RESUMO
The Organic Cation Transporter Novel 1 (OCTN1), also known as SLC22A4, is widely expressed in various human tissues, and involved in numerous physiological and pathological processes remains. It facilitates the transport of organic cations, zwitterions, with selectivity for positively charged solutes. Ergothioneine, an antioxidant compound, and acetylcholine (Ach) are among its substrates. Given the lack of experimentally solved structures of this protein, this study aimed at generating a reliable 3D model of OCTN1 to shed light on its substrate-binding preferences and the role of sodium in substrate recognition and transport. A chimeric model was built by grafting the large extracellular loop 1 (EL1) from an AlphaFold-generated model onto a homology model. Molecular dynamics simulations revealed domain-specific mobility, with EL1 exhibiting the highest impact on overall stability. Molecular docking simulations identified cytarabine and verapamil as highest affinity ligands, consistent with their known inhibitory effects on OCTN1. Furthermore, MM/GBSA analysis allowed the categorization of substrates into weak, good, and strong binders, with molecular weight strongly correlating with binding affinity to the recognition site. Key recognition residues, including Tyr211, Glu381, and Arg469, were identified through interaction analysis. Ach demonstrated a low interaction energy, supporting the hypothesis of its one-directional transport towards to outside of the membrane. Regarding the role of sodium, our model suggested the involvement of Glu381 in sodium binding. Molecular dynamics simulations of systems at increasing levels of Na+ concentrations revealed increased sodium occupancy around Glu381, supporting experimental data associating Na+ concentration to molecule transport. In conclusion, this study provides valuable insights into the 3D structure of OCTN1, its substrate-binding preferences, and the role of sodium in the recognition. These findings contribute to the understanding of OCTN1 involvement in various physiological and pathological processes and may have implications for drug development and disease management.
Assuntos
Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteínas de Transporte de Cátions Orgânicos , Humanos , Proteínas de Transporte de Cátions Orgânicos/química , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Proteínas de Transporte de Cátions Orgânicos/genética , Simportadores/química , Simportadores/metabolismo , Sítios de Ligação , Ligação Proteica , Ergotioneína/química , Ergotioneína/metabolismo , Sódio/metabolismo , Sódio/química , Simulação por Computador , Acetilcolina/metabolismo , Acetilcolina/química , LigantesRESUMO
Multidrug and toxin extrusion (MATE) family transporters excrete toxic compounds coupled to Na+/H+ influx. Although structures of MATE transporters are available, the mechanism by which substrate export is coupled to ion influx remains unknown. To address this issue, we conducted a structural analysis of Pyrococcus furiosus MATE (PfMATE) using solution nuclear magnetic resonance (NMR). The NMR analysis, along with thorough substitutions of all non-exposed acidic residues, confirmed that PfMATE is under an equilibrium between inward-facing (IF) and outward-facing (OF) conformations, dictated by the Glu163 protonation. Importantly, we found that only the IF conformation exhibits a mid-µM affinity for substrate recognition. In contrast, the OF conformation exhibited only weak mM substrate affinity, suitable for releasing substrate to the extracellular side. These results indicate that PfMATE is an affinity-directed H+ antiporter where substrates selectively bind to the protonated IF conformation in the equilibrium, and subsequent proton release mechanistically ensures H+-coupled substrate excretion by the transporter.
Assuntos
Proteínas Arqueais , Pyrococcus furiosus , Pyrococcus furiosus/metabolismo , Proteínas Arqueais/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/genética , Ligação Proteica , Especificidade por Substrato , Sítios de Ligação , Modelos Moleculares , Prótons , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Proteínas de Transporte de Cátions Orgânicos/química , Proteínas de Transporte de Cátions Orgânicos/genética , Ressonância Magnética Nuclear Biomolecular , Conformação ProteicaRESUMO
Organic Cation Transporter 1 (OCT1) plays a crucial role in hepatic metabolism by mediating the uptake of a range of metabolites and drugs. Genetic variations can alter the efficacy and safety of compounds transported by OCT1, such as those used for cardiovascular, oncological, and psychological indications. Despite its importance in drug pharmacokinetics, the substrate selectivity and underlying structural mechanisms of OCT1 remain poorly understood. Here, we present cryo-EM structures of full-length human OCT1 in the inward-open conformation, both ligand-free and drug-bound, indicating the basis for its broad substrate recognition. Comparison of our structures with those of outward-open OCTs provides molecular insight into the alternating access mechanism of OCTs. We observe that hydrophobic gates stabilize the inward-facing conformation, whereas charge neutralization in the binding pocket facilitates the release of cationic substrates. These findings provide a framework for understanding the structural basis of the promiscuity of drug binding and substrate translocation in OCT1.
