RESUMEN
Vacuolar type ATPase (V-ATPase) has recently emerged as a promising novel anticancer target based on extensive in vitro and in vivo studies with archazolids, complex polyketide macrolides, which present the most potent V-ATPase inhibitors known to date. Herein, we report a biomimetic, one-step preparation of archazolid F, the most potent and least abundant archazolid, the design and synthesis of five novel, carefully selected archazolid analogues, and the biological evaluation of these antiproliferative agents, leading to the discovery of a very potent but profoundly simplified archazolid analogue. Furthermore, the first general biological profiling of the archazolids against a broad range of more than 100 therapeutically relevant targets is reported, leading to the discovery of novel and important targets. Finally, first pharmacokinetic data of these natural products are disclosed. All of these data are relevant in the further preclinical development of the archazolids as well as the evaluation of V-ATPases as a novel and powerful class of anticancer targets.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Macrólidos/farmacología , Tiazoles/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/farmacocinética , Línea Celular Tumoral , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacocinética , Humanos , Macrólidos/síntesis química , Macrólidos/farmacocinética , Ratones , Ratas , Tiazoles/síntesis química , Tiazoles/farmacocinética , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidoresRESUMEN
The solute carrier 45 family (SLC45) was defined in the course of the Human Genome Project and consists of four members, A1-A4, which show only 20-30% identity of amino acid sequences among each other. All these members exhibit an identity of â¼20% to plant H+/sucrose cotransporters. Recently, we expressed members of the murine SLC45 family in yeast cells and demonstrated that they are, like their plant counterparts, H+/sucrose cotransporters. In contrast with the plant proteins, SLC45 transporters recognise also the monosaccharides glucose and fructose as physiological substrates and seem to be involved in alternative sugar supply as well as in osmoregulation of several mammalian tissues. In the present study, we provide novel insights into the regulation of SLC45 transporters. By screening for interaction partners, we found a 14-3-3 protein as a promising candidate for control of transport activity. Indeed, co-expression of the gamma isoform of murine 14-3-3 protein in yeast and Xenopus oocytes led to a significant decrease in transport rates of the murine SLC45 transporters as well as of the plant H+/sucrose transporter Sut1.
Asunto(s)
Proteínas 14-3-3/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas de Plantas/metabolismo , Sacarosa/metabolismo , Proteínas 14-3-3/genética , Secuencia de Aminoácidos , Animales , Femenino , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Ratones , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Plantas/genética , Xenopus laevisRESUMEN
In the present study, we describe the detection and analysis of a novel type of sugar transporter in mammalian spermatozoa. This transporter belongs to the SLC45 family for which two features are remarkable and distinguish it from other known families of sugar transporters. Firstly, SLC45 transporters recognise not only the monosaccharides glucose or fructose but also the disaccharide sucrose as a substrate. Secondly, the uptake of sugars is coupled to a proton gradient. Uptake experiments using radioactively labelled sucrose indicated a functional transporter of the SLC45 family in bull spermatozoa. Real-time PCR as well as Western blots demonstrated the occurrence of the SLC45 member A4 in mouse testis and sperms. Furthermore, immunocytochemical analysis of mouse tissues revealed that the signal of SLC45A4 was mainly located in the principle piece of spermatozoa. We postulate that the SLC45A4 transporter plays an important role in nutrition of spermatozoa during their maturation in the epididymis. Moreover, we suggest that knowledge about the presence of the SLC45A4 may be useful also for the methodical improvement of cryopreservation of mammalian spermatozoa.
