RESUMEN
The lysosomal degradation of macromolecules produces diverse small metabolites exported by specific transporters for reuse in biosynthetic pathways. Here we deorphanized the major facilitator superfamily domain containing 1 (MFSD1) protein, which forms a tight complex with the glycosylated lysosomal membrane protein (GLMP) in the lysosomal membrane. Untargeted metabolomics analysis of MFSD1-deficient mouse lysosomes revealed an increase in cationic dipeptides. Purified MFSD1 selectively bound diverse dipeptides, while electrophysiological, isotope tracer and fluorescence-based studies in Xenopus oocytes and proteoliposomes showed that MFSD1-GLMP acts as a uniporter for cationic, neutral and anionic dipeptides. Cryoelectron microscopy structure of the dipeptide-bound MFSD1-GLMP complex in outward-open conformation characterized the heterodimer interface and, in combination with molecular dynamics simulations, provided a structural basis for its selectivity towards diverse dipeptides. Together, our data identify MFSD1 as a general lysosomal dipeptide uniporter, providing an alternative route to recycle lysosomal proteolysis products when lysosomal amino acid exporters are overloaded.
Asunto(s)
Dipéptidos , Lisosomas , Lisosomas/metabolismo , Animales , Dipéptidos/metabolismo , Oocitos/metabolismo , Microscopía por Crioelectrón , Ratones , Xenopus laevis , Humanos , Ratones Noqueados , Simulación de Dinámica Molecular , Simportadores/metabolismo , Simportadores/genética , Simportadores/química , Femenino , Canales de Potencial de Receptor TransitorioRESUMEN
Lysosomes degrade excess or damaged cellular components and recycle their building blocks through membrane transporters. They also act as nutrient-sensing signaling hubs to coordinate cell responses. The membrane protein PQ-loop repeat-containing protein 2 (PQLC2; "picklock two") is implicated in both functions, as it exports cationic amino acids from lysosomes and serves as a receptor and amino acid sensor to recruit the C9orf72/SMCR8/WDR41 complex to lysosomes upon nutrient starvation. Its transport activity is essential for drug treatment of the rare disease cystinosis. Here, we quantitatively studied PQLC2 transport activity using electrophysiological and biochemical methods. Charge/substrate ratio, intracellular pH, and reversal potential measurements showed that it operates in a uniporter mode. Thus, PQLC2 is uncoupled from the steep lysosomal proton gradient, unlike many lysosomal transporters, enabling bidirectional cationic amino acid transport across the organelle membrane. Surprisingly, the specific presence of arginine, but not other substrates (lysine, histidine), in the discharge ("trans") compartment impaired PQLC2 transport. Kinetic modeling of the uniport cycle recapitulated the paradoxical substrate-yet-inhibitor behavior of arginine, assuming that bound arginine facilitates closing of the transporter's cytosolic gate. Arginine binding may thus tune PQLC2 gating to control its conformation, suggesting a potential mechanism for nutrient signaling by PQLC2 to its interaction partners.
Asunto(s)
Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Arginina/metabolismo , Sistemas de Transporte de Aminoácidos Básicos/genética , Animales , Arginina/farmacología , Citosol/metabolismo , Femenino , Células HEK293 , Humanos , Cinética , Lisina/metabolismo , Lisina/farmacología , Lisosomas/metabolismo , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Técnicas de Placa-Clamp , Xenopus , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMEN
Sialin, encoded by the SLC17A5 gene, is a lysosomal sialic acid transporter defective in Salla disease, a rare inherited leukodystrophy. It also enables metabolic incorporation of exogenous sialic acids, leading to autoantibodies against N-glycolylneuraminic acid in humans. Here, we identified a novel class of human sialin ligands by virtual screening and structure-activity relationship studies. The ligand scaffold is characterized by an amino acid backbone with a free carboxylate, an N-linked aromatic or heteroaromatic substituent, and a hydrophobic side chain. The most potent compound, 45 (LSP12-3129), inhibited N-acetylneuraminic acid 1 (Neu5Ac) transport in a non-competitive manner with IC50 ≈ 2.5 µM, a value 400-fold lower than the KM for Neu5Ac. In vitro and molecular docking studies attributed the non-competitive character to selective inhibitor binding to the Neu5Ac site in a cytosol-facing conformation. Moreover, compound 45 rescued the trafficking defect of the pathogenic mutant (R39C) causing Salla disease. This new class of cell-permeant inhibitors provides tools to investigate the physiological roles of sialin and help develop pharmacological chaperones for Salla disease.
