Human Monocarboxylate Transporter Research Articles

Role of Human Monocarboxylate Transporter 1 (hMCT1) and 4 (hMCT4) in Tumor Cells and the Tumor Microenvironment.

In recent years, the abnormal glucose metabolism of tumor cells has attracted increasing attention. Abnormal glucose metabolism is closely related to the occurrence and development of tumors. Monocarboxylate transporters (MCTs) transport the sugar metabolites lactic acid and pyruvate, which affect glucose metabolism and tumor progression in a variety of ways. Thus, research has recently focused on MCTs and their potential functions in cancer. The MCT superfamily consists of 14 members. MCT1 and MCT4 play a crucial role in the maintenance of intracellular pH in tumor cells by transporting monocarboxylic acids (such as lactate, pyruvate and butyrate). MCT1 and MCT4 are highly expressed in a variety of tumor cells and are involved the proliferation, invasion and migration of tumor cells, which are closely related to the prognosis of cancer. Because of their important functions in tumor cells, MCT1 and MCT4 have become potential targets for cancer treatment. In this review, we focus on the structure, function and regulation of MCT1 and MCT4 and discuss the developed inhibitors of MCT1 and MCT4 to provide more comprehensive information that might aid in the development of strategies targeting MCTs in cancer.

Atorvastatin Exerts More Selective Inhibitory Effects on hMCT2 than on hMCT1 and hMCT4.

Human monocarboxylate transporter 1 (hMCT1), hMCT2, and hMCT4 transport monocarboxylates, such as L-lactate and pyruvate, with pH dependency. They are often over-expressed in various cancer cells and mediate the energy balance and pH homeostasis. Therefore, hMCT inhibitors can potentially be used as anticancer drugs. However, isoform-selective inhibitors have not yet been well-characterized. In addition, several statins and 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors have been reported to inhibit hMCTs, but their selectivity has not yet been evaluated. In this study, we aimed to determine whether statins could inhibit hMCT1, hMCT2, and hMCT4. We expressed hMCT1, hMCT2, and hMCT4 in a heterologous expression system of Xenopus oocytes and performed inhibitory experiments with various statins (fluvastatin, atorvastatin, simvastatin, rosuvastatin, pravastatin, and pitavastatin). As the three-dimensional structure of hMCT2 has been recently reported, docking simulations of statins and their structures were also performed to estimate the inhibition site. All statins inhibited the transport activities of hMCT1, hMCT2, and hMCT4. In addition, atorvastatin was found to be a potent isoform-selective inhibitor of hMCT2. Docking simulation indicated that atorvastatin could interact with a site surrounded by transmembrane (TM)-2, TM11, and intracellular helix in the TM6/7loop. Therefore, targeting this site may lead to the discovery of more potent hMCT2-selective inhibitors. Atorvastatin exerts selective inhibitory effects on hMCT2. These findings provide insights into the inhibitory mechanism of statins against hMCT1, hMCT2, and hMCT4 and may aid in the development of novel anticancer agents.

Human monocarboxylate transporters accept and relay protons via the bound substrate for selectivity and activity at physiological pH.

Human monocarboxylate/H+ transporters, MCT, facilitate the transmembrane translocation of vital weak acid metabolites, mainly l-lactate. Tumors exhibiting a Warburg effect rely on MCT activity for l-lactate release. Recently, high-resolution MCT structures revealed binding sites for anticancer drug candidates and the substrate. Three charged residues, Lys 38, Asp 309, and Arg 313 (MCT1 numbering) are essential for substrate binding and initiation of the alternating access conformational change. However, the mechanism by which the proton cosubstrate binds and traverses MCTs remained elusive. Here, we report that substitution of Lys 38 by neutral residues maintained MCT functionality in principle, yet required strongly acidic pH conditions for wildtype-like transport velocity. We determined pH-dependent biophysical transport properties, Michaelis-Menten kinetics, and heavy water effects for MCT1 wildtype and Lys 38 mutants. Our experimental data provide evidence for the bound substrate itself to accept and shuttle a proton from Lys 38 to Asp 309 initiating transport. We have shown before that substrate protonation is a pivotal step in the mechanisms of other MCT-unrelated weak acid translocating proteins. In connection with this study, we conclude that utilization of the proton binding and transfer capabilities of the transporter-bound substrate is probably a universal theme for weak acid anion/H+ cotransport.