Asunto(s)
Mamíferos/metabolismo , Espermatozoides/metabolismo , Simportadores/metabolismo , Animales , Transporte Biológico/fisiología , Glucosa/metabolismo , Masculino , Proteínas de Transporte de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Protones , Sacarosa/metabolismo , Azúcares/metabolismoRESUMEN
The solute carrier family 45 a3 member (SLC45A3), known also as prostein, has been implicated with prostate cancer and the regulation of lipid metabolism in oligodendrocytes. Recently, we expressed SLC45A3 in yeast cells and characterised it as a proton-coupled sucrose symporter. However, the physiological functions of SLC45A3 were still unknown. Here, we report that SLC45A3 occurs in the kidney and is highly expressed in the medullary collecting duct (IMCD), a part of the kidney responsible for final urine concentration and faced to hyperosmotic environment. Moreover, messenger RNA (mRNA) expression of endogenous SLC45A3 in rat IMCD cells as well as in NRK52E cells increased up to four-fold under hyperosmotic conditions at 600 mOsmol/kg. Using NRK52E cells as an experimental model, we investigated the proton-coupled sugar transport and found that the uptake of sucrose or glucose was enhanced by hyperosmolarity. Down-regulation of expression by small interfering RNA (siRNA) decreased the osmotically inducible part of sucrose uptake and confirmed the involvement of SLC45A3 in this process. Furthermore, we observed an up to four-fold elevation of sucrose uptake triggered by hyperosmolarity across the apical membrane of NRK52E cells, while uptake across the basolateral membrane was not affected. Due to this finding, we conclude that SLC45A3 may occur at the luminal side of kidney epithelial cells and thus may take up solutes from the tubular fluid. Altogether, we show that SLC45A3 is a novel sugar transporter in kidney and hypothesise that the disaccharide sucrose, and probably the monosaccharides glucose and fructose, may serve as compatible osmolytes in urine.
Asunto(s)
Riñón/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Sacarosa/metabolismo , Simportadores/metabolismo , Animales , Membrana Celular/metabolismo , Células Cultivadas , Regulación hacia Abajo/fisiología , Transporte Iónico/fisiología , Túbulos Renales Colectores/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Concentración Osmolar , Protones , ARN Mensajero/metabolismo , RatasRESUMEN
Vacuolar-type H(+) -ATPases (V-ATPases) have gained recent attention as highly promising anticancer drug targets, and therefore detailed structural analyses and studies of inhibitor interactions are very important research objectives. Spin labeling of the V-ATPase holoenzyme from the tobacco hornworm Manduca sexta and V-ATPase in isolated yeast (Saccharomyces cerevisiae) vacuoles was accomplished by two novel methods involving the covalent binding of a (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) derivative of N,N'-dicyclohexylcarbodiimide (DCC) to the essential glutamate residue in the active site and the noncovalent interaction of a radical analogue of the highly potent inhibitor archazolid, a natural product from myxobacteria. Both complexes were evaluated in detail by electron paramagnetic resonance (EPR) spectroscopic studies and double electron-electron resonance (DEER) measurements, revealing insight into the inhibitor binding mode, dynamics, and stoichiometry as well as into the structure of the central functional subunitâ c of these medicinally important hetero-multimeric proton-translocating proteins. This study also demonstrates the usefulness of natural product derived spin labels as tools in medicinal chemistry.
Asunto(s)
Diciclohexilcarbodiimida/farmacología , Espectroscopía de Resonancia por Spin del Electrón , Inhibidores Enzimáticos/farmacología , Macrólidos/farmacología , Manduca/enzimología , Tiazoles/farmacología , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , ATPasas de Translocación de Protón Vacuolares/metabolismo , Animales , Sitios de Unión , Óxidos N-Cíclicos/química , Diciclohexilcarbodiimida/química , Inhibidores Enzimáticos/química , Macrólidos/química , Manduca/efectos de los fármacos , Modelos Moleculares , Marcadores de Spin , Tiazoles/química , ATPasas de Translocación de Protón Vacuolares/químicaRESUMEN
BACKGROUND: Granulomatous reactions to poly-L-lactic acid (PLLA)-based filler have been described previously. Neither the biological background of these partly late-onset reactions or the desired augmenting effect of PLLA has been studied to date. Histological studies have revealed foreign body reactions and foreign body giant cell formation. OBJECTIVE: The aim of this study was to increase our knowledge about the biological mechanisms behind the augmenting effect of PLLA-based filler. METHODS: We characterised the cell infiltrate and collagen type of PLLA-treated tissue by immunofluorescence staining. The expression of genes related to collagen metabolism was determined. RESULTS: CD68(+) macrophages were found next to PLLA. CD90(+) fibroblasts were found alongside. αSMA-positive structures indicated myofibroblasts and neovascularisation. Substantial collagen type III deposition was detected next to PLLA particles and collagen type I was found at the periphery of PLLA encapsulations. mRNA expression for collagen type I and III transcripts, as well as for TGFß1 and TIMP1, was upregulated significantly. CONCLUSION: PLLA-induced augmentation is most likely based on capsule formation orchestrating macrophages, (myo-)fibroblasts, and collagen type I and III fibres. We observed considerably slower degradation of PLLA particles than described previously. Thus PLLA particles were still retrievable 28 months after subcutaneous application.