Assuntos
Proteínas de Transporte de Cátions Orgânicos , Transportador 1 de Cátions Orgânicos , Humanos , Transportador 1 de Cátions Orgânicos/genética , Transportador 1 de Cátions Orgânicos/química , Transportador 1 de Cátions Orgânicos/metabolismo , Proteínas de Transporte de Cátions Orgânicos/química , Transporte Biológico , Transportador 2 de Cátion Orgânico/metabolismoRESUMO
The Novel Organic Cation Transporter, OCTN1, is the first member of the OCTN subfamily; it belongs to the wider Solute Carrier family SLC22, which counts many members including cation and anion organic transporters. The tertiary structure has not been resolved for any cation organic transporter. The functional role of OCNT1 is still not well assessed despite the many functional studies so far conducted. The lack of a definitive identification of OCTN1 function can be attributed to the different experimental systems and methodologies adopted for studying each of the proposed ligands. Apart from the contradictory data, the international scientific community agrees on a role of OCTN1 in protecting cells and tissues from oxidative and/or inflammatory damage. Moreover, the involvement of this transporter in drug interactions and delivery has been well clarified, even though the exact profile of the transported/interacting molecules is still somehow confusing. Therefore, OCTN1 continues to be a hot topic in terms of its functional role and structure. This review focuses on the most recent advances on OCTN1 in terms of functional aspects, physiological roles, substrate specificity, drug interactions, tissue expression, and relationships with pathology.
Assuntos
Biomarcadores , Suscetibilidade a Doenças , Interações Medicamentosas , Proteínas de Transporte de Cátions Orgânicos/genética , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Simportadores/genética , Simportadores/metabolismo , Acetilação , Animais , Sítios de Ligação , Transporte Biológico , Ergotioneína/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Modelos Moleculares , Conformação Molecular , Especificidade de Órgãos , Proteínas de Transporte de Cátions Orgânicos/química , Ligação Proteica , Relação Estrutura-Atividade , Simportadores/químicaRESUMO
A transporter of the multidrug and toxic compound extrusion (MATE) family, Nicotiana tabacum MATE2 (NtMATE2), is located in the vacuole membrane of the tobacco plant root and is involved in the transportation of nicotine, a secondary or specialized metabolic compound in Solanaceae. Here, we report the crystal structures of NtMATE2 in its outward-facing forms. The overall structure has a bilobate V-shape with pseudo-symmetrical assembly of the N- and C-lobes. In one crystal structure, the C-lobe cavity of NtMATE2 interacts with an unidentified molecule that may partially mimic a substrate. In addition, NtMATE2-specific conformational transitions imply that an unprecedented movement of the transmembrane α-helix 7 is related to the release of the substrate into the vacuolar lumen.
