Functional Characterization of 5-Oxoproline Transport via SLC16A1/MCT1

Thyrotropin-releasing hormone is a tripeptide that consists of 5-oxoproline, histidine, and proline. The peptide is rapidly metabolized by various enzymes. 5-Oxoproline is produced by enzymatic hydrolysis in a variety of peptides. Previous studies showed that 5-oxoproline could become a possible biomarker for autism spectrum disorders. Here we demonstrate the involvement of SLC16A1 in the transport of 5-oxoproline. An SLC16A1 polymorphism (rs1049434) was recently identified. However, there is no information about the effect of the polymorphism on SLC16A1 function. In this study, the polymorphism caused an observable change in 5-oxoproline and lactate transport via SLC16A1. The Michaelis constant (K-m) was increased in an SLC16A1 mutant compared with that in the wild type. In addition, the proton concentration required to produce half-maximal activation of transport activity (K-0.5, H(+)) was increased in the SLC16A1 mutant compared with that in the wild type. Furthermore, we examined the transport of 5-oxoproline in T98G cells as an astrocyte cell model. Despite the fact that 5-oxoproline is an amino acid derivative, Na+-dependent and amino acid transport systems scarcely contributed to 5-oxoproline transport. Based on our findings, we conclude that H+-coupled 5-oxoproline transport is mediated solely by SLC16A1 in the cells

Identification of a selective inhibitor of human monocarboxylate transporter 4

The human monocarboxylate transporters (hMCTs/SLC16As) mediate the uptake of various mono-carboxylates. Several isoforms of hMCTs are expressed in cancerous tissue as well as in normal tissue. In cancerous tissue, hypoxia induces the expression of hMCT4, which transports the energetic metabolite lactate across the plasma membrane. Since hMCT4 is involved in pH regulation and the transport of lactate in cancer cells, an hMCT4 inhibitor could function as an anticancer agent. Although several non specific hMCT inhibitors have been developed, a selective hMCT4 inhibitor has not yet been identified. The aim of this study was therefore to identify a selective hMCT4 inhibitor for use as a pharmacological tool for studying hMCT4. The heterologous expression system of the Xenopus oocyte was used to assess the effects of test compounds on hMCT4, whereupon isobutyrate derivatives, fibrates, and bindarit (2-[(1-benzyl-1H-indazol-3-yl)methoxy]-2-methylpropanoic acid) were demonstrated to exhibit selective inhibitory effects against this transporter. It is suggested that the structure formed from the joining of an isobutyrate moiety and two aromatic rings by appropriate linkers is important for acquiring the selective hMCT4-inhibiting activity. These findings provide novel insights into the ligand recognition of hMCT4, and contribute to the development of novel anticancer agents. (C) 2017 Published by Elsevier Inc

Involvement of Histidine Residue His382 in pH Regulation of MCT4 Activity

Monocarboxylate transporter 4 (MCT4) is a pH-dependent bi-directional lactate transporter. Transport of lactate via MCT4 is increased by extracellular acidification. We investigated the critical histidine residue involved in pH regulation of MCT4 function. Transport of lactate via MCT4 was measured by using a Xenopus laevis oocyte expression system. MCT4-mediated lactate transport was inhibited by Zn2+ in a pH physiological condition but not in an acidic condition. The histidine modifier DEPC (diethyl pyrocarbonate) reduced MCT4 activity but did not completely inactivate MCT4. After treatment with DEPC, pH regulation of MCT4 function was completely knocked out. Inhibitory effects of DEPC were reversed by hydroxylamine and suppressed in the presence of excess lactate and Zn2+. Therefore, we performed an experiment in which the extracellular histidine residue was replaced with alanine. Consequently, the pH regulation of MCT4-H382A function was also knocked out. Our findings demonstrate that the histidine residue His382 in the extracellular loop of the transporter is essential for pH regulation of MCT4-mediated substrate transport activity

Crucial residue involved in L-lactate recognition by human monocarboxylate transporter 4 (hMCT4).

BACKGROUND: Monocarboxylate transporters (MCTs) transport monocarboxylates such as lactate, pyruvate and ketone bodies. These transporters are very attractive therapeutic targets in cancer. Elucidations of the functions and structures of MCTs is necessary for the development of effective medicine which targeting these proteins. However, in comparison with MCT1, there is little information on location of the function moiety of MCT4 and which constituent amino acids govern the transport function of MCT4. The aim of the present work was to determine the molecular mechanism of L-lactate transport via hMCT4. EXPERIMENTAL APPROACH: Transport of L-lactate via hMCT4 was determined by using hMCT4 cRNA-injected Xenopus laevis oocytes. hMCT4 mediated L-lactate uptake in oocytes was measured in the absence and presence of chemical modification agents and 4,4'-diisothiocyanostilbene-2,2'-disulphonate (DIDS). In addition, L-lactate uptake was measured by hMCT4 arginine mutants. Immunohistochemistry studies revealed the localization of hMCT4. RESULTS: In hMCT4-expressing oocytes, treatment with phenylglyoxal (PGO), a compound specific for arginine residues, completely abolished the transport activity of hMCT4, although this abolishment was prevented by the presence of L-lactate. On the other hand, chemical modifications except for PGO treatment had no effect on the transport activity of hMCT4. The transporter has six conserved arginine residues, two in the transmembrane-spanning domains (TMDs) and four in the intracellular loops. In hMCT4-R278 mutants, the uptake of L-lactate is void of any transport activity without the alteration of hMCT4 localization. CONCLUSIONS: Our results suggest that Arg-278 in TMD8 is a critical residue involved in substrate, L-lactate recognition by hMCT4

Inhibitory effects of metals on MCT4-mediated lactate uptake.

<p>(A) MCT4-mediated uptake of 0.1 mM lactate (0.1 μCi/ml) was measured in a transport buffer of pH 7.5 containing 5 mM of each metal for 10 min. The inset shows the effect of 5 mM zinc under an acidic condition (pH 5.5). (B) MCT1-mediated uptake of 0.1 mM lactate (0.1 μCi/ml) was measured in a transport buffer of pH 7.5 containing 5 mM of each metal for 10 min. The background uptake values of water-injected oocytes were subtracted. Data are presented as means±S.E. of three experiments.</p