Structural aspects of the glucose and monocarboxylate transporters involved in the Warburg effect.

Cancer cells shift their glucose catabolism from aerobic respiration to lactic fermentation even in the presence of oxygen, and this is known as the "Warburg effect". To accommodate the high glucose demands and to avoid lactate accumulation, the expression levels of human glucose transporters (GLUTs) and human monocarboxylate transporters (MCTs) are elevated to maintain metabolic homeostasis. Therefore, inhibition of GLUTs and/or MCTs provides potential therapeutic strategies for cancer treatment. Here, we summarize recent advances in the structural characterization of GLUTs and MCTs, providing a comprehensive understanding of their transport and inhibition mechanisms to facilitate further development of anticancer therapies.

AAV9-MCT8 Delivery at Juvenile Stage Ameliorates Neurological and Behavioral Deficits in a Mouse Model of MCT8-Deficiency.

Background: Allan-Herndon-Dudley syndrome (AHDS) is a severe psychomotor disability disorder that also manifests characteristic abnormal thyroid hormone (TH) levels. AHDS is caused by inactivating mutations in monocarboxylate transporter 8 (MCT8), a specific TH plasma membrane transporter widely expressed in the central nervous system (CNS). MCT8 mutations cause impaired transport of TH across brain barriers, leading to insufficient neural TH supply. There is currently no successful therapy for the neurological symptoms. Earlier work has shown that intravenous (IV), but not intracerebroventricular adeno-associated virus serotype 9 (AAV9) -based gene therapy given to newborn Mct8 knockout (Mct8-/y) male mice increased triiodothyronine (T3) brain content and partially rescued TH-dependent gene expression, suggesting a promising approach to treat this neurological disorder. Methods: The potential of IV delivery of AAV9 carrying human MCT8 was tested in the well-established Mct8-/y/Organic anion-transporting polypeptide 1c1 (Oatp1c1)-/ - double knockout (dKO) mouse model of AHDS, which, unlike Mct8-/y mice, displays both neurological and TH phenotype. Further, as the condition is usually diagnosed during childhood, treatment was given intravenously to P30 mice and psychomotor tests were carried out blindly at P120-P140 after which tissues were collected and analyzed. Results: Systemic IV delivery of AAV9-MCT8 at a juvenile stage led to improved locomotor and cognitive functions at P120-P140, which was accompanied by a near normalization of T3 content and an increased response of positively regulated TH-dependent gene expression in different brain regions examined (thalamus, hippocampus, and parietal cortex). The effects on serum TH concentrations and peripheral tissues were less pronounced, showing only improvement in the serum T3/reverse T3 (rT3) ratio and in liver deiodinase 1 expression. Conclusion: IV administration of AAV9, carrying the human MCT8, to juvenile dKO mice manifesting AHDS has long-term beneficial effects, predominantly on the CNS. This preclinical study indicates that this gene therapy has the potential to ameliorate the devastating neurological symptoms in patients with AHDS.

Lactic Acid Permeability of Aquaporin-9 Enables Cytoplasmic Lactate Accumulation via an Ion Trap.

(1) Background: Human aquaporin-9 (AQP9) conducts several small uncharged metabolites, such as glycerol, urea, and lactic acid. Certain brain tumors were shown to upregulate AQP9 expression, and the putative increase in lactic acid permeability was assigned to severity. (2) Methods: We expressed AQP9 and human monocarboxylate transporter 1 (MCT1) in yeast to determine the uptake rates and accumulation of radiolabeled l-lactate/l-lactic acid in different external pH conditions. (3) Results: The AQP9-mediated uptake of l-lactic acid was slow compared to MCT1 at neutral and slightly acidic pH, due to low concentrations of the neutral substrate species. At a pH corresponding to the pKa of l-lactic acid, uptake via AQP9 was faster than via MCT1. Substrate accumulation was fundamentally different between AQP9 and MCT1. With MCT1, an equilibrium was reached, at which the intracellular and extracellular l-lactate/H+ concentrations were balanced. Uptake via AQP9 was linear, theoretically yielding orders of magnitude of higher substrate accumulation than MCT1. (4) Conclusions: The selectivity of AQP9 for neutral l-lactic acid establishes an ion trap for l-lactate after dissociation. This may be physiologically relevant if the transmembrane proton gradient is steep, and AQP9 acts as the sole uptake path on at least one side of a polarized cell.

