Molecular Mechanisms for the Selective Transport of Dichlorofluorescein by Human Organic Anion Transporting Polypeptide 1B1.

Human organic anion transporting polypeptide (OATP) 1B1 and 1B3 are two highly homologous liver-specific uptake transporters. However, 2',7'-dichlorofluorescein (DCF) is preferably transported by OATP1B1. In the present study, the molecular mechanisms for the selective transport of DCF by OATP1B1 were investigated by constructing and characterizing an array of OATP1B1/1B3 chimeras and site-directed mutagenesis. Our results show that transmembrane domain (TM) 10 is crucial for the surface expression and function of OATP1B1, in which Q541 and L545 play the most important roles in DCF transport. Replacement of TM10 in OATP1B1 with its OATP1B3 counterpart led to OATP1B1's complete intracellular retention. Q541 and L545 may interact with DCF directly via hydrogen bonding and hydrophobic interactions. The decrease of DCF uptake by Q541A and L545S was due to their reduced binding affinity for DCF as compared with OATP1B1. In addition, Q541 and L545 are also crucial for the transport of estradiol-17ß-glucuronide (E17ßG) but not for the transport of estrone-3-sulfate (E3S), indicating different interaction modes between DCF/E17ßG and E3S in OATP1B1. Taken together, Q541 and L545 in TM10 are critical for OATP1B1-mediated DCF uptake, but their effect is substrate-dependent. SIGNIFICANCE STATEMENT: The key TMs and amino acid residues for the selective transport of DCF by OATP1B1 were identified. TM10 is crucial for the surface expression and function of OATP1B1. Within TM10, Q541 and L545 played the most significant roles and affected the function of OATP1B1 in a substrate-dependent manner. This information is crucial for a better understanding of the mechanism of the multispecificity of OATP1B1 and as a consequence the mechanism of OATP1B1-mediated drug-drug interactions.

G45 and V386 in Transmembrane Domains 1 and 8 Are Critical for the Activation of OATP1B3-Mediated E17ßG Uptake by Clotrimazole.

Organic anion-transporting polypeptides (OATPs) 1B1 and 1B3 are two highly homologous transport proteins. However, OATP1B1- and 1B3-mediated estradiol-17ß-glucuronide (E17ßG) uptake can be differentially affected by clotrimazole. In this study, by functional characterization on chimeric transporters and single mutants, we find that G45 in transmembrane domain 1 (TM1) and V386 in TM8 are critical for the activation of OATP1B3-mediated E17ßG uptake by clotrimazole. However, the effect of clotrimazole on the function of OATP1B3 is substrate-dependent as clotrimazole does not stimulate OATP1B3-mediated uptake of 4',5'-dibromofluorescein (DBF) and rosuvastatin. In addition, clotrimazole is not transported by OATP1B3, but it can efficiently permeate the plasma membrane due to its lipophilic properties. Homology modeling and molecular docking indicate that E17ßG binds in a substrate binding pocket of OATP1B3 through hydrogen bonding and hydrophobic interactions, among which its sterol scaffold forms hydrophobic contacts with V386. In addition, a flexible glycine residue at position 45 is essential for the activation of OATP1B3. Finally, clotrimazole is predicted to bind at an allosteric site, which mainly consists of hydrophobic residues located at the cytoplasmic halves of TMs 4, 5, 10, and 11.

Recombinant expression and characterization of yeast Mrc1, a DNA replication checkpoint mediator.

In Saccharomyces cerevisiae, Mrc1 (homolog of human Claspin and mediator of replication checkpoint) is not only a part of the replication machine, but also participates in the replication stress response when DNA replication is blocked by hydroxyurea. Since Mrc1 is expressed in a small amount in cells and has many proteins interacting with it as a mediator, it is difficult to obtain Mrc1 with high concentration and purity. This article reports the purification of a stable truncation of Mrc1 and the full length Mrc1. High concentration and high purity of Mrc1 was obtained and the three-dimensional structure of Mrc1 was analyzed, which is a ring with a hole in the center. At the same time, we found that Mrc1 has an interaction with Rad24-RFC a clamp loader in the replication checkpoint, and can form a complex with it, implying that we can assemble large replication checkpoint complexes in vitro. These results initially reveal the ring structure of Mrc1 and its interaction with Rad24-RFC in replication checkpoints in S. cerevisiae.

Human organic anion transporting polypeptide 1B3 (OATP1B3) is more heavily N-glycosylated than OATP1B1 in extracellular loops 2 and 5.

Human organic anion transporting polypeptide 1B3 (OATP1B3) and 1B1 are two liver-specific and highly homologous uptake transporters, whose structures consist of 12 transmembrane domains. The present study showed that OATP1B3 is more heavily N-glycosylated than OATP1B1 in extracellular loop 2 (EL2) and EL5. OATP1B3 has six N-glycosylation sites, namely N134, N145, N151, N445, N503, and N516, which is twice of that of OATP1B1. Single removal of individual N-glycans seems to have minimal influence on the surface expression and function of OATP1B3. However, simultaneous removal of all N-glycans will lead to OATP1B3's large retention in the endoplasmic reticulum and cellular degradation and thus significantly disrupts its surface expression. While N-glycosylation plays a crucial role in the surface expression of OATP1B3, it also has some effect on the transport function of OATP1B3 per se, which is not due to a decrease of substrate binding affinity but due to a reduced transporter's turnover number. Taken together, N-glycosylation is essential for normal surface expression and function of OATP1B3. Its disruption by some liver diseases such as NASH might alter the pharmacokinetic/pharmacodynamic properties of OATP1B3's substrate drugs.

Development of a sensitive LC-MS/MS method for the quantification of theranostic agent cypate in mouse plasma and application to a pharmacokinetic study.

Cypate, a heptamethine cyanine dye, is a prototypic near-infrared (NIR) theranostic agent for optical imaging and photothermal therapy. In the present study, a selective, sensitive, and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantitation of cypate in mouse plasma. The chromatographic separation was achieved using a short C18 column (2.1 mm × 50 mm, 5 µm) with a run time of 5 min. The MS was operated in multiple reaction monitoring (MRM) mode via positive electrospray ionization. The ion transitions for cypate and internal standard IR-820 were m/z 626.3 â†’ 596.3 and m/z 827.4 â†’ 330.2, respectively. The method was linear over a concentration range of 1.0-500 ng/mL. The within-run and between-run precision was less than 14.4% with accuracy in the range of -13.4% ∼ 9.8%. The validated method was successfully applied to a pharmacokinetic study of cypate in mice following intravenous administration.