Asunto(s)
Aminoácidos/química , Aminoácidos/metabolismo , Lisosomas/metabolismo , Transportadores de Anión Orgánico/metabolismo , Simportadores/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Células HEK293 , Células HeLa , Humanos , Ligandos , Simulación del Acoplamiento Molecular/métodos , Estructura Secundaria de Proteína , RatasRESUMEN
Congenital disorders of glycosylation are severe inherited diseases in which aberrant protein glycosylation is a hallmark. Transmembrane protein 165 (TMEM165) is a novel Golgi transmembrane protein involved in type II congenital disorders of glycosylation. Although its biologic function is still a controversial issue, we have demonstrated that the Golgi glycosylation defect due to TMEM165 deficiency resulted from a Golgi Mn2+ homeostasis defect. The goal of this study was to delineate the cellular pathway by which extracellular Mn2+ rescues N-glycosylation in TMEM165 knockout (KO) cells. We first demonstrated that after extracellular exposure, Mn2+ uptake by HEK293 cells at the plasma membrane did not rely on endocytosis but was likely done by plasma membrane transporters. Second, we showed that the secretory pathway Ca2+-ATPase 1, also known to mediate the influx of cytosolic Mn2+ into the lumen of the Golgi apparatus, is not crucial for the Mn2+-induced rescue glycosylation of lysosomal-associated membrane protein 2 (LAMP2). In contrast, our results demonstrate the involvement of cyclopiazonic acid- and thapsigargin (Tg)-sensitive pumps in the rescue of TMEM165-associated glycosylation defects by Mn2+. Interestingly, overexpression of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) 2b isoform in TMEM165 KO cells partially rescues the observed LAMP2 glycosylation defect. Overall, this study indicates that the rescue of Golgi N-glycosylation defects in TMEM165 KO cells by extracellular Mn2+ involves the activity of Tg and cyclopiazonic acid-sensitive pumps, probably the SERCA pumps.-Houdou, M., Lebredonchel, E., Garat, A., Duvet, S., Legrand, D., Decool, V., Klein, A., Ouzzine, M., Gasnier, B., Potelle, S., Foulquier, F. Involvement of thapsigargin- and cyclopiazonic acid-sensitive pumps in the rescue of TMEM165-associated glycosylation defects by Mn2+.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Indoles/farmacología , Manganeso/farmacología , Proteínas de la Membrana/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Tapsigargina/farmacología , Antiportadores , Transporte Biológico , Calcio/metabolismo , Proteínas de Transporte de Catión , Trastornos Congénitos de Glicosilación/tratamiento farmacológico , Trastornos Congénitos de Glicosilación/metabolismo , Trastornos Congénitos de Glicosilación/patología , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Inhibidores Enzimáticos/farmacología , Glicosilación , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/metabolismo , Células HEK293 , Homeostasis , Humanos , Proteína 2 de la Membrana Asociada a los Lisosomas/genética , Proteína 2 de la Membrana Asociada a los Lisosomas/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genéticaRESUMEN
Vitamin B12 (cobalamin (Cbl)), in the cofactor forms methyl-Cbl and adenosyl-Cbl, is required for the function of the essential enzymes methionine synthase and methylmalonyl-CoA mutase, respectively. Cbl enters mammalian cells by receptor-mediated endocytosis of protein-bound Cbl followed by lysosomal export of free Cbl to the cytosol and further processing to these cofactor forms. The integral membrane proteins LMBD1 and ABCD4 are required for lysosomal release of Cbl, and mutations in the genes LMBRD1 and ABCD4 result in the cobalamin metabolism disorders cblF and cblJ. We report a new (fifth) patient with the cblJ disorder who presented at 7 days of age with poor feeding, hypotonia, methylmalonic aciduria, and elevated plasma homocysteine and harbored the mutations c.1667_1668delAG [p.Glu556Glyfs*27] and c.1295G>A [p.Arg432Gln] in the ABCD4 gene. Cbl cofactor forms are decreased in fibroblasts from this patient but could be rescued by overexpression of either ABCD4 or, unexpectedly, LMBD1. Using a sensitive live-cell FRET assay, we demonstrated selective interaction between ABCD4 and LMBD1 and decreased interaction when ABCD4 harbored the patient mutations p.Arg432Gln or p.Asn141Lys or when artificial mutations disrupted the ATPase domain. Finally, we showed that ABCD4 lysosomal targeting depends on co-expression of, and interaction with, LMBD1. These data broaden the patient and mutation spectrum of cblJ deficiency, establish a sensitive live-cell assay to detect the LMBD1-ABCD4 interaction, and confirm the importance of this interaction for proper intracellular targeting of ABCD4 and cobalamin cofactor synthesis.