Asunto(s)
Técnicas Cosméticas , Rellenos Dérmicos/administración & dosificación , Fibroblastos/efectos de los fármacos , Ácido Láctico/administración & dosificación , Macrófagos/efectos de los fármacos , Polímeros/administración & dosificación , Tejido Subcutáneo/efectos de los fármacos , Biopsia , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo III/genética , Colágeno Tipo III/metabolismo , Rellenos Dérmicos/efectos adversos , Rellenos Dérmicos/metabolismo , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Alemania , Humanos , Inmunohistoquímica , Inyecciones Subcutáneas , Ácido Láctico/efectos adversos , Ácido Láctico/metabolismo , Macrófagos/metabolismo , Macrófagos/patología , Persona de Mediana Edad , Miofibroblastos/efectos de los fármacos , Miofibroblastos/metabolismo , Miofibroblastos/patología , Poliésteres , Polímeros/efectos adversos , Polímeros/metabolismo , Estudios Prospectivos , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tejido Subcutáneo/metabolismo , Tejido Subcutáneo/patología , Factores de Tiempo , Inhibidor Tisular de Metaloproteinasa-1/genética , Factor de Crecimiento Transformador beta1/genética , Regulación hacia ArribaRESUMEN
Vacuolar H(+)-ATPases are multisubunit complexes that operate with rotary mechanics and are essential for membrane proton transport throughout eukaryotes. Here we report a â¼ 1 nm resolution reconstruction of a V-ATPase in a different conformational state from that previously reported for a lower-resolution yeast model. The stator network of the V-ATPase (and by implication that of other rotary ATPases) does not change conformation in different catalytic states, and hence must be relatively rigid. We also demonstrate that a conserved bearing in the catalytic domain is electrostatic, contributing to the extraordinarily high efficiency of rotary ATPases. Analysis of the rotor axle/membrane pump interface suggests how rotary ATPases accommodate different c ring stoichiometries while maintaining high efficiency. The model provides evidence for a half channel in the proton pump, supporting theoretical models of ion translocation. Our refined model therefore provides new insights into the structure and mechanics of the V-ATPases.
Asunto(s)
Proteínas de Insectos/química , Manduca/enzimología , ATPasas de Translocación de Protón Vacuolares/química , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Secuencia Conservada , Microscopía por Crioelectrón , Análisis de Fourier , Modelos Moleculares , Estructura Cuaternaria de Proteína , Estructura Secundaria de ProteínaRESUMEN
The members of the solute carrier 45 (SLC45) family have been implicated in the regulation of glucose homoeostasis in the brain (SLC45A1), with skin and hair pigmentation (SLC45A2), and with prostate cancer and myelination (SLC45A3). However, apart from SLC45A1, a proton-associated glucose transporter, the function of these proteins is still largely unknown, although sequence similarities to plant sucrose transporters mark them as a putative sucrose transporter family. Heterologous expression of the three members SLC45A2, SLC45A3 and SLC45A4 in Saccharomyces cerevisiae confirmed that they are indeed sucrose transporters. [(14)C]Sucrose-uptake measurements revealed intermediate transport affinities with Km values of approximately 5 mM. Transport activities were best under slightly acidic conditions and were inhibited by the protonophore carbonyl cyanide m-chlorophenylhydrazone, demonstrating an H(+)-coupled transport mechanism. Na(+), on the other hand, had no effect on sucrose transport. Competitive inhibition assays indicated a possible transport also of glucose and fructose. Real-time PCR of mouse tissues confirmed mRNA expression of SLC45A2 in eyes and skin and of SLC45A3 primarily in the prostate, but also in other tissues, whereas SLC45A4 showed a predominantly ubiquitous expression. Altogether the results provide new insights into the physiological significance of SLC45 family members and challenge existing concepts of mammalian sugar transport, as they (i) transport a disaccharide, and (ii) perform secondary active transport in a proton-dependent manner.
