Interaction of mammalian and plant H+/sucrose transporters with 14-3-3 proteins.

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 ∼20% 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.

Putative role of an SLC45 H+/sugar cotransporter in mammalian spermatozoa.

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.

Putative role of the H(+)/sucrose symporter SLC45A3 as an osmolyte transporter in the kidney.

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.

Proton-associated sucrose transport of mammalian solute carrier family 45: an analysis in Saccharomyces cerevisiae.

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.

Reversible disassembly of the yeast V-ATPase revisited under in vivo conditions.

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.

The binding site of the V-ATPase inhibitor apicularen is in the vicinity of those for bafilomycin and archazolid.

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.

Identification of an animal sucrose transporter.

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.

Protein kinase A-dependent and -independent activation of the V-ATPase in Malpighian tubules of Aedes aegypti.

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.

Synthesis and biological evaluation of a water-soluble derivative of the potent V-ATPase inhibitor archazolid.

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.

Understanding the inhibitory effect of highly potent and selective archazolides binding to the vacuolar ATPase.

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.

Archazolid A binds to the equatorial region of the c-ring of the vacuolar H+-ATPase.

The macrolactone archazolid is a novel, highly specific V-ATPase inhibitor with an IC(50) value in the low nanomolar range. The binding site of archazolid is presumed to overlap with the binding site of the established plecomacrolide V-ATPase inhibitors bafilomycin and concanamycin in subunit c of the membrane-integral V(O) complex. Using a semi-synthetic derivative of archazolid for photoaffinity labeling of the V(1)V(O) holoenzyme we confirmed binding of archazolid to the V(O) subunit c. For the plecomacrolide binding site a model has been published based on mutagenesis studies of the c subunit of Neurospora crassa, revealing 11 amino acids that are part of the binding pocket at the interface of two adjacent c subunits (Bowman, B. J., McCall, M. E., Baertsch, R., and Bowman, E. J. (2006) J. Biol. Chem. 281, 31885-31893). To investigate the contribution of these amino acids to the binding of archazolid, we established in Saccharomyces cerevisiae mutations that in N. crassa had changed the IC(50) value for bafilomycin 10-fold or more and showed that out of the amino acids forming the plecomacrolide binding pocket only one amino acid (tyrosine 142) contributes to the binding of archazolid. Using a fluorescent derivative of N,N'-dicyclohexylcarbodiimide, we found that the binding site for archazolid comprises the essential glutamate within helix 4 of subunit c. In conclusion the archazolid binding site resides within the equatorial region of the V(O) rotor subunit c. This hypothesis was supported by an additional subset of mutations within helix 4 that revealed that leucine 144 plays a role in archazolid binding.

Archazolid A-15-O-ß-D-glucopyranoside and iso-archazolid B: potent V-ATPase inhibitory polyketides from the myxobacteria Cystobacter violaceus and Archangium gephyra.

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.

Synthesis of Novel Potent Archazolids: Pharmacology of an Emerging Class of Anticancer Drugs.

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.

Influence of ATP and ADP on dissociation of the V-ATPase into its V(1) and V(O) complexes.

Although the reversible dissociation of the V(1)V(O) holoenzyme into its V(1) and V(O) complexes is a general mechanism for the regulation of V-ATPases, important aspects are still not understood. By analyzing the endogenous nucleotide content of the V(1)V(O) holoenzyme and of the V(1) complex, both purified from Manduca sexta larval midgut, we found that the V(1) complex contained 1.7 molec. of ADP, whereas only 0.3 molec. of ADP were bound to the V(1)V(O) holoenzyme. By contrast, both proteins contained only negligible amounts of ATP. Incubation of the V(1)V(O) holoenzyme with various adenine nucleotides revealed that ATP hydrolysis, leading to a state containing tightly bound ADP is necessary for its dissociation.

Cruentaren A, a highly cytotoxic benzolactone from Myxobacteria is a novel selective inhibitor of mitochondrial F1-ATPases.