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Errores Innatos del Metabolismo de los Aminoácidos/genética , Lisosomas/metabolismo , Errores Innatos del Metabolismo/genética , Modelos Moleculares , Mutación , Proteínas de Transporte Nucleocitoplasmático/genética , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/deficiencia , Transportadoras de Casetes de Unión a ATP/metabolismo , Errores Innatos del Metabolismo de los Aminoácidos/metabolismo , Errores Innatos del Metabolismo de los Aminoácidos/patología , Sustitución de Aminoácidos , Dominio Catalítico , Línea Celular Transformada , Células Cultivadas , Células HeLa , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Lisosomas/enzimología , Lisosomas/patología , Errores Innatos del Metabolismo/metabolismo , Errores Innatos del Metabolismo/patología , Simulación del Acoplamiento Molecular , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/deficiencia , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Transporte de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Homología Estructural de Proteína , Vitamina B 12/metabolismoRESUMEN
Lysosomes degrade cellular components sequestered by autophagy or extracellular material internalized by endocytosis and phagocytosis. The macromolecule building blocks released by lysosomal hydrolysis are then exported to the cytosol by lysosomal transporters, which remain undercharacterized. In this study, we designed an in situ assay of lysosomal amino acid export based on the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis that detects lysosomal storage. This assay was used to screen candidate lysosomal transporters, leading to the identification of sodium-coupled neutral amino acid transporter 7 (SNAT7), encoded by the SLC38A7 gene, as a lysosomal transporter highly selective for glutamine and asparagine. Cell fractionation confirmed the lysosomal localization of SNAT7, and flux measurements confirmed its substrate selectivity and showed a strong activation by the lysosomal pH gradient. Interestingly, gene silencing or editing experiments revealed that SNAT7 is the primary permeation pathway for glutamine across the lysosomal membrane and it is required for growth of cancer cells in a low free-glutamine environment, when macropinocytosis and lysosomal degradation of extracellular proteins are used as an alternative source of amino acids. SNAT7 may, thus, represent a novel target for glutamine-related anticancer therapies.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Glutamina/metabolismo , Membranas Intracelulares/metabolismo , Lisosomas/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Glutamina/genética , Células HeLa , Humanos , Lisosomas/genética , Lisosomas/patología , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patología , Microambiente TumoralRESUMEN
Potassium is the most abundant ion to face both plasma and organelle membranes. Extensive research over the past seven decades has characterized how K(+) permeates the plasma membrane to control fundamental processes such as secretion, neuronal communication, and heartbeat. However, how K(+) permeates organelles such as lysosomes and endosomes is unknown. Here, we directly recorded organelle K(+) conductance and discovered a major K(+)-selective channel KEL on endosomes and lysosomes. KEL is formed by TMEM175, a protein with unknown function. Unlike any of the â¼80 plasma membrane K(+) channels, TMEM175 has two repeats of 6-transmembrane-spanning segments and has no GYG K(+) channel sequence signature-containing, pore-forming P loop. Lysosomes lacking TMEM175 exhibit no K(+) conductance, have a markedly depolarized ΔΨ and little sensitivity to changes in [K(+)], and have compromised luminal pH stability and abnormal fusion with autophagosomes during autophagy. Thus, TMEM175 comprises a K(+) channel that underlies the molecular mechanism of lysosomal K(+) permeability.