A Holistic Evolutionary and 3D Pharmacophore Modelling Study Provides Insights into the Metabolism, Function, and Substrate Selectivity of the Human Monocarboxylate Transporter 4 (hMCT4).

Monocarboxylate transporters (MCTs) are of great research interest for their role in cancer cell metabolism and their potential ability to transport pharmacologically relevant compounds across the membrane. Each member of the MCT family could potentially provide novel therapeutic approaches to various diseases. The major differences among MCTs are related to each of their specific metabolic roles, their relative substrate and inhibitor affinities, the regulation of their expression, their intracellular localization, and their tissue distribution. MCT4 is the main mediator for the efflux of L-lactate produced in the cell. Thus, MCT4 maintains the glycolytic phenotype of the cancer cell by supplying the molecular resources for tumor cell proliferation and promotes the acidification of the extracellular microenvironment from the co-transport of protons. A promising therapeutic strategy in anti-cancer drug design is the selective inhibition of MCT4 for the glycolytic suppression of solid tumors. A small number of studies indicate molecules for dual inhibition of MCT1 and MCT4; however, no selective inhibitor with high-affinity for MCT4 has been identified. In this study, we attempt to approach the structural characteristics of MCT4 through an in silico pipeline for molecular modelling and pharmacophore elucidation towards the identification of specific inhibitors as a novel anti-cancer strategy.

Structural basis of human monocarboxylate transporter 1 inhibition by anti-cancer drug candidates

Proton-coupled monocarboxylate transporters MCT1-4 catalyze the transmembrane movement of metabolically essential monocarboxylates and have been targeted for cancer treatment because of their enhanced expression in various tumors. Here, we report five cryo-EM structures, at resolutions of 3.0-3.3Å, of human MCT1 bound to lactate or inhibitors in the presence of Basigin-2, a single transmembrane segment (TM)-containing chaperon. MCT1 exhibits similar outward-open conformations when complexed with lactate or the inhibitors BAY-8002 and AZD3965. In the presence of the inhibitor 7ACC2 or with the neutralization of the proton-coupling residue Asp309 by Asn, similar inward-open structures were captured. Complemented by structural-guided biochemical analyses, our studies reveal the substrate binding and transport mechanism of MCTs, elucidate the mode of action of three anti-cancer drug candidates, and identify the determinants for subtype-specific sensitivities to AZD3965 by MCT1 and MCT4. These findings lay out an important framework for structure-guided drug discovery targeting MCTs.

Identification of the essential extracellular aspartic acids conserved in human monocarboxylate transporters 1, 2, and 4

Human monocarboxylate transporters (hMCTs) 1–4 transport monocarboxylates, such as l-lactate and pyruvate, as well as H+ across the plasma membrane. hMCT1, 2, and 4 play important roles in energy balance, pH homeostasis. However, the molecular mechanism of these transporters, especially their pH dependency, remains unknown. The aim of this study was to identify the residues involved in the pH dependence of hMCT1, 2, and 4. Firstly, we focused on the effects of extracellular acids of hMCT1. l-Lactate uptake assay and site-directed mutagenesis revealed that the aspartic acid of hMCT1 (hMCT1 D414) was an important residue conserved in MCT1, 2, and 4 (hMCT2 D398 and hMCT4 D379). Because the functional characteristic of hMCT2-mediated l-lactate transport has not been reported, we built a hMCT2-expressing system using Xenopus laevis oocytes. The transport activity of hMCT2 was enhanced by co-expression with embigin, an ancillary protein, and kinetic analysis of hMCT2-mediated l-lactate uptake revealed that the apparent Km value (0.32 ± 0.02 mM) was lower than that mediated by hMCT1 and 4. Finally, we investigated the conserved aspartic acids of hMCT2 and 4, and revealed that these residues were essential for l-lactate transport. These findings suggested that the extracellular aspartic acids conserved in hMCT1, 2, and 4 played important roles in transport activity and pH dependency, and can function as a first step of substrate and H+ recognition and transport from the extracellular to the intracellular region. These findings contributed to enhance our understanding of the transport process of hMCT1, 2, and 4.

