 by increasing pipecolic acid and salicylic acid, while I. aquatica injected with SrAl₂O₄ products 2 and 3 showed a decrease in salicylic acid around 0.005 and 0.061-fold, respectively, compared to control plants. and an excess accumulation of MDA around 10.00–12.00 μmol g⁻¹ FW. A 15 % concentration of SrAl₂O₄ can be used for LEPs development, enabling photoemission 18-fold for 50 min. SrAl₂O₄ product 1 has the potential to be a material for LEPs.