Asunto(s)
Transporte Biológico/genética , Proteínas de la Membrana/biosíntesis , Proteínas de Transporte de Monosacáridos/biosíntesis , Sacarosa/metabolismo , Simportadores/biosíntesis , Secuencia de Aminoácidos , Animales , Ojo/metabolismo , Regulación Fúngica de la Expresión Génica , Ratones , Proteínas de Transporte de Monosacáridos/metabolismo , Protones , Saccharomyces cerevisiae , Piel/metabolismo , Sacarosa/químicaRESUMEN
Primary active proton transport by eukaryotic V-ATPases (vacuolar ATPases) is regulated via the reversible disassembly of the V1Vo holoenzyme into its peripheral catalytic V1 complex and its membrane-bound proton-translocating Vo complex. This nutrient-dependent phenomenon had been first detected in the midgut epithelium of non-feeding moulting tobacco hornworms (Manduca sexta) and in glucose-deprived yeast cells (Saccharomyces cerevisiae). Since reversible disassembly to date had been investigated mostly in vitro, we wanted to test this phenomenon under in vivo conditions. We used living yeast cells with V-ATPase subunits fused to green, yellow or cyan fluorescent protein and found that only the V1 subunit C (Vma5) was released into the cytosol after substitution of extracellular glucose with galactose, whereas the other V1 subunits remained at or near the membrane. FRET analysis demonstrated close proximity between V1 and Vo even under glucose-starvation conditions. Disassembly, but not reassembly, depended on functional microtubules. Results from overlay blots, pull-down assays and bimolecular fluorescence complementation support the assumption that subunit C interacts directly with microtubules without involvement of linker proteins.
Asunto(s)
Subunidades de Proteína/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Citosol/metabolismo , ATPasas de Translocación de Protón/metabolismo , Saccharomyces cerevisiae/enzimologíaRESUMEN
The vacuolar H(+)-ATPase (V-ATPase) is an ATP-driven proton pump essential to the function of eukaryotic cells. Its cytoplasmic V1 domain is an ATPase, normally coupled to membrane-bound proton pump Vo via a rotary mechanism. How these asymmetric motors are coupled remains poorly understood. Low energy status can trigger release of V1 from the membrane and curtail ATP hydrolysis. To investigate the molecular basis for these processes, we have carried out cryo-electron microscopy three-dimensional reconstruction of deactivated V1 from Manduca sexta. In the resulting model, three peripheral stalks that are parts of the mechanical stator of the V-ATPase are clearly resolved as unsupported filaments in the same conformations as in the holoenzyme. They are likely therefore to have inherent stiffness consistent with a role as flexible rods in buffering elastic power transmission between the domains of the V-ATPase. Inactivated V1 adopted a homogeneous resting state with one open active site adjacent to the stator filament normally linked to the H subunit. Although present at 1:1 stoichiometry with V1, both recombinant subunit C reconstituted with V1 and its endogenous subunit H were poorly resolved in three-dimensional reconstructions, suggesting structural heterogeneity in the region at the base of V1 that could indicate positional variability. If the position of H can vary, existing mechanistic models of deactivation in which it binds to and locks the axle of the V-ATPase rotary motor would need to be re-evaluated.
Asunto(s)
Proteínas de Insectos/metabolismo , Proteínas de Insectos/ultraestructura , Manduca/enzimología , ATPasas de Translocación de Protón Vacuolares/metabolismo , ATPasas de Translocación de Protón Vacuolares/ultraestructura , Animales , Microscopía por Crioelectrón , Imagenología Tridimensional , Modelos Moleculares , Unión Proteica , Conformación Proteica , Subunidades de Proteína/química , Subunidades de Proteína/metabolismoRESUMEN
The V-ATPase is a membrane-bound protein complex which pumps protons across the membrane to generate a large proton motive force through the coupling of an ATP-driven 3-stroke rotary motor (V1) to a multistroke proton pump (Vo). This is done with near 100% efficiency, which is achieved in part by flexibility within the central rotor axle and stator connections, allowing the system to flex to minimise the free energy loss of conformational changes during catalysis. We have used electron microscopy to reveal distinctive bending along the V-ATPase complex, leading to angular displacement of the V1 domain relative to the Vo domain to a maximum of ~30°. This has been complemented by elastic network normal mode analysis that shows both flexing and twisting with the compliance being located in the rotor axle, stator filaments, or both. This study provides direct evidence of flexibility within the V-ATPase and by implication in related rotary ATPases, a feature predicted to be important for regulation and their high energetic efficiencies.