Asunto(s)
Endosomas/metabolismo , Lisosomas/metabolismo , Canales de Potasio/metabolismo , Potasio/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Técnicas de Inactivación de Genes , Humanos , Membranas Intracelulares/metabolismo , Ratones , Datos de Secuencia Molecular , Técnicas de Placa-Clamp , Fagosomas/metabolismo , Canales de Potasio/química , Canales de Potasio/genética , Alineación de SecuenciaRESUMEN
Secondary transporters driven by a V-type Hâº-ATPase accumulate nonpeptide neurotransmitters into synaptic vesicles. Distinct transporter families are involved depending on the neurotransmitter. Monoamines and acetylcholine on the one hand, and glutamate and ATP on the other hand, are accumulated by SLC18 and SLC17 transporters, respectively, which belong to the major facilitator superfamily (MFS). GABA and glycine accumulate through a common SLC32 transporter from the amino acid/polyamine/organocation (APC) superfamily. Although crystallographic structures are not yet available for any vesicular transporter, homology modeling studies of MFS-type vesicular transporters based on distantly related bacterial structures recently provided significant advances, such as the characterization of substrate-binding pockets or the identification of spatial clusters acting as hinge points during the alternating-access cycle. However, several basic issues, such as the ion stoichiometry of vesicular amino acid transporters, remain unsettled.
Asunto(s)
Proteínas Transportadoras Vesiculares de Neurotransmisores/metabolismo , Animales , Transporte Biológico , Cloruros/metabolismo , Humanos , Ligandos , Proteínas Transportadoras Vesiculares de Neurotransmisores/antagonistas & inhibidores , Proteínas Transportadoras Vesiculares de Neurotransmisores/química , Proteínas Transportadoras Vesiculares de Neurotransmisores/clasificaciónRESUMEN
Glial cells are increasingly recognized as active players that profoundly influence neuronal synaptic transmission by specialized signaling pathways. In particular, astrocytes have been shown recently to release small molecules, such as the amino acids l-glutamate and d-serine as "gliotransmitters," which directly control the efficacy of adjacent synapses. However, it is still controversial whether gliotransmitters are released from a cytosolic pool or by Ca(2+)-dependent exocytosis from secretory vesicles, i.e., by a mechanism similar to the release of synaptic vesicles in synapses. Here we report that rat cortical astrocytes contain storage vesicles that display morphological and biochemical features similar to neuronal synaptic vesicles. These vesicles share some, but not all, membrane proteins with synaptic vesicles, including the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) synaptobrevin 2, and contain both l-glutamate and d-serine. Furthermore, they show uptake of l-glutamate and d-serine that is driven by a proton electrochemical gradient. d-Serine uptake is associated with vesicle acidification and is dependent on chloride. Whereas l-serine is not transported, serine racemase, the synthesizing enzyme for d-serine, is anchored to the membrane of the vesicles, allowing local generation of d-serine. Finally, we reveal a previously unexpected mutual vesicular synergy between d-serine and l-glutamate filling in glia vesicles. We conclude that astrocytes contain vesicles capable of storing and releasing d-serine, l-glutamate, and most likely other neuromodulators in an activity-dependent manner.