Cooperative transport mechanism of human monocarboxylate transporter 2

Proton-linked monocarboxylate transporters (MCTs) must transport monocarboxylate efficiently to facilitate monocarboxylate efflux in glycolytically active cells, and transport monocarboxylate slowly or even shut down to maintain a physiological monocarboxylate concentration in glycolytically inactive cells. To discover how MCTs solve this fundamental aspect of intracellular monocarboxylate homeostasis in the context of multicellular organisms, we analyzed pyruvate transport activity of human monocarboxylate transporter 2 (MCT2). Here we show that MCT2 transport activity exhibits steep dependence on substrate concentration. This property allows MCTs to turn on almost like a switch, which is physiologically crucial to the operation of MCTs in the cellular context. We further determined the cryo-electron microscopy structure of the human MCT2, demonstrating that the concentration sensitivity of MCT2 arises from the strong inter-subunit cooperativity of the MCT2 dimer during transport. These data establish definitively a clear example of evolutionary optimization of protein function.

Spatiotemporal Changes of Cerebral Monocarboxylate Transporter 8 Expression.

Background: Mutations of monocarboxylate transporter 8 (MCT8), a thyroid hormone (TH)-specific transmembrane transporter, cause a severe neurodevelopmental disorder, the Allan-Herndon-Dudley syndrome. In MCT8 deficiency, TH is not able to reach those areas of the brain where TH uptake depends on MCT8. Currently, therapeutic options for MCT8-deficient patients are missing, as TH treatment is not successful in improving neurological deficits. Available data on MCT8 protein and transcript levels indicate complex expression patterns in neural tissue depending on species, brain region, sex, and age. However, information on human MCT8 expression is still scattered and additional efforts are needed to map sites of MCT8 expression in neurovascular units and neural tissue. This is of importance because new therapeutic strategies for this disease are urgently needed. Methods: To identify regions and time windows of MCT8 expression, we used highly specific antibodies against MCT8 to perform immunofluorescence labeling of postnatal murine brains, adult human brain tissue, and human cerebral organoids. Results: Qualitative and quantitative analyses of murine brain samples revealed stable levels of MCT8 protein expression in endothelial cells of the blood-brain barrier (BBB), choroid plexus epithelial cells, and tanycytes during postnatal development. Conversely, the neuronal MCT8 protein expression that was robustly detectable in specific brain regions of young mice strongly declined with age. Similarly, MCT8 immunoreactivity in adult human brain tissue was largely confined to endothelial cells of the BBB. Recently, cerebral organoids emerged as promising models of human neural development and our first analyses of forebrain-like organoids revealed MCT8 expression in early neuronal progenitor cell populations. Conclusions: With respect to MCT8-deficient conditions, our analyses not only strongly support the contention that the BBB presents a lifelong barrier to TH uptake but also highlight the need to decipher the TH transport role of MCT8 in early neuronal cell populations in more detail. Improving the understanding of the spatiotemporal expression in latter barriers will be critical for therapeutic strategies addressing MCT8 deficiency in the future.

Molecular characterization of the orphan transporter SLC16A9, an extracellular pH- and Na+-sensitive creatine transporter

Human monocarboxylate transporters (hMCTs) mediate the transport of monocarboxylates across plasma membranes. One such transporter, hMCT9, has been shown to be related to serum uric acid levels and the risk of renal overload gout. However, the functional characteristics of hMCT9 remain unknown. The aim of this study was to investigate the expression and localization of hMCT9 using a Xenopus laevis oocyte heterologous expression system and characterize its transport properties. Kinetic analysis of hMCT9-mediated creatine uptake revealed that uptake consisted of two components, with apparent Km values of 237 mm (low-affinity) and 23.7 mm (high-affinity), respectively. The transport activity of hMCT9 was dependent on the extracellular pH and activity sharply increased with increasing pH. Under Na+-free conditions, hMCT9-mediated creatine uptake was reduced by one-half, indicating that hMCT9 is a Na+-sensitive transporter. Moreover, carbonyl cyanide 3-chlorophenylhydrazone (a protonophore) inhibited hMCT9 activity, whereas valinomycin (a K+-ionophore) did not inhibit the transporter. These results suggest that hMCT9 is susceptible to changes in H+ gradients. A cis-inhibition assay of hMCT9-and hMCT12-mediated creatine transport revealed that cyclocreatine, creatine, guanidineacetate, and 3-guanidinopropionate are recognized by the transporter, and 4-guanidinobutyrate and guanidinoethyl sulfonate selectively inhibited hMCT9 activity. These findings demonstrate that hMCT9 is an extracellular pH- and Na+-sensitive creatine transporter.

