Plasmalemmal vacuolar-type H+-ATPase in cancer biology

Vacuolar-type H⁺-adenosine triphosphatase (V-ATPase) is one of the most fundamental enzymes in nature. V-ATPases are responsible for the regulation of proton concentration in the intracellular acidic compartments. It has similar structure with the mitochondrial F₀F₁-ATP synthase (F-ATPase).^† The V-ATPases are composed of multiple subunits and have various physiological functions, including membrane and organelle protein sorting, neurotransmitter uptake, cellular degradative processes, and cytosolic pH regulation. The V-ATPases have been involved in multidrug resistance. Recently, plasma membrane V-ATPases have been involved in regulation of extracellular acidity, essential for cellular invasiveness and proliferation in tumor metastasis. The current knowledge regarding the structure and function of V-ATPase and its role in cancer biology is discussed. F in F₀F₁ ATPase is the coupling energy factor.     

CryoEM of V-ATPases: Assembly,disassembly, and inhibition

Vacuolar-type ATPases (V-ATPases) are responsible for the acidification of intracellular compartments in almost all eukaryotic cells, while in some specialized cells they acidify the extracellular environment. As ubiquitous proton pumps, these large membrane-embedded enzymes are involved in many fundamental cellular processes that require tight control of pH. Consequently, V-ATPase malfunction or aberrant activity has been linked to numerous diseases. In the past ten years, electron cryomicroscopy (cryoEM) of yeast V-ATPases has revealed the architecture and rotary catalytic mechanism of these macromolecular machines. More recently, studies have revealed the structures of V-ATPases in animals and plants, uncovered aspects of how V-ATPases are assembled and regulated by reversible dissociation, and shown how V-ATPase activity can be modulated by proteins and small molecule inhibitors. In this review, we highlight these recent developments.     

Saccharomyces cerevisiae Vacuolar H+-ATPase Regulation by Disassembly and Reassembly: One Structure and Multiple Signals

Vacuolar H⁺-ATPases (V-ATPases) are highly conserved ATP-driven proton pumps responsible for acidification of intracellular compartments. V-ATPase proton transport energizes secondary transport systems and is essential for lysosomal/vacuolar and endosomal functions. These dynamic molecular motors are composed of multiple subunits regulated in part by reversible disassembly, which reversibly inactivates them. Reversible disassembly is intertwined with glycolysis, the RAS/cyclic AMP (cAMP)/protein kinase A (PKA) pathway, and phosphoinositides, but the mechanisms involved are elusive. The atomic- and pseudo-atomic-resolution structures of the V-ATPases are shedding light on the molecular dynamics that regulate V-ATPase assembly. Although all eukaryotic V-ATPases may be built with an inherent capacity to reversibly disassemble, not all do so. V-ATPase subunit isoforms and their interactions with membrane lipids and a V-ATPase-exclusive chaperone influence V-ATPase assembly. This minireview reports on the mechanisms governing reversible disassembly in the yeast Saccharomyces cerevisiae, keeping in perspective our present understanding of the V-ATPase architecture and its alignment with the cellular processes and signals involved.     

V-ATPases as Drug Targets

V-ATPases are large, complex enzymes responsible for acidification of many internal compartments in eukaryotic cells. They also occur on plasma membranes of specialized cells, where they acidify the surrounding milieu. Numerous physiological processes depend on the activity of V-ATPases, and V-ATPases are implicated as a contributing factor in multiple diseases, including osteoporosis, deafness, and cancer. Three classes of natural products have been identified as potent inhibitors of V-ATPases. The bafilomycins and concanamycins, which inhibit all known eukaryotic V-ATPases, are the most extensively studied inhibitors. They bind the Vo subunit c and may inhibit the enzyme by preventing rotation of the c subunit ring. The salicylihalamides and lobatamides show remarkable specificity for animal V-ATPases. The chondropsins preferentially inhibit the fungal V-ATPase. Because of the variety of processes and diseases associated with V-ATPases and the possibility of designing selective inhibitors, the V-ATPases are attractive targets for development of therapeutic agents.     

Diversity of mouse proton-translocating ATPase: presence of multiple isoforms of the C,d and G subunits

Vacuolar-type proton-translocating ATPases (V-ATPases), multimeric proton pumps, are involved in a wide variety of physiological processes. For their diverse functions, V-ATPases utilize a specific subunit isoform(s). Here, we reported the molecular cloning and characterization of three novel subunit isoforms, C2, d2 and G3, of mouse V-ATPase. These isoforms were expressed in a tissue-specific manner, in contrast to the ubiquitously expressed C1, d1 and G1 isoforms. C2 was expressed predominantly in lung and kidney, and d2 and G3 specifically in kidney. We introduced these isoforms into yeasts lacking the corresponding genes. Although the G3 and d2 did not rescue the vmaDelta phenotype, d1 and the two C isoforms functionally complemented the Deltavma6 and Deltavma5, respectively, indicating that they are bona fide subunits of V-ATPase.     

