A substrate translocation trajectory in a cytoplasm-facing topological model of the monocarboxylate/H+ symporter Jen1p

Previous mutational analysis of Jen1p, a Saccharomyces cerevisiae monocarboxylate/H+ symporter of the Major Facilitator Superfamily, has suggested that the consensus sequence 379NXX[S/T]HX[S/T]QD387 in transmembrane segment VII (TMS-VII) is part of the substrate translocation pathway. Here, we rationally design, analyse and show that several novel mutations in TMS-V and TMS-XI directly modify Jen1p function. Among the residues studied, F270 (TMS-V) and Q498 (TMS-XI) are critical specificity determinants for the distinction of mono- from di-carboxylates, and N501 (TMS-XI) is a critical residue for function. Using a model created on the basis of Jen1p similarity with the GlpT permease, we show that all polar residues critical for function within TMS-VII and TMS-XI (N379, H383, D387, Q498, N501) are perfectly aligned in an imaginary axis that lies parallel to a protein pore. This model and subsequent mutational analysis further reveal that an additional polar residue facing the pore, R188 (TMS-II), is irreplaceable for function. Our model also justifies the role of F270 and Q498 in substrate specificity. Finally, docking approaches reveal a ‘trajectory-like’ substrate displacement within the Jen1p pore, where R188 plays a major dynamic role mediating the orderly relocation of the substrate by subsequent H-bond interactions involving itself and residues H383, N501 and Q498.Fundação para a Ciência e a Tecnologia (FCT)fellowship SFRH/BPD/22976/2005 (ISS) and SFRH/BD/61530/2009 (JSP

MCT1 genetic polymorphism influence in high intensity circuit training: A pilot study

Monocarboxylate Transporter 1 (MCT1) mediates the transport of the main fraction of lactate across the sarcolemma. A common polymorphic MCT1 variant has been identified, but its role in high intensity exercise performance has not been defined. We investigated the influence of MCT1 A1470T polymorphism (rs1049434) on lactate accumulation after high intensity circuit training. Ten men aged 20–26 performed three controlled circuit training (CWT) sessions at 60%, 70%, and 80% of the 15 repetition maximum (15 RM), in non-consecutive days. CWT included three sets of a circuit of eight exercises, obtaining lactate measurements immediately after each set had been completed. Two independent variables were analysed: MTC1 genotypes according to the presence or absence of the A1470T polymorphism, and the intensity of circuit training. Genotype distributions were in Hardy–Weinberg equilibrium, being 30% wild-type, 50% heterozygotes, and 20% mutated homozygotes. Mean lactate concentration at 80% of 15 RM were significantly higher than the mean lactate values at the other intensities (p < 0.01). Significant differences between genetic groups were found in the lactate accumulation slope at 80% of 15 RM (p = 0.02) and in the maximal lactate concentration reached by all subjects in the study (Lmax) (p = 0.03). The carriers of the A1470T polymorphism in the MTC1 gene seem to exhibit a worse lactate transport capability into the less active muscle cells for oxidation

Role of the monocarboxylate transporter MCT1 in the uptake of lactate during active recovery

Purpose We assessed the role of monocarboxylate transporter 1 (MCT1) on lactate clearance during an active recovery after high-intensity exercise, by comparing genetic groups based on the T1470A (rs1049434) MCT1 polymorphism, whose influence on lactate transport has been proven. Methods Sixteen young male elite field hockey players participated in this study. All of them completed two 400 m maximal run tests performed on different days, followed by 40 min of active or passive recovery. Lactate samples were measured immediately after the tests, and at min 10, 20, 30 and 40 of the recoveries. Blood lactate decreases were calculated for each 10-min period. Participants were distributed into three groups according to the T1470A polymorphism (TT, TA and AA). Results TT group had a lower blood lactate decrease than AA group during the 10?20 min period of the active recovery (p = 0.018). This period had the highest blood lactate for the whole sample, significantly differing from the other periods (p ? 0.003). During the passive recovery, lactate declines were constant except for the 0?10-min period (p ? 0.003), suggesting that liver uptake is similar in all the genetic groups, and that the difference seen during the active recovery is mainly due to muscle lactate uptake. Conclusions These differences according to the polymorphic variant T1470A suggest that MCT1 affects the plasma lactate decrease during a crucial period of active recovery, where the maximal lactate amount is cleared (i.e. 10?20 min period)

Expression QTL Modules as Functional Components Underlying Higher-Order Phenotypes

Systems genetics studies often involve the mapping of numerous regulatory relations between genetic loci and expression traits. These regulatory relations form a bipartite network consisting of genetic loci and expression phenotypes. Modular network organizations may arise from the pleiotropic and polygenic regulation of gene expression. Here we analyzed the expression QTL (eQTL) networks derived from expression genetic data of yeast and mouse liver and found 65 and 98 modules respectively. Computer simulation result showed that such modules rarely occurred in randomized networks with the same number of nodes and edges and same degree distribution. We also found significant within-module functional coherence. The analysis of genetic overlaps and the evidences from biomedical literature have linked some eQTL modules to physiological phenotypes. Functional coherence within the eQTL modules and genetic overlaps between the modules and physiological phenotypes suggests that eQTL modules may act as functional units underlying the higher-order phenotypes