Extracellular lysine 38 plays a crucial role in pH-dependent transport via human monocarboxylate transporter 1

Human monocarboxylate transporters (hMCTs) are expressed in many tissues and mediate the transport of various substrates across the plasma membrane. Among hMCTs, hMCT1–4 cotransport H+ with monocarboxylates such as pyruvate and l-lactate, implying that these proteins recognize both substrate and H+. However, the mechanism of translocation, and particularly that of hMCT1 pH-dependent transport, remains largely unknown. This study aimed at identifying residues involved in the pH dependence of hMCT1 using a combination of amino acid-modifying reagents, site-directed mutagenesis in a Xenopus laevis oocyte expression system, and homology modeling. We showed that diethyl pyrocarbonate (DEPC), phenylglyoxal (PGO), and 4,4′-diisothiocyanato-2,2′-stilbenedisulfonic acid disodium salt (DIDS), which react with histidine, arginine, and lysine residues respectively, all inhibited hMCT1 activity. Since DEPC, PGO, and DIDS are membrane impermeable reagents, we mutated to other residues individual histidine, arginine, and lysine residues located within the extracellular regions of hMCT1. Analyses of these mutants demonstrated that except for K38, the extracellular basic residues of hMCT1 were not involved in its transport activity and pH dependence. Moreover, analyses of various mutants in which K38 was substituted for another residue and of an hMCT1 homology model focusing on the location of K38 in the three-dimensional structure delineated the mechanism of hMCT1 pH dependence. Collectively, our data indicate that K38 plays an essential role in hMCT1 transport activity. We would like to propose a mechanism whereby K38 is positioned within a hydrophobic and narrow cavity that is part of the transport pathway, and regulates pH-dependent gating of hMCT1.

In Vitro Characterization of Human, Mouse, and Zebrafish MCT8 Orthologues

Background: Mutations in the thyroid hormone (TH) transporter monocarboxylate transporter 8 (MCT8) cause MCT8 deficiency, characterized by severe intellectual and motor disability and abnormal serum thyroid function tests. Various Mct8 knock-out mouse models as well as mct8 knock-out and knockdown zebrafish models are used as a disease model for MCT8 deficiency. Although important for model eligibility, little is known about the functional characteristics of the MCT8 orthologues in these species. Therefore, we here compared the functional characteristics of mouse (mm) MCT8 and zebrafish (dr) Mct8 to human (hs) MCT8. Methods: We performed extensive transport studies in COS-1 and JEG-3 cells transiently transfected with hsMCT8, drMct8, and mmMCT8. Protein expression levels and subcellular localization were assessed by immunoblotting, surface biotinylation, and immunocytochemistry. Sequence alignment and structural modeling were used to interpret functional differences between the orthologues. Results: hsMCT8, drMct8, and mmMCT8 all facilitated the uptake and efflux of 3,3'-diiodothyronine (3,3'-T2), rT3, triiodothyronine (T3), and thyroxine (T4), although the initial uptake rates of drMct8 were 1.5-4.0-fold higher than for hsMCT8 and mmMCT8. drMct8 exhibited 3-50-fold lower apparent IC50 values than hsMCT8 and mmMCT8 for all tested substrates, and substrate preference of drMct8 (3,3'-T2, T3 > T4 > rT3) differed from hsMCT8 and mmMCT8 (T3 > T4 > rT3, 3,3'-T2). Compared with hsMCT8 and mmMCT8, cis-inhibition studies showed that T3 uptake by drMct8 was inhibited at a lower concentration and by a broader spectrum of TH metabolites. Total and cell surface expression levels of drMct8 and hsMCT8 were equal and both significantly exceeded those of mmMCT8. Structural modeling located most non-conserved residues outside the substrate pore, except for H192 in hsMCT8, which is replaced by a glutamine in drMct8. However, a H192Q substituent of hsMCT8 did not alter its transporter characteristics. Conclusion: Our studies substantiate the eligibility of mice and zebrafish models for human MCT8 deficiency. However, differences in the intrinsic transporter properties of MCT8 orthologues may exist, which should be realized when comparing MCT8 deficiency in different in vivo models. Moreover, our findings may indicate that the protein domains outside the substrate channel may play a role in substrate selection and protein stability.