Function, structure and regulation of the vacuolar (H+)-ATPases

The vacuolar ATPases (or V-ATPases) are ATP-driven proton pumps that function to both acidify intracellular compartments and to transport protons across the plasma membrane. Intracellular V-ATPases function in such normal cellular processes as receptor-mediated endocytosis, intracellular membrane traffic, prohormone processing, protein degradation and neurotransmitter uptake, as well as in disease processes, including infection by influenza and other viruses and killing of cells by anthrax and diphtheria toxin. Plasma membrane V-ATPases are important in such physiological processes as urinary acidification, bone resorption and sperm maturation as well as in human diseases, including osteopetrosis, renal tubular acidosis and tumor metastasis. V-ATPases are large multi-subunit complexes composed of a peripheral domain (V₁) responsible for hydrolysis of ATP and an integral domain (V₀) that carries out proton transport. Proton transport is coupled to ATP hydrolysis by a rotary mechanism. V-ATPase activity is regulated in vivo using a number of mechanisms, including reversible dissociation of the V₁ and V₀ domains, changes in coupling efficiency of proton transport and ATP hydrolysis and changes in pump density through reversible fusion of V-ATPase containing vesicles. V-ATPases are emerging as potential drug targets in treating a number of human diseases including osteoporosis and cancer.     

Physical interaction between aldolase and vacuolar H+-ATPase is essential for the assembly and activity of the proton pump

Vacuolar proton-translocating ATPases (V-ATPases) are a family of highly conserved proton pumps that couple hydrolysis of cytosolic ATP to proton transport out of the cytosol. Although V-ATPases are involved in a number of cellular processes, how the proton pumps are regulated under physiological conditions is not well understood. We have reported that the glycolytic enzyme aldolase mediates V-ATPase assembly and activity by physical association with the proton pump (Lu, M., Holliday, L. S., Zhang, L., Dunn, W. A., and Gluck, S. L. (2001) J. Biol. Chem. 276, 30407-30413 and Lu, M., Sautin, Y., Holliday, L. S., and Gluck, S. L. (2004) J. Biol. Chem. 279, 8732-8739). In this study, we generate aldolase mutants that lack binding to the B subunit of V-ATPase but retain normal catalytic activities. Functional analysis of the aldolase mutants shows that disruption of binding between aldolase and the B subunit of V-ATPase results in disassembly and malfunction of V-ATPase. In contrast, aldolase enzymatic activity is not required for V-ATPase assembly. Taken together, these findings strongly suggest an important role for physical association between aldolase and V-ATPase in the regulation of the proton pump.     

Vacuolar (H)-ATPases in Caenorhabditis elegans: What can we learn about giant H pumps from tiny worms?

Vacuolar (H⁺)-ATPases, also called V-ATPases, are ATP-driven proton pumps that are highly phylogenetically conserved. Early biochemical and cell biological studies have revealed many details of the molecular mechanism of proton pumping and of the structure of the multi-subunit membrane complex, including the stoichiometry of subunit composition. In addition, yeast and mouse genetics have broadened our understanding of the physiological consequences of defective vacuolar acidification and its related disease etiologies. Recently, phenotypic investigation of V-ATPase mutants in Caenorhabditis elegans has revealed unexpected new roles of V-ATPases in both cellular function and early development. In this review, we discuss the functions of the V-ATPases discovered in C. elegans.     

The blowfly salivary gland - a model system for analyzing the regulation of plasma membrane V-ATPase

Vacuolar H(+)-ATPases (V-ATPases) are heteromultimeric proteins that use the energy of ATP hydrolysis for the electrogenic transport of protons across membranes. They are common to all eukaryotic cells and are located in the plasma membrane or in membranes of acid organelles. In many insect epithelia, V-ATPase molecules reside in large numbers in the apical plasma membrane and create an electrochemical proton gradient that is used for the acidification or alkalinization of the extracellular space, the secretion or reabsorption of ions and fluids, the import of nutrients, and diverse other cellular activities. Here, we summarize our results on the functions and regulation of V-ATPase in the tubular salivary gland of the blowfly Calliphora vicina. In this gland, V-ATPase activity energizes the secretion of a KCl-rich saliva in response to the neurohormone serotonin (5-HT). Because of particular morphological and physiological features, the blowfly salivary glands are a superior and exemplary system for the analysis of the intracellular signaling pathways and mechanisms that modulate V-ATPase activity and solute transport in an insect epithelium.     