Assuntos
Nicotiana/metabolismo , Nicotina/química , Proteínas de Transporte de Cátions Orgânicos/química , Proteínas de Plantas/química , Vacúolos/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Transporte Biológico , Clonagem Molecular , Cristalografia por Raios X , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Modelos Moleculares , Nicotina/metabolismo , Proteínas de Transporte de Cátions Orgânicos/genética , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Células Vegetais/química , Células Vegetais/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Nicotiana/genética , Vacúolos/químicaRESUMO
In both prokaryotes and eukaryotes, multidrug and toxic-compound extrusion (MATE) transporters catalyze the efflux of a broad range of cytotoxic compounds, including human-made antibiotics and anticancer drugs. MATEs are secondary-active antiporters, i.e., their drug-efflux activity is coupled to, and powered by, the uptake of ions down a preexisting transmembrane electrochemical gradient. Key aspects of this mechanism, however, remain to be delineated, such as its ion specificity and stoichiometry. We previously revealed the existence of a Na+-binding site in a MATE transporter from Pyroccocus furiosus (PfMATE) and hypothesized that this site might be broadly conserved among prokaryotic MATEs. Here, we evaluate this hypothesis by analyzing VcmN and ClbM, which along with PfMATE are the only three prokaryotic MATEs whose molecular structures have been determined at atomic resolution, i.e. better than 3 Å. Reinterpretation of existing crystallographic data and molecular dynamics simulations indeed reveal an occupied Na+-binding site in the N-terminal lobe of both structures, analogous to that identified in PfMATE. We likewise find this site to be strongly selective against K+, suggesting it is mechanistically significant. Consistent with these computational results, DEER spectroscopy measurements for multiple doubly-spin-labeled VcmN constructs demonstrate Na+-dependent changes in protein conformation. The existence of this binding site in three MATE orthologs implicates Na+ in the ion-coupled drug-efflux mechanisms of this class of transporters. These results also imply that observations of H+-dependent activity likely stem either from a site elsewhere in the structure, or from H+ displacing Na+ under certain laboratory conditions, as has been noted for other Na+-driven transport systems.
Assuntos
Antiporters/química , Proteínas de Transporte de Cátions Orgânicos/química , Conformação Proteica/efeitos dos fármacos , Sódio/química , Antibacterianos/efeitos adversos , Antibacterianos/farmacologia , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacologia , Antiporters/ultraestrutura , Sítios de Ligação/efeitos dos fármacos , Cristalografia por Raios X , Humanos , Íons/química , Modelos Moleculares , Simulação de Dinâmica Molecular , Proteínas de Transporte de Cátions Orgânicos/ultraestrutura , Células Procarióticas/química , Células Procarióticas/ultraestrutura , Domínios Proteicos/efeitos dos fármacosRESUMO
Ablation of Slc22a14 causes male infertility in mice, but the underlying mechanisms remain unknown. Here, we show that SLC22A14 is a riboflavin transporter localized at the inner mitochondrial membrane of the spermatozoa mid-piece and show by genetic, biochemical, multi-omic, and nutritional evidence that riboflavin transport deficiency suppresses the oxidative phosphorylation and reprograms spermatozoa energy metabolism by disrupting flavoenzyme functions. Specifically, we find that fatty acid ß-oxidation (FAO) is defective with significantly reduced levels of acyl-carnitines and metabolites from the TCA cycle (the citric acid cycle) but accumulated triglycerides and free fatty acids in Slc22a14 knockout spermatozoa. We demonstrate that Slc22a14-mediated FAO is essential for spermatozoa energy generation and motility. Furthermore, sperm from wild-type mice treated with a riboflavin-deficient diet mimics those in Slc22a14 knockout mice, confirming that an altered riboflavin level causes spermatozoa morphological and bioenergetic defects. Beyond substantially advancing our understanding of spermatozoa energy metabolism, our study provides an attractive target for the development of male contraceptives.