Asunto(s)
ATPasas de Translocación de Protón Vacuolares/metabolismo , Animales , Microscopía por Crioelectrón , Manduca/enzimología , Saccharomyces/enzimología , ATPasas de Translocación de Protón Vacuolares/ultraestructuraRESUMEN
According to the classic point of view, transport of sugars across animal plasma membranes is performed by two families of transporters. Secondary active transport occurs via Na(+) symporters of the SLC5 gene family, while passive transport occurs via facilitative transporters of the SLC2 family. In recent years a new family appeared in the scenery which was called the SLC45 gene family of putative sugar transporters, mainly because of obvious similarities to plant sucrose transporters. The SLC45 family consists of only four members that have been denominated A1-A4. These members apparently have counterparts in all vertebrates. Moreover, their amino acid sequences reveal close homologies also to respective invertebrate proteins such as a recently detected sucrose transporter in Drosophila, and suggest a phylogenetic relationship also to corresponding proteins from plants, fungi and bacteria. This minireview describes the molecular features of its members with a focus on their possible role as sugar transporters.
Asunto(s)
Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/fisiología , Modelos Moleculares , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Transporte de Monosacáridos/fisiología , Familia de Multigenes/genética , Conformación Proteica , Humanos , Proteínas de Transporte de Membrana/metabolismo , Modelos Biológicos , Proteínas de Transporte de Monosacáridos/metabolismo , Monosacáridos/metabolismo , Filogenia , Especificidad de la EspecieRESUMEN
Transepithelial ion transport in insect Malpighian tubules is energized by an apical V-ATPase. In hematophagous insects, a blood meal during which the animal ingests huge amounts of salt and water stimulates transepithelial transport processes linked to V-ATPase activation, but how this is accomplished is still unclear. Here we report that membrane-permeant derivatives of cAMP increase the bafilomycin-sensitive ATPase activity in Malpighian tubules of Aedes aegypti twofold and activate ATP-dependent transport processes. In parallel, membrane association of the V(1) subunits C and D increases, consistent with the assembly of the holoenzyme. The protein kinase A inhibitor H-89 abolishes all cAMP-induced effects, consistent with protein kinase A (PKA) being involved in V-ATPase activation. Metabolic inhibition induced by KCN, azide and 2,4-dinitrophenol, respectively, also induces assembly of functional V-ATPases at the membrane without PKA involvement, indicating a phosphorylation-independent activation mechanism.
Asunto(s)
Aedes/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Túbulos de Malpighi/enzimología , ATPasas de Translocación de Protón Vacuolares/metabolismo , Animales , Diuresis , Femenino , Túbulos de Malpighi/metabolismo , Serotonina/metabolismoRESUMEN
The water-solubility of the highly potent V-ATPase inhibitors archazolid A and the glucosylated derivative archazolid C was studied in the presence of a wide range of cosolvents, revealing very low solubilites. The first water-soluble analogue was then designed, synthesized, and evaluated for V-ATPase inhibitory activity in vitro.
Asunto(s)
Macrólidos/farmacología , Tiazoles/farmacología , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Macrólidos/síntesis química , Macrólidos/química , Estructura Molecular , Solubilidad , Relación Estructura-Actividad , Tiazoles/síntesis química , Tiazoles/química , ATPasas de Translocación de Protón Vacuolares/metabolismo , Agua/químicaRESUMEN
Vacuolar ATPases are a potential therapeutic target because of their involvement in a variety of severe diseases such as osteoporosis or cancer. Archazolide A (1) and related analogs have been previously identified as selective inhibitors of V-ATPases with potency down to the subnanomolar range. Herein we report on the determination of the ligand binding mode by a combination of molecular docking, molecular dynamics simulations, and biochemical experiments, resulting in a sound model for the inhibitory mechanism of this class of putative anticancer agents. The binding site of archazolides was confirmed to be located in the equatorial region of the membrane-embedded V(O)-rotor, as recently proposed on the basis of site-directed mutagenesis. Quantification of the bioactivity of a series of archazolide derivatives, together with the docking-derived binding mode of archazolides to the V-ATPase, revealed favorable ligand profiles, which can guide the development of a simplified archazolide analog with potential therapeutic relevance.