Identification of inhibitors of vacuolar proton-translocating ATPase pumps in yeast by high-throughput screening flow cytometry

Fluorescence intensity of the pH-sensitive carboxyfluorescein derivative 2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) was monitored by high-throughput flow cytometry in living yeast cells. We measured fluorescence intensity of BCECF trapped in yeast vacuoles, acidic compartments equivalent to lysosomes where vacuolar proton-translocating ATPases (V-ATPases) are abundant. Because V-ATPases maintain a low pH in the vacuolar lumen, V-ATPase inhibition by concanamycin A alkalinized the vacuole and increased BCECF fluorescence. Likewise, V-ATPase-deficient mutant cells had greater fluorescence intensity than wild-type cells. Thus, we detected an increase of fluorescence intensity after short- and long-term inhibition of V-ATPase function. We used yeast cells loaded with BCECF to screen a small chemical library of structurally diverse compounds to identify V-ATPase inhibitors. One compound, disulfiram, enhanced BCECF fluorescence intensity (although to a degree beyond that anticipated for pH changes alone in the mutant cells). Once confirmed by dose-response assays (EC₅₀ = 26 μM), we verified V-ATPase inhibition by disulfiram in secondary assays that measured ATP hydrolysis in vacuolar membranes. The inhibitory action of disulfiram against V-ATPase pumps revealed a novel effect previously unknown for this compound. Because V-ATPases are highly conserved, new inhibitors identified could be used as research and therapeutic tools in cancer, viral infections, and other diseases where V-ATPases are involved.     

Structure of the vacuolar adenosine triphosphatases

Vacuolar adenosine triphosphatases (V-ATPases) represent an important class of proton pumps found in endomembrane systems of eucaryotic cells, where they are involved in pH regulation. Progress has been made in the structure determination of this large, membrane-bound multisubunit enzyme complex. Electron microscopy of the V-ATPase has revealed a ball-and-stalk-like structure similar to F1F0-type ATP synthase, to which the V-ATPase is evolutionary related. Aside from the overall structural similarity of the V-ATPase and F-ATP synthase, a number of distinct structural differences exist between the two related enzymes, giving clues to their different function and regulation in the organism.     

Interaction between vacuolar H(+)-ATPase and microfilaments during osteoclast activation.

Vacuolar H(+)-ATPases (V-ATPases) are multisubunit enzymes that acidify compartments of the vacuolar system of all eukaryotic cells. In osteoclasts, the cells that degrade bone, V-ATPases, are recruited from intracellular membrane compartments to the ruffled membrane, a specialized domain of the plasma membrane, where they are maintained at high densities, serving to acidify the resorption bay at the osteoclast attachment site on bone (Blair, H. C., Teitelbaum, S. L., Ghiselli, R., and Gluck, S. L. (1989) Science 249, 855-857). Here, we describe a new mechanism involved in controlling the activity of the bone-resorptive cell. V-ATPase in osteoclasts cultured in vitro was found to form a detergent-insoluble complex with actin and myosin II through direct binding of V-ATPase to actin filaments. Plating bone marrow cells onto dentine slices, a physiologic stimulus that activates osteoclast resorption, produced a profound change in the association of the V-ATPase with actin, assayed by coimmunoprecipitation and immunocytochemical colocalization of actin filaments and V-ATPase in osteoclasts. Mouse marrow and bovine kidney V-ATPase bound rabbit muscle F-actin directly with a maximum stoichiometry of 1 mol of V-ATPase per 8 mol of F-actin and an apparent affinity of 0.05 microM. Electron microscopy of negatively stained samples confirmed the binding interaction. These findings link transport of V-ATPase to reorganization of the actin cytoskeleton during osteoclast activation.     

Close-Up and Genomic Views of the Yeast Vacuolar H<Superscript>+</Superscript>-ATPase

The yeast V-ATPase has emerged as an excellent model for other eukaryotic V-ATPases. In this review, recent biochemical and genomic studies of the yeast V-ATPase are described, with a focus on: 1) the role of V₁ subunit H in coupling ATP hydrolysis and proton pumping and 2) identification of the full set of yeast haploid deletion mutants that exhibit the pH and calcium-sensitive growth characteristic of loss of V-ATPase activity. The combination of “close-up” biochemical views of V-ATPase structure and mechanism and “geomic” views of its functional reach promises to provide new insights into the physiological of V-ATPases.     