Assuntos
Ciclo do Ácido Cítrico/genética , Fertilidade/genética , Infertilidade Masculina/genética , Proteínas de Transporte de Cátions Orgânicos/genética , Riboflavina/metabolismo , Espermatozoides/metabolismo , Animais , Carnitina/análogos & derivados , Carnitina/metabolismo , Ciclo do Ácido Cítrico/efeitos dos fármacos , Dieta/métodos , Ácidos Graxos/metabolismo , Feminino , Fertilização in vitro , Expressão Gênica , Humanos , Infertilidade Masculina/dietoterapia , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Masculino , Metaboloma , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Modelos Moleculares , Proteínas de Transporte de Cátions Orgânicos/química , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Riboflavina/farmacologia , Motilidade dos Espermatozoides/efeitos dos fármacos , Motilidade dos Espermatozoides/genética , Espermatozoides/efeitos dos fármacos , Espermatozoides/patologiaRESUMO
Multidrug and toxic compound extrusion (MATE) transporters are primarily expressed in the kidneys and liver, where they contribute to the excretion of organic cations. Our previous study suggested that pig MATE2 (class III) participates in testosterone secretion from Leydig cells. In humans, it is unclear which MATE class is involved in testosterone transport. In this study, we aimed to clarify whether human MATE1 (hMATE1) or human MATE2K (hMATE2K) mediates testosterone transport. To confirm that testosterone inhibits transporter-mediated tetraethylammonium (TEA) uptake, a cis-inhibition assay was performed using cells that stably expressed hMATE1 or hMATE2K. Docking simulations were performed to characterize differences in the binding of hMATE1 and hMATE2K to testosterone. Transport experiments in LLC-PK1 cells that stably expressed hMATE1 were used to test whether hMATE1 mediates testosterone transport. We detected differences between the amino acid sequences of the substrate-binding sites of hMATE1 and hMATE2K that could potentially be involved in testosterone binding. Testosterone and estradiol inhibited TEA uptake mediated by hMATE1 but not that mediated by hMATE2K. Transport experiments in LLC-PK1 cells indicated that testosterone might be transported via hMATE1. This study suggested that hMATE1, but not hMATE2K, is involved in human testosterone transport.
Assuntos
Proteínas de Transporte de Cátions Orgânicos/metabolismo , Testosterona/farmacologia , Animais , Cimetidina/farmacologia , Estradiol/farmacologia , Células HEK293 , Humanos , Células LLC-PK1 , Modelos Moleculares , Proteínas de Transporte de Cátions Orgânicos/química , Suínos , Tetraetilamônio/metabolismoRESUMO
The multidrug and toxin extrusion (MATE) transporters catalyze active efflux of a broad range of chemically- and structurally-diverse compounds including antimicrobials and chemotherapeutics, thus contributing to multidrug resistance in pathogenic bacteria and cancers. Multiple methodological approaches have been taken to investigate the structural basis of energy transduction and substrate translocation in MATE transporters. Crystal structures representing members from all three MATE subfamilies have been interpreted within the context of an alternating access mechanism that postulates occupation of distinct structural intermediates in a conformational cycle powered by electrochemical ion gradients. Here we review the structural biology of MATE transporters, integrating the crystallographic models with biophysical and computational studies to define the molecular determinants that shape the transport energy landscape. This holistic analysis highlights both shared and disparate structural and functional features within the MATE family, which underpin an emerging theme of mechanistic diversity within the framework of a conserved structural scaffold.
Assuntos
Proteínas de Transporte de Cátions Orgânicos/fisiologia , Animais , Sequência Conservada , Resistência a Medicamentos/genética , Humanos , Modelos Moleculares , Proteínas de Transporte de Cátions Orgânicos/química , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
The membrane phospholipids phosphatidylcholine and phosphatidylethanolamine (PE) are synthesized de novo by the CDP-choline and CDP-ethanolamine (Kennedy) pathway, in which the extracellular substrates choline and ethanolamine are transported into the cell, phosphorylated, and coupled with diacylglycerol to form the final phospholipid product. Although multiple transport systems have been established for choline, ethanolamine transport is poorly characterized and there is no single protein assigned a transport function for ethanolamine. The solute carriers 44A (SLC44A) known as choline transporter-like proteins-1 and -2 (CTL1 and CTL2) are choline transporter at the plasma membrane and mitochondria. We report a novel function of CTL1 and CTL2 in ethanolamine transport. Using the lack or the gain of gene function in combination with specific antibodies and transport inhibitors we established two distinct ethanolamine transport systems of a high affinity, mediated by CTL1, and of a low affinity, mediated by CTL2. Both transporters are Na+-independent ethanolamine/H+ antiporters. Primary human fibroblasts with separate frameshift mutations in the CTL1 gene (M1= SLC44A1ΔAsp517 and M2= SLC44A1ΔSer126) are devoid of CTL1 ethanolamine transport but maintain unaffected CTL2 transport. The lack of CTL1 in M2 cells reduced the ethanolamine transport, the flux through the CDP-ethanolamine Kennedy pathway, and PE synthesis. In contrast, overexpression of CTL1 in M2 cells improved ethanolamine transport and PE synthesis. These data firmly establish that CTL1 and CTL2 are the first identified ethanolamine transporters in whole cells and mitochondria, with intrinsic roles in de novo PE synthesis by the Kennedy pathway and intracellular redistribution of ethanolamine.
