V-ATPase subunit interactions: the long road to therapeutic targeting

Over the last three decades, V-ATPases have emerged from the obscurity of poorly understood membrane proton transport phenomena to being recognized as ubiquitous proton pumps that underlie vital cellular processes in all eukaryotic and many prokaryotic cells. These exquisitely complex molecular motors also engage in diverse specialized roles contributing to development, tissue function and pH homeostasis within complex organisms. Increasingly, mutations and misappropriation of V-ATPase function have been linked to diseases, ranging from sclerosing bone pathologies and renal tubular acidosis to bone-loss disorders and cancer metastasis. Much remains to be learned about the details of V-ATPase cell and molecular biology; nevertheless, interest in V-ATPases as potential therapeutic targets has burgeoned in recent years. In this review, we present a history of our involvement and contributions to the understanding of V-ATPase structure and function and our nascent and ongoing contributions to translating the knowledge gained from basic research on the nature of V-ATPases into tools for drug discovery. We focus here primarily on the treatment of bone-loss pathologies, like osteoporosis, and present proof-of-concept for a drug screening strategy based on targeting a3-B2 subunit interactions within the V-ATPase complex.     

Novel insights into V-ATPase functioning: distinct roles for its accessory subunits ATP6AP1/Ac45 and ATP6AP2/(pro) renin receptor

The vacuolar (H+)-ATPase (V-ATPase) is a universal proton pump and its activity is required for a variety of cell-biological processes such as membrane trafficking, receptor-mediated endocytosis, lysosomal protein degradation, osteoclastic bone resorption and maintenance of acid-base homeostasis by renal intercalated cells. In neuronal and neuroendocrine cells, the V-ATPase is the major regulator of intragranular acidification which is indispensable for correct prohormone processing and neurotransmitter uptake. In these specialized cells, the V-ATPase is equipped with the accessory subunits ATP6AP1/Ac45 and ATP6AP2/(pro) renin receptor. Recent studies have shown that Ac45 interacts with the V0- sector of the V-ATPase complex, thereby regulating the intragranular pH and Ca2+-dependent exocytotic membrane fusion. Thus, Ac45 can be considered as a V-ATPase regulator in the neuroendocrine secretory pathway. ATP6AP2 has recently been found to be identical to the (pro) renin receptor and has a dual role: (i) in the renin-angiotensin system that also regulates V-ATPase activity; (ii) acting as an adapter by binding to both the V-ATPase and the Wnt receptor complex, thereby recruiting the receptor complex into an acidic microenvironment. We here provide an overview of the two V-ATPase accessory subunits as novel key players in V-ATPase regulation. We argue that the accessory subunits are candidate genes for V-ATPase-related human disorders and promising targets for manipulating V-ATPase functioning in vivo.     

Rational identification of enoxacin as a novel V-ATPase-directed osteoclast inhibitor

Binding between vacuolar H+-ATPases (V-ATPases) and microfilaments is mediated by an actin binding domain in the B-subunit. Both isoforms of mammalian B-subunit bind microfilaments with high affinity. A similar actinbinding activity has been demonstrated in the B-subunit of yeast. A conserved "profilin-like" domain in the B-subunit mediates this actin-binding activity, named due to its sequence and structural similarity to an actin-binding surface of the canonical actin binding protein profilin. Subtle mutations in the "profilin-like" domain eliminate actin binding activity without disrupting the ability of the altered protein to associate with the other subunits of V-ATPase to form a functional proton pump. Analysis of these mutated B-subunits suggests that the actin-binding activity is not required for the "housekeeping" functions of V-ATPases, but is important for certain specialized roles. In osteoclasts, the actin-binding activity is required for transport of V-ATPases to the plasma membrane, a prerequisite for bone resorption. A virtual screen led to the identification of enoxacin as a small molecule that bound to the actin-binding surface of the B2-subunit and competitively inhibited B2-subunit and actin interaction. Enoxacin disrupted osteoclastic bone resorption in vitro, but did not affect osteoblast formation or mineralization. Recently, enoxacin was identified as an inhibitor of the virulence of Candida albicans and more importantly of cancer growth and metastasis. Efforts are underway to determine the mechanisms by which enoxacin and other small molecule inhibitors of B2 and microfilament binding interaction selectively block bone resorption, the virulence of Candida, cancer growth, and metastasis.     