Asunto(s)
Astrocitos/metabolismo , Neuroglía/metabolismo , Serina/metabolismo , Transmisión Sináptica/fisiología , Vesículas Sinápticas/metabolismo , Animales , Astrocitos/ultraestructura , Transporte Biológico Activo/fisiología , Células Cultivadas , Femenino , Masculino , Neuroglía/fisiología , Neuroglía/ultraestructura , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Vesículas Sinápticas/ultraestructuraRESUMEN
Lysosomes are membrane-bound endocytic organelles that play a major role in degrading cell macromolecules and recycling their building blocks. A comprehensive knowledge of the lysosome function requires an extensive description of its content, an issue partially addressed by previous proteomic analyses. However, the proteins underlying many lysosomal membrane functions, including numerous membrane transporters, remain unidentified. We performed a comparative, semi-quantitative proteomic analysis of rat liver lysosome-enriched and lysosome-nonenriched membranes and used spectral counts to evaluate the relative abundance of proteins. Among a total of 2,385 identified proteins, 734 proteins were significantly enriched in the lysosomal fraction, including 207 proteins already known or predicted as endo-lysosomal and 94 proteins without any known or predicted subcellular localization. The remaining 433 proteins had been previously assigned to other subcellular compartments but may in fact reside on lysosomes either predominantly or as a secondary location. Many membrane-associated complexes implicated in diverse processes such as degradation, membrane trafficking, lysosome biogenesis, lysosome acidification, signaling, and nutrient sensing were enriched in the lysosomal fraction. They were identified to an unprecedented extent as most, if not all, of their subunits were found and retained by our screen. Numerous transporters were also identified, including 46 novel potentially lysosomal proteins. We expressed 12 candidates in HeLa cells and observed that most of them colocalized with the lysosomal marker LAMP1, thus confirming their lysosomal residency. This list of candidate lysosomal proteins substantially increases our knowledge of the lysosomal membrane and provides a basis for further characterization of lysosomal functions.
Asunto(s)
Hepatocitos/metabolismo , Membranas Intracelulares/química , Hígado/metabolismo , Lisosomas/química , Proteínas de Transporte de Membrana/aislamiento & purificación , Proteoma/aislamiento & purificación , Animales , Biomarcadores/metabolismo , Expresión Génica , Células HeLa , Hepatocitos/química , Humanos , Hígado/química , Proteínas de Membrana de los Lisosomas/genética , Proteínas de Membrana de los Lisosomas/metabolismo , Masculino , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Microscopía Fluorescente , Anotación de Secuencia Molecular , Proteoma/genética , Proteoma/metabolismo , Ratas , Ratas WistarRESUMEN
Cystinosin, the lysosomal cystine exporter defective in cystinosis, is the founding member of a family of heptahelical membrane proteins related to bacteriorhodopsin and characterized by a duplicated motif termed the PQ loop. PQ-loop proteins are more frequent in eukaryotes than in prokaryotes; except for cystinosin, their molecular function remains elusive. In this study, we report that three yeast PQ-loop proteins of unknown function, Ypq1, Ypq2, and Ypq3, localize to the vacuolar membrane and are involved in homeostasis of cationic amino acids (CAAs). We also show that PQLC2, a mammalian PQ-loop protein closely related to yeast Ypq proteins, localizes to lysosomes and catalyzes a robust, electrogenic transport that is selective for CAAs and strongly activated at low extracytosolic pH. Heterologous expression of PQLC2 at the yeast vacuole rescues the resistance phenotype of an ypq2 mutant to canavanine, a toxic analog of arginine efficiently transported by PQLC2. Finally, PQLC2 transports a lysine-like mixed disulfide that serves as a chemical intermediate in cysteamine therapy of cystinosis, and PQLC2 gene silencing trapped this intermediate in cystinotic cells. We conclude that PQLC2 and Ypq1-3 proteins are lysosomal/vacuolar exporters of CAAs and suggest that small-molecule transport is a conserved feature of the PQ-loop protein family, in agreement with its distant similarity to SWEET sugar transporters and to the mitochondrial pyruvate carrier. The elucidation of PQLC2 function may help improve cysteamine therapy. It may also clarify the origin of CAA abnormalities in Batten disease.