Assuntos
Antígenos CD/metabolismo , Membrana Celular/metabolismo , Etanolamina/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Animais , Antígenos CD/química , Transporte Biológico , Linhagem Celular , Humanos , Glicoproteínas de Membrana/química , Proteínas de Membrana Transportadoras/química , Modelos Moleculares , Proteínas de Transporte de Cátions Orgânicos/química , Conformação ProteicaRESUMO
BACKGROUND: Insufficient renal urate excretion and/or overproduction of uric acid (UA) are the dominant causes of hyperuricemia. Baicalein (BAL) is widely distributed in dietary plants and has extensive biological activities, including antioxidative, anti-inflammatory and antihypertensive activities. PURPOSE: To investigate the anti-hyperuricemic effects of BAL and the underlying mechanisms in vitro and in vivo. METHODS: We investigated the inhibitory effects of BAL on GLUT9 and URAT1 in vitro through electrophysiological experiments and 14C-urate uptake assays. To evaluate the impact of BAL on serum and urine UA, the expression of GLUT9 and URAT1, and the activity of xanthine oxidase (XOD), we developed a mouse hyperuricemia model by potassium oxonate (PO) injection. Molecular docking analysis based on homology modeling was performed to explain the predominant efficacy of BAL compared with the other test compounds. RESULTS: BAL dose-dependently inhibited GLUT9 and URAT1 in a noncompetitive manner with IC50 values of 30.17 ± 8.68 µM and 31.56 ± 1.37 µM, respectively. BAL (200 mg/kg) significantly decreased serum UA and enhanced renal urate excretion in PO-induced hyperuricemic mice. Moreover, the expression of GLUT9 and URAT1 in the kidney was downregulated, and XOD activity in the serum and liver was suppressed. The docking analysis revealed that BAL potently interacted with Trp336, Asp462, Tyr71 and Gln328 of GLUT9 and Ser35 and Phe241 of URAT1. CONCLUSION: These results indicated that BAL exerts potent antihyperuricemic efects through renal UA excretal promotion and serum UA production. Thus, we propose that BAL may be a promising treatment for the prevention of hyperuricemia owing to its multitargeted inhibitory activity.
Assuntos
Flavanonas/farmacologia , Hiperuricemia/tratamento farmacológico , Ácido Úrico/urina , Xantina Oxidase/antagonistas & inibidores , Animais , Antioxidantes/farmacologia , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Flavanonas/química , Flavanonas/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/química , Proteínas Facilitadoras de Transporte de Glucose/genética , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Células HEK293 , Humanos , Hiperuricemia/induzido quimicamente , Rim/efeitos dos fármacos , Rim/metabolismo , Fígado/efeitos dos fármacos , Masculino , Camundongos , Simulação de Acoplamento Molecular , Transportadores de Ânions Orgânicos/química , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo , Proteínas de Transporte de Cátions Orgânicos/química , Proteínas de Transporte de Cátions Orgânicos/genética , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Ácido Oxônico/toxicidade , Ácido Úrico/sangueRESUMO
Multidrug and toxic compound extrusion (MATE) transporters are ubiquitous ion-coupled antiporters that extrude structurally and chemically dissimilar cytotoxic compounds and have been implicated in conferring multidrug resistance. Here, we integrate double electron-electron resonance (DEER) with functional assays and site-directed mutagenesis of conserved residues to illuminate principles of ligand-dependent alternating access of PfMATE, a proton-coupled MATE from the hyperthermophilic archaeon Pyrococcus furiosus Pairs of spin labels monitoring the two sides of the transporter reconstituted into nanodiscs reveal large-amplitude movement of helices that alter the orientation of a putative substrate binding cavity. We found that acidic pH favors formation of an inward-facing (IF) conformation, whereas elevated pH (>7) and the substrate rhodamine 6G stabilizes an outward-facing (OF) conformation. The lipid-dependent PfMATE isomerization between OF and IF conformation is driven by protonation of a previously unidentified intracellular glutamate residue that is critical for drug resistance. Our results can be framed in a mechanistic model of transport that addresses central aspects of ligand coupling and alternating access.