Autophagy and bacterial infectious diseases

Autophagy is a housekeeping process that maintains cellular homeostasis through recycling of nutrients and degradation of damaged or aged cytoplasmic constituents. Over the past several years, accumulating evidence has suggested that autophagy can function as an intracellular innate defense pathway in response to infection with a variety of bacteria and viruses. Autophagy plays a role as a specialized immunologic effector and regulates innate immunity to exert antimicrobial defense mechanisms. Numerous bacterial pathogens have developed the ability to invade host cells or to subvert host autophagy to establish a persistent infection. In this review, we have summarized the recent advances in our understanding of the interaction between antibacterial autophagy (xenophagy) and different bacterial pathogens.     

Symmetrical gas-phase dissociation of noncovalent protein complexes via surface collisions

Previous gas-phase dissociation experiments of protein-protein complexes have resulted in product ion distributions that are asymmetric by charge and mass, providing limited insight into the chemical nature of subunit organization and interaction. In these experiments, a symmetric charge distribution results from an "energy sudden" collision of protein-protein complexes with a surface, indicating that it may be possible to probe the suboligomeric structure of noncovalent complexes in the gas phase. It is proposed that energy sudden surface activation of cytochrome C homodimers results in dissociation without significant unfolding of one of the monomeric subunits. Previously proposed mechanisms for the dissociation of protein-protein complexes are discussed in the context of these results. These experiments demonstrate the potential to preserve the structural details of subunit interaction within a protein-protein complex and help elucidate the asymmetric nature of macromolecular dissociation in the gas phase.     

The V-ATPase as a target for antifungal drugs

The ubiquitous and essential V-ATPase is a worthy chemotherapeutic target in the escalating battle against invasive fungal infections. Pathogenic fungi require optimum V-ATPase function for secretion of virulence factors, induction of stress response pathways, hyphal morphology and homeostasis of pH and other cations in order to successfully survive within and colonize the host. This review discusses why impairment of V-ATPase activity confers multidrug sensitivity and loss of virulence. Recent evidence points to the V-ATPase as a novel downstream target of the azole class of antifungals that inhibit the biogenesis of ergosterol. Depletion of ergosterol from vacuolar membranes led to progressive alkalization of yeast vacuoles, loss of V-ATPase activity and growth inhibition that could be rescued by exogenous ergosterol feeding. Other studies point to a critical role for sphingolipids, phospholipids and cardiolipin in V-ATPase function. Thus, drugs that inhibit the V-ATPase directly, or indirectly by modulating the membrane milieu, can profoundly affect fungal viability and virulence. These findings justify a systematic screen for fungal specific V-ATPase inhibitors or membrane active compounds that can be used in antifungal chemotherapy.     

Mass spectrometric identification of mitochondrial oxidative phosphorylation subunits separated by two-dimensional blue-native polyacrylamide gel electrophoresis

Blue-native polyacrylamide gel electrophoresis is a powerful tool for the separation of intact membrane protein complexes mainly applied to the analysis of the enzymes of the mitochondrial oxidative phosphorylation system (OXPHOS). Combined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), it reveals a two-dimensional pattern showing the individual subunits of the five OXPHOS multi-enzyme complexes. This pattern is useful in the diagnostic analysis of several diseases related to disorders in the oxidative phosphorylation system. However, in order to use this method for systematic diagnostic purposes and to be able to link disease with absence or reduced expression of specific subunits, an unambiguous identification of the individual subunits is necessary. In this study, we completed this task, implementing peptide mass fingerprinting and mass spectrometric sequence analysis. In the course of these analyses, we discovered a novel variant of a cytochrome c oxidase subunit VIc.     

AMP-activated protein kinase: structure and regulation

Mammalian AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that acts as a sensor of cellular energy status. It is activated by a large variety of cellular stresses that increase cellular AMP and decrease ATP levels and also by physiological stimuli, such as muscle contraction, or by hormones such as leptin and adiponectin. AMPK modulates multiple metabolic pathways. As a result, it has become a target for the development of new drugs for the treatment of type II diabetes, obesity or even cancer. In fact, it has been recently reported that drugs used in the treatment of diabetes, such as metformin and thiazolidinediones (TZDs), exert their beneficial effects through the activation of AMPK. AMPK is a heterotrimeric complex composed of a catalytic subunit (AMPK-alpha) and two regulatory subunits (AMPK-beta and AMPK-gamma). Functional orthologues of this kinase complex are found throughout eukaryotic kingdom, from yeast to humans, indicating that the function of this complex is evolutionarily conserved. This review summarizes the recent studies on the structure and regulation of the AMPK heterotrimeric complex.     