Cruentaren A, a new antifungal benzolactone produced by the myxobacterium Byssovorax cruenta, proved to be highly cytotoxic against various human cell lines. It inhibited the proliferation of different cancer cell lines including a multidrug-resistant KB line at low nanomolar levels. It arrested human histocytic lymphoma cells (U-937) in G(0/1) phase, but did not trigger an apoptotic process. Studies to uncover the molecular target of cruentaren A showed that the novel compound, despite its structural similarity to the benzolactone enamides apicularen and salicylihalamide, was no V-ATPase inhibitor. In contrast, cruentaren specifically inhibited mitochondrial F(O)F(1)-ATPases with IC50 values of 15-30 nM. Although the exact binding site of cruentaren remains undefined, inhibition was shown to occur by interaction with the catalytic F(1) domain. Since mitochondrial ATPases play a crucial role in the pathophysiology of several human disorders including cancer, cruentaren or synthetic derivatives thereof could form the basis of future therapeutic strategies.

The first hydroxylated archazolid from the myxobacterium Cystobacter violaceus: isolation, structural elucidation and V-ATPase inhibition.

The novel macrocyclic polyketide, 10-hydroxymethyl-archazolid-7-O-beta-D-glucopyranoside (archazolid D), was obtained from the myxobacterium Cystobacter violaceus. The structure of this first hydroxylated archazolid was determined by spectroscopic analysis, in particular by HMBC, HMQC, and ROESY NMR investigations, and by degradation. This novel metabolite was evaluated for growth inhibition of murine connective tissue cells and V-ATPase inhibition in comparison to other known archazolids.

Cruentaren, a new antifungal salicylate-type macrolide from Byssovorax cruenta (myxobacteria) with inhibitory effect on mitochondrial ATPase activity. Fermentation and biological properties.

The novel macrolide cruentaren A was produced at levels up to 3.2 mg/liter by cultures of the myxobacterium Byssovorax cruenta. The new compound strongly inhibited the growth of yeasts and filamentous fungi and showed high cytotoxicity against L929 mouse fibroblast cells. A minor co-metabolite of cruentaren A, named cruentaren B, and identified as a six-membered lactone isomer of cruentaren A, showed only marginal cytotoxicity and no antifungal activity. Cruentaren A inhibited F0F1 mitochondrial ATP-hydrolysis in submitochondrial particles of yeasts and beef heart.

EPR Studies of V-ATPase with Spin-Labeled Inhibitors DCC and Archazolid: Interaction Dynamics with Proton Translocating Subunit†c.

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 subunit 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.

Archazolid and apicularen: novel specific V-ATPase inhibitors.

BACKGROUND: V-ATPases constitute a ubiquitous family of heteromultimeric, proton translocating proteins. According to their localization in a multitude of eukaryotic membranes, they energize many different transport processes. Since their malfunction is correlated with various diseases in humans, the elucidation of the properties of this enzyme for the development of selective inhibitors and drugs is one of the challenges in V-ATPase research. RESULTS: Archazolid A and B, two recently discovered cytotoxic macrolactones produced by the myxobacterium Archangium gephyra, and apicularen A and B, two novel benzolactone enamides produced by different species of the myxobacterium Chondromyces, exerted a similar inhibitory efficacy on a wide range of mammalian cell lines as the well established plecomacrolidic type V-ATPase inhibitors concanamycin and bafilomycin. Like the plecomacrolides both new macrolides also prevented the lysosomal acidification in cells and inhibited the V-ATPase purified from the midgut of the tobacco hornworm, Manduca sexta, with IC50 values of 20-60 nM. However, they did not influence the activity of mitochondrial F-ATPase or that of the Na+/K+-ATPase. To define the binding sites of these new inhibitors we used a semi-synthetic radioactively labelled derivative of concanamycin which exclusively binds to the membrane Vo subunit c. Whereas archazolid A prevented, like the plecomacrolides concanamycin A, bafilomycin A1 and B1, labelling of subunit c by the radioactive I-concanolide A, the benzolactone enamide apicularen A did not compete with the plecomacrolide derivative. CONCLUSION: The myxobacterial antibiotics archazolid and apicularen are highly efficient and specific novel inhibitors of V-ATPases. While archazolid at least partly shares a common binding site with the plecomacrolides bafilomycin and concanamycin, apicularen adheres to an independent binding site.

The biological basis for poly-L-lactic acid-induced augmentation.

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.