Assuntos
Antiporters/química , Antiporters/metabolismo , Proteínas de Transporte de Cátions Orgânicos/química , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Antiporters/genética , Resistência a Múltiplos Medicamentos , Espectroscopia de Ressonância de Spin Eletrônica , Ligantes , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas de Transporte de Cátions Orgânicos/genética , Conformação Proteica , Prótons , Pyrococcus furiosus/metabolismoRESUMO
Metformin, a drug frequently used by diabetic patients as the first-line treatment worldwide, is positively charged and is transported into the cell through human organic cation transporter (hOCT 1-3) proteins. We aimed to mimic the cellular uptake of metformin by hOCT1-3 with various bioinformatics methods and tools. 3D structure of OCT1-3 proteins was predicted by considering the structures and function of these proteins. We predicted functional regions (active and ligand binding sites) of OCT1-3 and performed comparative bioinformatics analysis. The predicted structure of hOCT1-3 was then analyzed in the Blind Docking server and the results were confirmed with predicted binding site residues and conserved domain regions. We simulated the OCT1-3 and metformin docking and also validated the docking procedure with other substrates of HOCT1-3 proteins. We selected the best poses of metformin docking simulations as per binding energy (-5.27 to -4.60 kcal/mol). Lastly, we validated the static description of protein-ligand (OCT-Metformin) interactions by performing molecular dynamics simulation. Eventually, we obtained stable simulation of OCT-metformin interaction.
Assuntos
Metformina/metabolismo , Fator 1 de Transcrição de Octâmero/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Transportador 2 de Cátion Orgânico/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Biologia Computacional , Humanos , Metformina/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Fator 1 de Transcrição de Octâmero/química , Proteínas de Transporte de Cátions Orgânicos/química , Transportador 2 de Cátion Orgânico/química , Ligação ProteicaRESUMO
Xenobiotic and metabolite extrusion is an important process for the proper functions of cells and their compartments, including acidic organelles. MATE (multidrug and toxic compound extrusion) is a large family of secondary active transporters involved in the transport of various compounds across cellular and organellar membranes, and is present in the three domains of life. The major substrates of the bacterial MATE transporters are cationic compounds, including clinically important antibiotics, and thereby MATE transporters confer multi-drug resistance to pathogenic bacteria. The plant MATE transporters are important for the accumulation of various metabolites in organelles, including vacuoles. The human MATE transporters are expressed in the brush-border membrane of the kidney, and are involved in the clearance of cationic drugs from the body. During the past decade, progress in structural biology has clarified the transport mechanism of these MATE transporters in atomic detail. The present review summarizes the reported structures of MATE family transporters, along with their structure-guided functional analyses. This integrated view of the structures of MATE transporters provides novel insights into their transport mechanism.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Proteínas de Plantas/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/classificação , Archaea/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/classificação , Proteínas Arqueais/metabolismo , Humanos , Simulação de Dinâmica Molecular , Proteínas de Transporte de Cátions Orgânicos/química , Proteínas de Transporte de Cátions Orgânicos/classificação , Proteínas de Plantas/química , Proteínas de Plantas/classificação , Plantas/metabolismo , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
Bacteria use a number of mechanisms to defend themselves from antimicrobial drugs. One important defense strategy is the ability to export drugs by multidrug transporters. One class of multidrug transporter, the so-called multidrug and toxic compound extrusion (MATE) transporters, extrude a variety of antibiotic compounds from the bacterial cytoplasm. These MATE transporters are driven by a Na+, H+, or combined Na+/H+ gradient, and act as antiporters to drive a conformational change in the transporter from the outward to the inward-facing conformation. In the inward-facing conformation, a chemical compound (drug) binds to the protein, resulting in a switch to the opposite conformation, thereby extruding the drug. Using molecular dynamics simulations, we now report the structural basis for Na+ and H+ binding in the dual ion coupled MATE transporter ClbM from Escherichia coli, which is connected to colibactin-induced genotoxicity, yielding novel insights into the ion/drug translocation mechanism of this bacterial transporter.