Disruption of the V-ATPase functionality as a way to uncouple bone formation and resorption - a novel target for treatment of osteoporosis

The unique ability of the osteoclasts to resorb the calcified bone matrix is dependent on secretion of hydrochloric acid. This process is mediated by a vacuolar H+ ATPase (V-ATPase) and a chloride-proton antiporter. The structural subunit of the V-ATPase, a3, is highly specific for osteoclasts, and mutations in a3 lead to infantile malignant osteopetrosis, a phenomenon characterized by increased bone mass, an increased number of non-resorbing osteoclasts, and a complete lack of bone resorption. Importantly, these individuals have normal or even increased osteoblast numbers and bone formation suggesting that the osteoclasts, but not their resorptive capability, relay an anabolic signal, and, hence, that bone formation can be uncoupled from bone resorption when the a3 subunit is eliminated by mutations, or possibly by pharmacological intervention. The pharmacological profile of the a3 subunit as a highly specific target with a mode of action profile augmenting uncoupling and sustained bone formation, as derived from osteopetrotic patients and mice, highlights the relevance of the V-ATPase in future osteoporosis drug development. However, as illustrated by numerous attempts at developing specific inhibitors of the osteoclastic V-ATPase it is a very difficult target to work with, and an inhibitor possessing the desired profile remains elusive, although highly promising approaches recently have been launched.     

Formation of functional heterologous complexes using subunits from the picromycin, erythromycin and oleandomycin polyketide synthases

BACKGROUND: Recently developed tools for the genetic manipulation of modular polyketide synthases (PKSs) have advanced the development of combinatorial biosynthesis technologies for drug discovery. Although many of the current techniques involve engineering individual domains or modules of the PKS, few experiments have addressed the ability to combine entire protein subunits from different modular PKSs to create hybrid polyketide pathways. We investigated this possibility by in vivo assembly of heterologous PKS complexes using natural and altered subunits from related macrolide PKSs. RESULTS: The pikAI and pikAII genes encoding subunits 1 and 2 (modules 1-4) of the picromycin PKS (PikPKS) and the eryAIII gene encoding subunit 3 (modules 5-6) of the 6-deoxyerythronolide B synthase (DEBS) were cloned in two compatible Streptomyces expression vectors. A strain of Streptomyces lividans co-transformed with the two vectors produced the hybrid macrolactone 3-hydroxynarbonolide. Co-expression of the same pik genes with the gene for subunit 3 of the oleandomycin PKS (OlePKS) was also successful. A series of hybrid polyketide pathways was then constructed by combining PikPKS subunits 1 and 2 with modified DEBS3 subunits containing engineered domains in modules 5 or 6. We also report the effect of junction location in a set of DEBS-PikPKS fusions. CONCLUSIONS: We show that natural as well as engineered protein subunits from heterologous modular PKSs can be functionally assembled to create hybrid polyketide pathways. This work represents a new strategy that complements earlier domain engineering approaches for combinatorial biosynthesis in which complete modules or PKS protein subunits, in addition to individual enzymatic domains, are used as building blocks for PKS engineering.     

A comprehensive characterization of the T-cell antigen receptor complex composition by microcapillary liquid chromatography-tandem mass spectrometry

It has become apparent that many intracellular signaling processes involve the dynamic reorganization of cellular proteins into complex signaling assemblies that have a specific subunit composition, function, and subcellular location. Since the elements of such assemblies interact physically, multiprotein signaling complexes can be isolated and analyzed. Recent technical advances in highly sensitive protein identification by electrospray-tandem mass spectrometry have dramatically increased the sensitivity with which such analyses can be performed. The T-cell antigen receptor (TCR) is an oligomeric transmembrane protein complex that is essential to T-cell recognition and function. The extracellular protein domains are responsible for ligand binding while intracellular domains generate and transduce signals in response to specific receptor-ligand interactions. We used microbore capillary chromatography-tandem mass spectrometry to investigate the composition of the TCR protein complex isolated from resting and activated cells of the murine T-cell line CD11.3. We identified all the previously known subunits of the TCR/CD3 complex as well as proteins previously not known to associate with the TCR. The catalytic activities of some of these proteins could potentially be used to interfere pharmacologically with TCR signaling.     