Asunto(s)
Sistemas de Transporte de Aminoácidos Básicos/química , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Cisteamina/uso terapéutico , Cistinosis/tratamiento farmacológico , Cistinosis/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sistemas de Transporte de Aminoácidos Básicos/genética , Animales , Secuencia de Bases , Proteínas de Caenorhabditis elegans/genética , Canavanina/metabolismo , ARN Helicasas DEAD-box , ADN Complementario/genética , Proteínas de Drosophila , Fenómenos Electrofisiológicos , Femenino , Genes Fúngicos , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Oocitos/metabolismo , Estructura Secundaria de Proteína , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidad de la Especie , Vacuolas/metabolismo , Xenopus laevisRESUMEN
In human skin fibroblasts, a lysosomal transport system specific for cationic amino acids has been described and named system c. We asked if SLC7A14 (solute carrier family 7 member A14), an orphan protein assigned to the SLC7 subfamily of cationic amino acid transporters (CATs) due to sequence homology, may represent system c. Fusion proteins between SLC7A14 and enhanced GFP localized to intracellular vesicles, co-staining with the lysosomal marker LysoTracker(®). To perform transport studies, we first tried to redirect SLC7A14 to the plasma membrane (by mutating putative lysosomal targeting motifs) but without success. We then created a chimera carrying the backbone of human (h) CAT-2 and the protein domain of SLC7A14 corresponding to the so-called "functional domain" of the hCAT proteins, a protein stretch of 81 amino acids that determines the apparent substrate affinity, sensitivity to trans-stimulation, and (as revealed in this study) pH dependence. The chimera mediated arginine transport and exhibited characteristics similar but not identical to hCAT-2A (the low affinity hCAT-2 isoform). Western blot and microscopic analyses confirmed localization of the chimera in the plasma membrane of Xenopus laevis oocytes. Noticeably, arginine transport by the hCAT-2/SLC7A14 chimera was pH-dependent, trans-stimulated, and inhibited by α-trimethyl-L-lysine, properties assigned to lysosomal transport system c in human skin fibroblasts. Expression analysis showed strong expression of SLC7A14 mRNA in these cells. Taken together, these data strongly suggest that SLC7A14 is a lysosomal transporter for cationic amino acids.
Asunto(s)
Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Arginina/metabolismo , Fibroblastos/metabolismo , Lisosomas/metabolismo , Piel/metabolismo , Sistemas de Transporte de Aminoácidos Básicos/genética , Animales , Arginina/genética , Transporte Biológico Activo/fisiología , Línea Celular , Fibroblastos/citología , Humanos , Concentración de Iones de Hidrógeno , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Piel/citología , Xenopus laevisRESUMEN
Secondary active transporters from the SLC17 protein family are required for excitatory and purinergic synaptic transmission, sialic acid metabolism, and renal function, and several members are associated with inherited neurological or metabolic diseases. However, molecular tools to investigate their function or correct their genetic defects are limited or absent. Using structure-activity, homology modeling, molecular docking, and mutagenesis studies, we have located the substrate-binding site of sialin (SLC17A5), a lysosomal sialic acid exporter also recently implicated in exocytotic release of aspartate. Human sialin is defective in two inherited sialic acid storage diseases and is responsible for metabolic incorporation of the dietary nonhuman sialic acid N-glycolylneuraminic acid. We built cytosol-open and lumen-open three-dimensional models of sialin based on weak, but significant, sequence similarity with the glycerol-3-phosphate and fucose permeases from Escherichia coli, respectively. Molecular docking of 31 synthetic sialic acid analogues to both models was consistent with inhibition studies. Narrowing the sialic acid-binding site in the cytosol-open state by two phenylalanine to tyrosine mutations abrogated recognition of the most active analogue without impairing neuraminic acid transport. Moreover, a pilot virtual high-throughput screening of the cytosol-open model could identify a pseudopeptide competitive inhibitor showing >100-fold higher affinity than the natural substrate. This validated model of human sialin and sialin-guided models of other SLC17 transporters should pave the way for the identification of inhibitors, glycoengineering tools, pharmacological chaperones, and fluorescent false neurotransmitters targeted to these proteins.