Assuntos
Proteínas de Escherichia coli/química , Simulação de Dinâmica Molecular , Proteínas de Transporte de Cátions Orgânicos/química , Antibacterianos/química , Sítios de Ligação , Proteínas de Escherichia coli/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Ligação Proteica , Prótons , Sódio/metabolismoRESUMO
hURAT1 (human urate transporter 1) is a successful target for hyperuricemia. Recently, the modification work on hURAT1 inhibitors showed that the flexible linkers would benefit biological activity. The study aimed to investigate the contribution of the linkers and give modification strategies on this kind of structures based on QSAR models (HQSAR and topomer CoMFA). The most effective HQSAR and topomer CoMFA models were generated by applying the training set containing 63 compounds, with the cross-validated q2 values of 0.869/0.818 and the non-cross-validated correlation coefficients r2 of 0.951/0.978, respectively. The Y-randomization test was applied to ensure the robustness of the models. The external predictive correlation coefficient (rpred2) grounded on the external test set (21 compounds) of two models was 0.910 and 0.907, respectively. In addition, the models were validated by Golbraikh-Tropsha and Roy methods, as well as other statistical metrics. The results showed that both models were reliable. Topomer CoMFA steric/electrostatic contours and HQSAR atomic contribution maps illustrated the structural features which governed their inhibitory potency. The dependable results could provide important insights to guide the designing of more potential hURAT1 inhibitors.
Assuntos
Descoberta de Drogas , Transportadores de Ânions Orgânicos/química , Proteínas de Transporte de Cátions Orgânicos/química , Relação Quantitativa Estrutura-Atividade , Algoritmos , Descoberta de Drogas/métodos , Humanos , Modelos Químicos , Modelos Moleculares , Conformação Molecular , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Proteínas de Transporte de Cátions Orgânicos/antagonistas & inibidoresRESUMO
Variation in steroid hormone levels has wide implications for health and disease. The genes encoding the proteins involved in steroid disposition represent key determinants of interindividual variation in steroid levels and ultimately, their effects. Beginning with metabolomic data from genome-wide association studies (GWAS), we observed that genetic variants in the orphan transporter, SLC22A24 were significantly associated with levels of androsterone glucuronide and etiocholanolone glucuronide (sentinel SNPs p-value <1x10-30). In cells over-expressing human or various mammalian orthologs of SLC22A24, we showed that steroid conjugates and bile acids were substrates of the transporter. Phylogenetic, genomic, and transcriptomic analyses suggested that SLC22A24 has a specialized role in the kidney and appears to function in the reabsorption of organic anions, and in particular, anionic steroids. Phenome-wide analysis showed that functional variants of SLC22A24 are associated with human disease such as cardiovascular diseases and acne, which have been linked to dysregulated steroid metabolism. Collectively, these functional genomic studies reveal a previously uncharacterized protein involved in steroid homeostasis, opening up new possibilities for SLC22A24 as a pharmacological target for regulating steroid levels.