Asunto(s)
Macrólidos/metabolismo , Macrólidos/farmacología , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Tiazoles/metabolismo , Tiazoles/farmacología , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , ATPasas de Translocación de Protón Vacuolares/metabolismo , Animales , Línea Celular , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Concentración 50 Inhibidora , Macrólidos/química , Ratones , Unión Proteica , Estructura Secundaria de Proteína , Reproducibilidad de los Resultados , Saccharomyces cerevisiae/enzimología , Especificidad por Sustrato , Tiazoles/química , ATPasas de Translocación de Protón Vacuolares/químicaRESUMEN
The investigation of V-ATPases as potential therapeutic drug targets and hence of their specific inhibitors is a promising approach in osteoporosis and cancer treatment because the occurrence of these diseases is interrelated to the function of the V-ATPase. Apicularen belongs to the novel inhibitor family of the benzolactone enamides, which are highly potent but feature the unique characteristic of not inhibiting V-ATPases from fungal sources. In this study we specify, for the first time, the binding site of apicularen within the membrane spanning V(O) complex. By photoaffinity labeling using derivatives of apicularen and of the plecomacrolides bafilomycin and concanamycin, each coupled to (14)C-labeled 4-(3-trifluoromethyldiazirin-3-yl)benzoic acid, we verified that apicularen binds at the interface of the V(O) subunits a and c. The binding site is in the vicinity to those of the plecomacrolides and of the archazolids, a third family of V-ATPase inhibitors. Expression of subunit c homologues from Homo sapiens and Manduca sexta, both species sensitive to benzolactone enamides, in a Saccharomyces cerevisiae strain lacking the corresponding intrinsic gene did not transfer this sensitivity to yeast. Therefore, the binding site of benzolactone enamides cannot be formed exclusively by subunit c. Apparently, subunit a substantially contributes to the binding of the benzolactone enamides.
Asunto(s)
Compuestos Bicíclicos Heterocíclicos con Puentes/química , Inhibidores Enzimáticos/farmacología , Macrólidos/química , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Tiazoles/química , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , Animales , Sitios de Unión , Unión Competitiva , Macrólidos/farmacología , Manduca , Conformación Molecular , Mutación , Plásmidos/metabolismo , Estructura Terciaria de Proteína , Tiazoles/farmacología , ATPasas de Translocación de Protón Vacuolares/químicaRESUMEN
V-ATPases in eukaryotes are heteromultimeric, H(+)-transporting proteins. They are localized in a multitude of different membranes and energize many different transport processes. Unique features of V-ATPases are, on the one hand, their ability to regulate enzymatic and ion transporting activity by the reversible dissociation of the catalytic V(1) complex from the membrane bound proton translocating V(0) complex and, on the other hand, their high sensitivity to specific macrolides such as bafilomycin and concanamycin from streptomycetes or archazolid and apicularen from myxomycetes. Both features require distinct intramolecular as well as intermolecular interactions. Here we will summarize our own results together with newer developments in both of these research areas.
Asunto(s)
ATPasas de Translocación de Protón Vacuolares/metabolismo , Humanos , Unión Proteica , Vacuolas/enzimologíaRESUMEN
According to a classic tenet, sugar transport across animal membranes is restricted to monosaccharides. Here, we present the first report of an animal sucrose transporter, SCRT, which we detected in Drosophila melanogaster at each developmental stage. We localized the protein in apical membranes of the late embryonic hindgut as well as in vesicular membranes of ovarian follicle cells. The fact that knockdown of SCRT expression results in significantly increased lethality demonstrates an essential function for the protein. Experiments with Saccharomyces cerevisiae as a heterologous expression system revealed that sucrose is a transported substrate. Because the knockout of SLC45A2, a highly similar protein belonging to the mammalian solute carrier family 45 (SLC45) causes oculocutaneous albinism and because the vesicular structures in which SCRT is located appear to contain melanin, we propose that these organelles are melanosome-like structures and that the transporter is necessary for balancing the osmotic equilibrium during the polymerization process of melanin by the import of a compatible osmolyte. In the hindgut epithelial cells, sucrose might also serve as a compatible osmolyte, but we cannot exclude the possibility that transport of this disaccharide also serves nutritional adequacy.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Sacarosa/metabolismo , Animales , Animales Modificados Genéticamente , Transporte Biológico , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genéticaRESUMEN
Two structurally novel analogues of the macrolides archazolids A and B, archazolid A-15-O-ß-D-glucopyranoside (archazolid E, 5) and iso-archazolid B (archazolid F, 6), were isolated from the myxobacterium Cystobacter violaceus and Archangium gephyra, respectively. Macrolactone 5 represents the first 15-O-glycoside of the archazolids. iso-Archazolid B (6) incorporates a C-3 alkene and presents the first constitutional isomer reported for this natural product class. The structures of these polyketides were determined by spectroscopic analysis, in particular by HMBC, HMQC, and ROESY NMR investigations and by chemical degradation. iso-Archazolid B (6) demonstrated extremely high antiproliferative and V-ATPase inhibitory effects, with IC(50) values in the picomolar range, while only moderate activity was observed for glycoside 5. iso-Archazolid B presents the most potent archazolid known.