Asunto(s)
Biología Computacional , Transportadores de Anión Orgánico/química , Transportadores de Anión Orgánico/metabolismo , Simportadores/química , Simportadores/metabolismo , Azepinas/metabolismo , Sitios de Unión , Evaluación Preclínica de Medicamentos , Células HEK293 , Humanos , Indoles/metabolismo , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación Missense , Transportadores de Anión Orgánico/genética , Proyectos Piloto , Unión Proteica , Conformación Proteica , Homología de Secuencia de Aminoácido , Ácidos Siálicos/química , Ácidos Siálicos/metabolismo , Relación Estructura-Actividad , Simportadores/genéticaRESUMEN
Secondary active transporters use electrochemical gradients provided by primary ion pumps to translocate metabolites or drugs "uphill" across membranes. Here we report the ion-coupling mechanism of cystinosin, an unusual eukaryotic, proton-driven transporter distantly related to the proton pump bacteriorhodopsin. In humans, cystinosin exports the proteolysis-derived dimeric amino acid cystine from lysosomes and is impaired in cystinosis. Using voltage-dependence analysis of steady-state and transient currents elicited by cystine and neutralization-scanning mutagenesis of conserved protonatable residues, we show that cystine binding is coupled to protonation of a clinically relevant aspartate buried in the membrane. Deuterium isotope substitution experiments are consistent with an access of this aspartate from the lysosomal lumen through a deep proton channel. This aspartate lies in one of the two PQ-loop motifs shared by cystinosin with a set of eukaryotic membrane proteins of unknown function and is conserved in about half of them, thus suggesting that other PQ-loop proteins may translocate protons.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Lisosomas/metabolismo , Secuencia de Aminoácidos , Sistemas de Transporte de Aminoácidos Neutros/química , Sistemas de Transporte de Aminoácidos Neutros/genética , Animales , Sitios de Unión , Humanos , Datos de Secuencia Molecular , Mutagénesis , Protones , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
DIRC2 (Disrupted in renal carcinoma 2) has been initially identified as a breakpoint-spanning gene in a chromosomal translocation putatively associated with the development of renal cancer. The DIRC2 protein belongs to the MFS (major facilitator superfamily) and has been previously detected by organellar proteomics as a tentative constituent of lysosomal membranes. In the present study, lysosomal residence of overexpressed as well as endogenous DIRC2 was shown by several approaches. DIRC2 is proteolytically processed into a N-glycosylated N-terminal and a non-glycosylated C-terminal fragment respectively. Proteolytic cleavage occurs in lysosomal compartments and critically depends on the activity of cathepsin L which was found to be indispensable for this process in murine embryonic fibroblasts. The cleavage site within DIRC2 was mapped between amino acid residues 214 and 261 using internal epitope tags, and is presumably located within the tentative fifth intralysosomal loop, assuming the typical MFS topology. Lysosomal targeting of DIRC2 was demonstrated to be mediated by a N-terminal dileucine motif. By disrupting this motif, DIRC2 can be redirected to the plasma membrane. Finally, in a whole-cell electrophysiological assay based on heterologous expression of the targeting mutant at the plasma membrane of Xenopus oocytes, the application of a complex metabolic mixture evokes an outward current associated with the surface expression of full-length DIRC2. Taken together, these data strongly support the idea that DIRC2 is an electrogenic lysosomal metabolite transporter which is subjected to and presumably modulated by limited proteolytic processing.