Assuntos
Proteínas de Transporte de Cátions Orgânicos/metabolismo , Esteroides/metabolismo , Simportadores/metabolismo , Androsterona/análogos & derivados , Androsterona/genética , Androsterona/metabolismo , Animais , Transporte Biológico , Estudo de Associação Genômica Ampla/métodos , Células HEK293 , Humanos , Metabolômica/métodos , Modelos Moleculares , Proteínas de Transporte de Cátions Orgânicos/química , Proteínas de Transporte de Cátions Orgânicos/genética , Filogenia , Polimorfismo de Nucleotídeo Único , Simportadores/química , Simportadores/genéticaRESUMO
The manual classification of protein domains is approaching its 20th anniversary. ECOD is our mixed manual-automatic domain classification. Over time, the types of proteins which require manual curation has changed. Depositions with complex multidomain and multichain arrangements are commonplace. Transmembrane domains are regularly classified. Repeatedly, domains which are initially believed to be novel are found to have homologous links to existing classified domains. Here we present a brief summary of recent manual curation efforts in ECOD generally combined with specific case studies of transmembrane and multidomain proteins wherein manual curation was useful for discovering new homologous relationships. We present a new taxonomy for the classification of ABC transporter transmembrane domains. We examine alternate topologies of the leucine-specific (LS) domain of Leucine tRNA-synthetase. Finally, we elaborate on a distant homologous links between two helical dimerization domains.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/classificação , Domínios Proteicos , Homologia Estrutural de Proteína , Proteínas de Transporte/química , Proteínas de Ciclo Celular/química , Cristalografia por Raios X , Bases de Dados de Proteínas , Endopeptidases/química , Escherichia coli/química , Humanos , Leucina-tRNA Ligase/química , Proteínas de Membrana/química , Proteínas de Transporte de Cátions Orgânicos/química , Multimerização Proteica , Estrutura Secundária de Proteína , Proteínas ras/químicaRESUMO
BACKGROUND: Despite all the efforts made up to now, the reasons that facilitate a protein becoming an allergen have not been elucidated yet. Alt a 1 protein is the major fungal allergen responsible for chronic asthma, but little is known about its immunological activity. Our main purpose was to investigate the ligand-dependent interactions of Alt a 1 in the human airway epithelium. METHODS: Alt a 1 with and without its ligand (holo- and apo- forms) was incubated with the pulmonary epithelial monolayer model, Calu-3 cells. Allergen transport and cytokine production were measured. Pull-down and immunofluorescence assays were employed to identify the receptor of Alt a 1 using the epithelial cell model and mouse tissues. Receptor-allergen-ligand interactions were analyzed by computational modeling. RESULTS: The holo-form could activate human monocytes, PBMCs, and polarized airway epithelial (Calu-3) cell lines. The allergen was also transported through the monolayer, without any alteration of the epithelial integrity (TEER). Alt a 1 also induced the production of proinflammatory IL8 and specific epithelial cytokines (IL33 and IL25) by Calu-3 cells. The interaction between epithelial cells and holo-Alt a 1 was found to be mediated by the SLC22A17 receptor, and its recognition of Alt a 1 was explained in structural terms. CONCLUSIONS: Our findings identified the Alt a 1 ligand as a central player in the interaction of the allergen with airway mucosa, shedding light into its potential role in the immunological response, while unveiling its potential as a new target for therapy intervention.
Assuntos
Antígenos de Fungos/imunologia , Antígenos de Fungos/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Apresentação de Antígeno/imunologia , Antígenos de Fungos/química , Biomarcadores , Linhagem Celular , Humanos , Leucócitos Mononucleares , Ligantes , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Modelos Moleculares , Proteínas de Transporte de Cátions Orgânicos/química , Proteínas de Transporte de Cátions Orgânicos/genética , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/imunologia , Relação Estrutura-AtividadeRESUMO
OCTN is a small subfamily of membrane transport proteins that belongs to the larger SLC22 family. Two of the three members of the subfamily, namely, OCTN2 and OCTN1, are present in humans. OCTN2 plays a crucial role in the absorption of carnitine from diet and in its distribution to tissues, as demonstrated by the occurrence of severe pathologies caused by malfunctioning or altered expression of this transporter. These findings suggest avoiding a strict vegetarian diet during pregnancy and in childhood. Other roles of OCTN2 are related to the traffic of carnitine derivatives in many tissues. The role of OCTN1 is still unclear, despite the identification of some substrates such as ergothioneine, acetylcholine, and choline. Plausibly, the transporter acts on the control of inflammation and oxidative stress, even though knockout mice do not display phenotypes. A clear role of both transporters has been revealed in drug interaction and delivery. The polyspecificity of the OCTNs is at the base of the interactions with drugs. Interestingly, OCTN2 has been recently exploited in the prodrug approach and in diagnostics. A promising application derives from the localization of OCTN2 in exosomes that represent a noninvasive diagnostic tool.