Monocarboxylate transporters (MCTs) in gliomas: expression and exploitation as therapeutic targets

Background. Gliomas exhibit high glycolytic rates, and monocarboxylate transporters (MCTs) play a major role in the maintenance of the glycolytic metabolism through the proton-linked transmembrane transport of lactate. However, their role in gliomas is poorly studied. Thus, we aimed to characterize the expression of MCT1, MCT4, and their chaperone CD 147 and to assess the therapeutic impact of MCT inhibition in gliomas. Methods. MCTs and CD 147 expressions were characterized by immunohistochemistry in nonneoplastic brain and glioma samples. The effect of CHC (MCT inhibitor) and MCT1 silencing was assessed in in vitro and in vivo glioblastoma models. Results. MCT1, MCT4, and CD 147 were overexpressed in the plasma membrane of glioblastomas, compared with diffuse astrocytomas and nonneoplastic brain. CHC decreased glycolytic metabolism, migration, and invasion and induced cell death in U251 cells (more glycolytic) but only affected proliferation in SW1088 (more oxidative). The effectiveness of CHC in glioma cells appears to be dependent on MCT membrane expression. MCT1 downregulation showed similar effects on different glioma cells, supporting CHC as an MCT1 inhibitor. There was a synergistic effect when combining CHC with temozolomide treatment in U251 cells. In the CAM in vivo model, CHC decreased the size of tumors and the number of blood vessels formed. Conclusions. This is the most comprehensive study reporting the expression of MCTs and CD 147 in gliomas. The MCT1 inhibitor CHC exhibited anti-tumoral and anti-angiogenic activity in gliomas and, of importance, enhanced the effect of temozolomide. Thus, our results suggest that development of therapeutic approaches targeting MCT1 may be a promising strategy in glioblastoma treatment.Life and Health Sciences Research Institute, University of Minho, Portugal; Fundacao para a Ciencia e Tecnologia [SFRH/BI/33503/2008, SFRH/BPD/69479/2010, SFRH/BD/36463/2007]info:eu-repo/semantics/publishedVersio

Pyruvate: immunonutritional effects on neutrophil intracellular amino or alpha-keto acid profiles and reactive oxygen species production

For the first time the immunonutritional role of pyruvate on neutrophils (PMN), free α-keto and amino acid profiles, important reactive oxygen species (ROS) produced [superoxide anion (O2−), hydrogen peroxide (H2O2)] as well as released myeloperoxidase (MPO) acitivity has been investigated. Exogenous pyruvate significantly increased PMN pyruvate, α-ketoglutarate, asparagine, glutamine, aspartate, glutamate, arginine, citrulline, alanine, glycine and serine in a dose as well as duration of exposure dependent manner. Moreover, increases in O2− formation, H2O2-generation and MPO acitivity in parallel with intracellular pyruvate changes have also been detected. Regarding the interesting findings presented here we believe, that pyruvate fulfils considerably the criteria for a potent immunonutritional molecule in the regulation of the PMN dynamic α-keto and amino acid pools. Moreover it also plays an important role in parallel modulation of the granulocyte-dependent innate immune regulation. Although further research is necessary to clarify pyruvate’s sole therapeutical role in critically ill patients’ immunonutrition, the first scientific successes seem to be very promising

Monocarboxylate transporters, Past, present, and future

We review here the 14 members of the Monocarboxylate transporter family (MCTs), their relationship based on sequence homology. The range of substrates transported by different members of this family extends from the standard monocarboxylate metabolites, lactic and pyruvic acids, to aromatic amino acids and thyroid hormones. The family is denoted Solute Carrier Family 16, or SLC16, among 43 SLC families constituting more than 300 members, which are annotated regularly at the website http://www.bioparadigms.org/slc/intro.htm. MCTs classically transport metabolites across plasma membranes with direction controlled by proton and metabolite concentrations independently of energy input, but they may also function in subcellular membranes. Their regulation may be complex, and they are implicated in leukocyte-mediated immunity, hypoxia induced cellular responses, and partitioning of the energy supply in several tissues. We focus here on histologic evidence (involving human tissue where available) and the first four ‘classical’ members; but we do annotate all 14, and note several candidate or proven genetic diseases that have arisen from MCT mutations. The review progresses through the following sections: (1) MCT1-4: genetics, kinetics, and modulation; (2) Chaperonins and targeting cofactors; (3) Tissue distribution of MCTs; (4) Intercellular lactate/pyruvate shuttles; (5) Transcriptional and translational regulation of MCTs; (6) Properties of other MCTs; and (7) Subcellular localization of MCTs and some future considerations. Along the way we posit questions or suggestions for future research