Adult Mice Lacking Mct8 and Dio2 Proteins Present Alterations in Peripheral Thyroid Hormone Levels and Severe Brain and Motor Skill Impairments.

Background: Mutations in the thyroid hormone (TH) transporter monocarboxylate transporter 8 (MCT8) lead to peripheral hyperthyroidism and profound psychomotor alterations in humans. Mice lacking Mct8 present peripheral hyperthyroidism but no gross neurological abnormalities due to brain compensatory mechanisms involving the enzyme deiodinase type 2 (Dio2). Methods: Here we have analyzed the endocrine and neurologic phenotype of mice lacking both Mct8 and Dio2 at three and six months of age. Thyroxine (T4) and 3,5,3' triiodothyronine (T3) levels/content were measured by specific radioimmunoassays; motor skill performance was evaluated by the footprint, rotarod, four limb hanging wire, and balance beam tests; and brain histological analysis was performed by immunostaining for neurofilament and parvalbumin. Results: We have found that this mouse model presents peripheral hyperthyroidism and brain hypothyroidism. Interestingly, the severity of the brain hypothyroidism seems permanent and varies across regions, with the striatum being a particularly affected area. We have also found brain alterations at the histological level compatible with TH deficiency and impaired motor skills. Conclusions: These findings indicate the potential of Mct8/Dio2-deficient mice to represent a model for human MCT8 deficiency, to understand the mechanisms underlying its pathophysiology, and ultimately design therapeutic interventions for human patients.

Homology modeling and site-directed mutagenesis identify amino acid residues underlying the substrate selection mechanism of human monocarboxylate transporters 1 (hMCT1) and 4 (hMCT4).

Human monocarboxylate transporters (hMCTs/SLC16As) mediate the transport of monocarboxylic compounds across plasma membranes. Among the hMCTs, hMCT1 and hMCT4 are expressed in various tissues, and transport substrates involved in energy metabolism. Both transporters mediate L-lactate transport, but, although hMCT1 also transports L-5-oxoproline (L-OPro), this compound is minimally transported by hMCT4. Thus, we were interested in the molecular mechanism responsible for the difference in substrate specificity between hMCT1 and hMCT4. Therefore, we generated 3D structure models of hMCT1 and hMCT4 to identify amino acid residues involved in the substrate specificity of these transporters. We found that the substrate specificity of hMCT1 was regulated by residues involved in turnover number (M69) and substrate affinity (F367), and these residues were responsible for recognizing (directly or indirectly) the -NH- moiety of L-OPro. Furthermore, our homology model of hMCT1 predicted that M69 and F367 participate in hydrophobic interactions with another region of hMCT1, emphasizing its potentially important role in the binding and translocation cycle of L-OPro. Mutagenesis experiments supported this model, showing that efficient L-OPro transport required a hydrophobic, long linear structure at position 69 and a hydrophobic, γ-branched structure at position 367. Our work demonstrated that the amino acid residues, M69 and F367, are key molecular elements for the transport of L-OPro by hMCT1. These two residues may be involved in substrate recognition and/or substrate-induced conformational changes.

Engineering Synthetic Chromosomes by Sequential Loading of Multiple Genomic Payloads over 100 Kilobase Pairs in Size

Gene delivery vehicles currently in the clinic for treatment of monogenic disorders lack sufficient carrying capacity to efficiently address complex polygenic diseases. Thus, to engineer multifaceted genetic circuits for bioengineering human cells as a therapeutic option for polygenic diseases, we require new tools that are currently in their infancy. Mammalian artificial chromosomes, or synthetic chromosomes, represent a viable approach for delivery of large genetic payloads that are mitotically stable and remain independent of the host genome. Previously, we described a mammalian synthetic chromosome platform, termed the ACE system, that requires a single unidirectional integrase for the introduction of multiple genes onto the ACE platform chromosome. In this report, we provide a proof of concept that the ACE synthetic chromosome bioengineering platform is amenable to sequential delivery of off-the-shelf large genomic fragments. Specifically, large genomic clones spanning the human solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1 or GLUT1, 169 kbp), and human monocarboxylate transporter 1 (SLC16A1 or MCT1, 144 kbp) genetic loci were engineered onto the ACE platform and demonstrated to express and correctly splice both gene transcripts. Thus, the ACE system provides a facile and tractable engineering platform for the development of gene-based therapeutic agents targeting polygenic diseases.

