RESUMO
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.
Assuntos
Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Arginina/metabolismo , Sistemas de Transporte de Aminoácidos Básicos/genética , Animais , Arginina/farmacologia , Citosol/metabolismo , Feminino , Células HEK293 , Humanos , Cinética , Lisina/metabolismo , Lisina/farmacologia , Lisossomos/metabolismo , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Xenopus , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMO
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.
Assuntos
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Lisossomos/metabolismo , Sequência de Aminoácidos , Sistemas de Transporte de Aminoácidos Neutros/química , Sistemas de Transporte de Aminoácidos Neutros/genética , Animais , Sítios de Ligação , Humanos , Dados de Sequência Molecular , Mutagênese , Prótons , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
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.
Assuntos
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 terapêutico , Cistinose/tratamento farmacológico , Cistinose/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sistemas de Transporte de Aminoácidos Básicos/genética , Animais , Sequência de Bases , Proteínas de Caenorhabditis elegans/genética , Canavanina/metabolismo , RNA Helicases DEAD-box , DNA Complementar/genética , Proteínas de Drosophila , Fenômenos Eletrofisiológicos , Feminino , Genes Fúngicos , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Oócitos/metabolismo , Estrutura Secundária de Proteína , Ratos , 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 , Especificidade da Espécie , Vacúolos/metabolismo , Xenopus laevisRESUMO
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.
Assuntos
Dipeptídeos , Lisossomos , Lisossomos/metabolismo , Animais , Dipeptídeos/metabolismo , Oócitos/metabolismo , Microscopia Crioeletrônica , Camundongos , Xenopus laevis , Humanos , Camundongos Knockout , Simulação de Dinâmica Molecular , Simportadores/metabolismo , Simportadores/genética , Simportadores/química , Feminino , Canais de Potencial de Receptor TransitórioRESUMO
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.
Assuntos
Biologia Computacional , Transportadores de Ânions Orgânicos/química , Transportadores de Ânions Orgânicos/metabolismo , Simportadores/química , Simportadores/metabolismo , Azepinas/metabolismo , Sítios de Ligação , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Humanos , Indóis/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Transportadores de Ânions Orgânicos/genética , Projetos Piloto , Ligação Proteica , Conformação Proteica , Homologia de Sequência de Aminoácidos , Ácidos Siálicos/química , Ácidos Siálicos/metabolismo , Relação Estrutura-Atividade , Simportadores/genéticaRESUMO
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.
Assuntos
Catepsina L/metabolismo , Proteínas de Membrana Lisossomal/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Catepsina L/genética , Biologia Computacional , Eletrofisiologia , Técnica Indireta de Fluorescência para Anticorpo , Células HeLa , Humanos , Immunoblotting , Imunoprecipitação , Proteínas de Membrana Lisossomal/genética , Proteínas de Membrana Transportadoras/genética , Camundongos , Proteínas de Neoplasias/genética , Ligação Proteica , XenopusRESUMO
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.
Assuntos
Aminoácidos/química , Aminoácidos/metabolismo , Lisossomos/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Simportadores/metabolismo , Animais , Relação Dose-Resposta a Droga , Células HEK293 , Células HeLa , Humanos , Ligantes , Simulação de Acoplamento Molecular/métodos , Estrutura Secundária de Proteína , RatosRESUMO
BACKGROUND INFORMATION: Free sialic acid storage diseases are caused by mutations of a lysosomal sialic acid transporter called sialin. We showed recently that the milder clinical form, Salla disease, and a related non-Finish case, are characterized by residual transport, whereas sialin mutants found in lethal infantile cases are inactive. In the present study, we have characterized the molecular effects of a putative polymorphism (M316I) and of four pathogenic mutations associated with either infantile (G127E and R57C) or Salla-like (G409E) phenotypes, or both (G328E). The transport activity of human sialin was analysed using a novel assay that was based on a construct without the functional lysosomal sorting motif, which is expressed at the plasma membrane. RESULTS: The lysosomal localization of human sialin was not (M316I and G328E) or only partially (R57C, G127E and G409E) affected by the missense mutations. In contrast, all pathogenic mutations abolished transport, whereas the putative M316I polymorphism induced an approx. 5-fold decrease of sialic acid transport. CONCLUSIONS: The molecular effects of the R57C and G127E mutations strengthen the conclusion that the infantile phenotype is caused by loss-of-function mutations. On the other hand, the milder severity of the heterozygous G409E patient may reflect an incomplete expression of the splicing mutation present on the second allele. In the case of the G328E mutation, found in the homozygous state in a clinically heterogeneous family, the apparent severity of the transport phenotype suggests that the genetic or environmental factors underlying this clinical heterogeneity might be protective.
Assuntos
Mutação de Sentido Incorreto , Transportadores de Ânions Orgânicos/genética , Polimorfismo Genético , Doença do Armazenamento de Ácido Siálico/genética , Simportadores/genética , Linhagem Celular , Criança , Humanos , Lisossomos/química , Lisossomos/metabolismo , Modelos Moleculares , Transportadores de Ânions Orgânicos/química , Transportadores de Ânions Orgânicos/metabolismo , Doença do Armazenamento de Ácido Siálico/metabolismo , Simportadores/química , Simportadores/metabolismoRESUMO
We previously isolated, from the earliest population of CD34+ hematopoietic progenitors that form in the aorta of the human embryo, a partial DNA complementary to RNA (cDNA) sequence that was later identified as the human homologue of rat sucrose non-fermenting protein (SNF-1) related kinase (rSNRK), a novel SNF-1-related kinase previously characterized in the rat. In the present study we report the cloning of the complete human SNF-1 related kinase (hSNRK) cDNA and show that the gene spans 39.8 kb at region 3p21 and contains six exons. Recombinant expression of the hSNRK coding sequence in Escherichia coli led to the production of a functional protein kinase of 85 kDa. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of hSNRK expression in fetal CD34+ hematopoietic progenitors revealed its continuous expression throughout human development with higher levels in highly dividing CD34+ CD38+ cells compared to quiescent CD34+ CD38- cells. This observation, together with the expression of hSNRK in numerous human leukemic cell lines, may reflect an implication of hSNRK protein in hematopoietic cell proliferation or differentiation. In the mouse, the SNRK cDNA is 4.6-kb-long and encodes a protein of 748 amino acids with a predicted molecular mass of 81,930 Da. The proteins from human, rat and mouse are strongly conserved and are characterized by the presence of a serine/threonine kinase catalytic domain, a bipartite nuclear targeting signal and an ubiquitin-associated domain. In situ hybridization and RT-PCR analysis of the pattern of mSNRK expression in the mouse reveals that it is temporally and spatially regulated during embryogenesis, and widespread expressed in adult tissues.