Identification of a new chondropsin class of antitumor compound that selectively inhibits V-ATPases

We identify a new naturally occurring class of inhibitor of vacuolar H+-ATPases (V-ATPases) isolated from vacuolar membranes of Neurospora crassa and from chromaffin granule membranes of Bos taurus. To date, the new class includes six chondropsins and poecillastrin A, large polyketide-derived macrolide lactams with 33-37 membered rings. In the National Cancer Institute's 60-cell screen the chondropsin class showed a tumor cell growth inhibitory fingerprint essentially indistinguishable from that of the bafilomycin/concanamycin and the salicylihalamide/lobatamide classes of well-established V-ATPase inhibitors. Half-maximal inhibition of V-ATPase activity in vitro occurred at 0.04-0.7 microM for the fungal vacuolar V-ATPase and at 0.4 to >10 microM for the chromaffin granule V-ATPase. Thus, the new inhibitors are somewhat less potent than the other two classes, which typically have Ki values of <10 nM for V-ATPases, and the new inhibitors differ from the other two classes in their specificity. The bafilomycin class inhibits all eucaryotic V-ATPases, the salicylihalamide class inhibits mammalian V-ATPases but not fungal V-ATPases, and the new chondropsin class inhibits the N. crassa V-ATPase better than the chromaffin granule V-ATPase. Two mutations in the N. crassa V-ATPase that affect the binding of bafilomycin had small but reproducible effects on the affinity of chondropsins for the V-ATPase, suggesting the possibility of a similar mechanism of inhibition.     

A Journey from Mammals to Yeast with Vacuolar H+-ATPase (V-ATPase)

The vacuolar H⁺-ATPase (V-ATPase) is one of the most fundamental enzymes in nature. It functions in almost every eukaryotic cell and energizes a wide variety of organelles and membranes. V-ATPase has a structure and mechanism of action similar to F-ATPase and several of their subunits probably evolved from common ancestors. In eukaryotic cells, F-ATPase is confined to the semiautonomous organelles, chloroplasts and mitochondria, which contain their own genes that encode some of the F-ATPase subunits. In contrast to F-ATPases, whose primary function in eukaryotic cells is to form ATP at the expense of the protonmotive force (pmf), V-ATPases function exclusively as ATP-dependent proton pumps. The pmf generated by V-ATPases in organelles and membranes of eukaryotic cells is utilized as a driving force for numerous secondary transport processes. It was the survival of the yeast mutant without the active enzyme and yeast genetics that allowed the identification of genuine subunits of the V-ATPase. It also revealed special properties of individual subunits, factors that are involved in the enzyme's biogenesis and assembly, as well as the involvement of V-ATPase in the secretory pathway, endocytosis, and respiration. It may be the insect V-ATPase that unconventionally resides in the plasma membrane of their midgut, that will give the first structure resolution of this complex.     

The vacuolar (H+)-ATPases--nature's most versatile proton pumps

The pH of intracellular compartments in eukaryotic cells is a carefully controlled parameter that affects many cellular processes, including intracellular membrane transport, prohormone processing and transport of neurotransmitters, as well as the entry of many viruses into cells. The transporters responsible for controlling this crucial parameter in many intracellular compartments are the vacuolar (H+)-ATPases (V-ATPases). Recent advances in our understanding of the structure and regulation of the V-ATPases, together with the mapping of human genetic defects to genes that encode V-ATPase subunits, have led to tremendous excitement in this field.     

Iejimalides show anti-osteoclast activity via V-ATPase inhibition

Iejimalides (IEJLs), 24-membered macrolides, are potent antitumor compounds, but their molecular targets remain to be revealed. In the course of screening, we identified IEJLs as potent osteoclast inhibitors. Since it is known that osteoclasts are sensitive to vacuolar H(+)-ATPase (V-ATPase) inhibitor, we investigated the effect of IEJLs on V-ATPases. IEJLs inhibited the V-ATPases of both mammalian and yeast cells in situ, and of yeast V-ATPases in vitro. A bafilomycin-resistant yeast mutant conferred IEJL resistance, suggesting that IEJLs bind a site similar to the bafilomycins/concanamycins-binding site. These results indicate that IEJLs are novel V-ATPase inhibitors, and that antitumor and antiosteporotic activities are exerted via V-ATPase inhibition.     

V-ATPases的功能及其抑制剂研究进展

V-ATPases作为一类酶,在真核细胞中广泛存在。V-ATPases是一个由多个亚基组成的复合物,主要有两个结构域,分别是位于外周的V1结构域和跨膜的V0结构域。V1结构域可以通过水解ATP供能;而V0结构域是质子的通道。它们发挥作用主要是通过水解ATP供能,泵运H＋进入囊泡腔中或泵H＋出细胞外。V-ATPases定位于细胞器膜及某些特殊细胞的细胞质膜,参与骨吸收、肿瘤的侵袭及耐药等生理及病理过程,因而V-ATPases是治疗骨质疏松、糖尿病及肿瘤等人类疾病的候选分子靶标。目前有许多研究致力于发现新的潜在的特异的V-ATPase抑制剂。