Modeling the Biochemical Phenotype of MCT8 Mutations In Vitro: Resolving a Troubling Inconsistency.

Allan-Herndon-Dudley syndrome (AHDS) is a severe genetic disease caused by mutations in the monocarboxylate transporter 8 (MCT8) gene. MCT8 mediates transport of thyroid hormones in and out of cells, which is thought to play a pivotal role for embryonic and postnatal development of the human brain. Disconcertingly, MCT8R271H leads to a severe form of AHDS but shows residual transport activity when expressed in several types of cultured cells. Here we try to determine the mechanism behind the transport function of MCT8R271H found in overexpressing cell systems. Mutations of Arg271 were introduced into human MCT8 and stably transfected into Madin-Darby canine kidney cells and the human-derived cell line JEG1. Radioactive thyroid hormone-uptake experiments were performed to analyze the pH-dependent effect of the mutation on transport activity. Arg271His transports thyroid hormones in and out of cells in a pH-dependent manner. Its transport activity increases below pH 7.3 and is clearly diminished at physiological pH. The Michaelis constant of the mutant is unaltered, whereas the maximum velocity is reduced. The expression of Arg271His in JEG1 cells leads to an almost nonfunctional transporter at physiological pH replicating the human phenotype for this mutant in vitro and demonstrates, again, that mutant MCT8 activity depends on cellular background. The protonation of His271 at acidic pH restores activity of the mutant protein, which is not active in its deprotonated form at physiological pH. Thus, experimental parameters must be controlled carefully when modeling MCT8 deficiency in cells.

Involvement of human monocarboxylate transporters (MCTs) in the uptake of L-lactate in human astrocytes and identification of a selective inhibitor of MCTs

Involvement of human monocarboxylate transporters (MCTs) in the uptake of L-lactate in human astrocytes and identification of a selective inhibitor of MCTs

Functional analysis of monocarboxylate transporter 8 mutations in Japanese Allan-Herndon-Dudley syndrome patients

Monocarboxylate transporter 8 (MCT8) facilitates T3 uptake into cells. Mutations in MCT8 lead to Allan-Herndon-Dudley syndrome (AHDS), which is characterized by severe psychomotor retardation and abnormal thyroid hormone profile. Nine uncharacterized MCT8 mutations in Japanese patients with severe neurocognitive impairment and elevated serum T3 levels were studied regarding the transport of T3. Human MCT8 (hMCT8) function was studied in wild-type (WT) or mutant hMCT8-transfected human placental choriocarcinoma cells (JEG3) by visualizing the locations of the proteins in the cells, detecting specific proteins, and measuring T3 uptake. We identified 6 missense (p.Arg445Ser, p.Asp498Asn, p.Gly276Arg, p.Gly196Glu, p.Gly401Arg, and p.Gly312Arg), 2 frameshift (p.Arg355Profs*64 and p.Tyr550Serfs*17), and 1 deletion (p.Pro561del) mutation(s) in the hMCT8 gene. All patients exhibited clinical characteristics of AHDS with high free T3, low-normal free T4, and normal-elevated TSH levels. All tested mutants were expressed at the protein level, except p.Arg355Profs*64 and p.Tyr550Serfs*17, which were truncated, and were inactive in T3 uptake, excluding p.Arg445Ser and p.Pro561del mutants, compared with WT-hMCT8. Immunocytochemistry revealed plasma membrane localization of p.Arg445Ser and p.Pro561del mutants similar with WT-hMCT8. The other mutants failed to localize in significant amount(s) in the plasma membrane and instead localized in the cytoplasm. These data indicate that p.Arg445Ser and p.Pro561del mutants preserve residual function, whereas p.Asp498Asn, p.Gly276Arg, p.Gly196Glu, p.Gly401Arg, p.Gly312Arg, p.Arg355Profs*64, and p.Tyr550Serfs*17 mutants lack function. These findings suggest that the mutations in MCT8 cause loss of function by reducing protein expression, impairing trafficking of protein to plasma membrane, and disrupting substrate channel.