Asunto(s)
Catepsina L/metabolismo , Proteínas de Membrana de los Lisosomas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Animales , Catepsina L/genética , Biología Computacional , Electrofisiología , Técnica del Anticuerpo Fluorescente Indirecta , Células HeLa , Humanos , Immunoblotting , Inmunoprecipitación , Proteínas de Membrana de los Lisosomas/genética , Proteínas de Transporte de Membrana/genética , Ratones , Proteínas de Neoplasias/genética , Unión Proteica , XenopusRESUMEN
Three different subtypes of H(+)-dependent carriers (named VGLUT1-3) concentrate glutamate into synaptic vesicles before its exocytotic release. Neurons using other neurotransmitter than glutamate (such as cholinergic striatal interneurons and 5-HT neurons) express VGLUT3. It was recently reported that VGLUT3 increases acetylcholine vesicular filling, thereby, stimulating cholinergic transmission. This new regulatory mechanism is herein designated as vesicular-filling synergy (or vesicular synergy). In the present report, we found that deletion of VGLUT3 increased several anxiety-related behaviors in adult and in newborn mice as early as 8 d after birth. This precocious involvement of a vesicular glutamate transporter in anxiety led us to examine the underlying functional implications of VGLUT3 in 5-HT neurons. On one hand, VGLUT3 deletion caused a significant decrease of 5-HT(1A)-mediated neurotransmission in raphe nuclei. On the other hand, VGLUT3 positively modulated 5-HT transmission of a specific subset of 5-HT terminals from the hippocampus and the cerebral cortex. VGLUT3- and VMAT2-positive serotonergic fibers show little or no 5-HT reuptake transporter. These results unravel the existence of a novel subset of 5-HT terminals in limbic areas that might play a crucial role in anxiety-like behaviors. In summary, VGLUT3 accelerates 5-HT transmission at the level of specific 5-HT terminals and can exert an inhibitory control at the raphe level. Furthermore, our results suggest that the loss of VGLUT3 expression leads to anxiety-associated behaviors and should be considered as a potential new target for the treatment of this disorder.
Asunto(s)
Sistemas de Transporte de Aminoácidos Acídicos/fisiología , Ansiedad/fisiopatología , Serotonina/fisiología , Sistemas de Transporte de Aminoácidos Acídicos/genética , Animales , Ansiedad/metabolismo , Autorreceptores/fisiología , Corteza Cerebral/fisiopatología , Hipocampo/fisiopatología , Ratones , Ratones Noqueados , Terminales Presinápticos/metabolismo , Núcleos del Rafe/fisiopatología , Receptor de Serotonina 5-HT1A/fisiología , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Transmisión Sináptica , Proteínas de Transporte Vesicular de Monoaminas/metabolismoRESUMEN
Vitamin B(12) (cobalamin) is essential in animals and humans for metabolism of methylmalonic acid, for the remethylation of homocysteine to methionine and, consequently, for all S-adenosylmethionine-dependent methylation reactions, including DNA synthesis. In man, cobalamin deficiency leads to anemia and neurologic and cognitive impairment. In the cblF inborn error of vitamin B(12) metabolism, free vitamin accumulates in lysosomes and cannot be converted to cofactors for mitochondrial methylmalonyl-CoA mutase and cytosolic methionine synthase. Recent work has shown that this defect is caused by mutations in the lysosomal membrane protein LMBD1, which shows significant homology to lipocalin membrane receptors, thereby indicating that LMBD1 is a lysosomal membrane exporter for cobalamin.