Membrane proteomics: use of additive main effects with multiplicative interaction model to classify plasma membrane proteins according to their solubility and electrophoretic properties

Recent efforts at the proteomic level were employed to describe the protein equipment of the plasma membrane of the model plant Arabidopsis thaliana. These studies had revealed that the plasma membrane is rich in extrinsic proteins but came up against two major problems: (i) few hydrophobic proteins were recovered in two-dimensional electrophoresis gels, and (ii) many plasma membrane proteins had no known function or were unknown in the database despite extensive sequencing of the Arabidopsis genome. In this paper, several methods expected to enrich a membrane sample in hydrophobic proteins were compared. The optimization of solubilization procedures revealed that the detergent to be used depends on the lipid content of the sample. The corresponding proteomes were compared with the statistical model AMMI (additive main effects with multiplicative interaction) that aimed at regrouping proteins according to their solubility and electrophoretic properties. Distinct groups emerged from this analysis and the identification of proteins in each group allowed us to assign specific features to several of them. For instance, two of these groups regrouped very hydrophobic proteins, one group contained V-ATPase subunits, another group contained proteins with one transmembrane domain as well as proteins known to interact with membrane proteins. This study provides methodological tools to study particular classes of plasma membrane proteins and should be applicable to other cellular membranes.     

An Electrostatic Charge Partitioning Model for the Dissociation of Protein Complexes in the Gas Phase

Electrosprayed multi-protein complexes can be dissociated by collisional activation in the gas phase. Typically, these processes follow a mechanism whereby a single subunit gets ejected with a disproportionately high amount of charge relative to its mass. This asymmetric behavior suggests that the departing subunit undergoes some degree of unfolding prior to being separated from the residual complex. These structural changes occur concomitantly with charge (proton) transfer towards the subunit that is being unraveled. Charge accumulation takes place up to the point where the subunit loses physical contact with the residual complex. This work develops a simple electrostatic model for studying the relationship between conformational changes and charge enrichment during collisional activation. Folded subunits are described as spheres that carry continuum surface charge. The unfolded chain is envisioned as random coil bead string. Simulations are guided by the principle that the system will adopt the charge configuration with the lowest potential energy for any backbone conformation. A finite-difference gradient algorithm is used to determine the charge on each subunit throughout the dissociation process. Both dimeric and tetrameric protein complexes are investigated. The model reproduces the occurrence of asymmetric charge partitioning for dissociation events that are preceded by subunit unfolding. Quantitative comparisons of experimental MS/MS data with model predictions yield estimates of the structural changes that occur during collisional activation. Our findings suggest that subunit separation can occur over a wide range of scission point structures that correspond to different degrees of unfolding.     

Signal transfer in haloarchaeal sensory rhodopsin- transducer complexes

Membrane-inserted complexes consisting of two photochemically reactive sensory rhodopsin (SR) subunits flanking a homodimer of a transducing protein subunit (Htr) are used by halophilic archaea for sensing light gradients to modulate their swimming behavior (phototaxis). The SR-Htr complexes extend into the cytoplasm where the Htr subunits bind a his-kinase that controls a phosphorylation system that regulates the flagellar motors. This review focuses on current progress primarily on the mechanism of signal relay within the SRII-HtrII complexes from Natronomonas pharaonis and Halobacterium salinarum. The recent elucidation of a photoactive site steric trigger crucial for signal relay, advances in understanding the role of proton transfer from the chromophore to the protein in SRII activation, and the localization of signal relay to the membrane-embedded portion of the SRII-HtrII interface, are beginning to produce a clear picture of the signal transfer process. The SR-Htr complexes offer unprecedented opportunities to resolve first examples of the chemistry of signal relay between membrane proteins at the atomic level, which would provide a major contribution to the general understanding of dynamic interactions between integral membrane proteins.     

New chemical crosslinking methods for the identification of transient protein-protein interactions with multiprotein complexes

Most proteins function as multiprotein complexes or interact with multiprotein complexes. Identification of protein-protein interactions in the context of their physiologically relevant complexes is therefore key to fully understand the cellular machinery. Here I discuss advances in chemical crosslinking methods that allow investigators to map direct subunit contacts in transient interactions with multimeric complexes. Methods discussed fall into two categories: (i) in vitro approaches with localized, inducible crosslinking reagents and (ii) in vivo approaches with unlocalized crosslinkers.     

Partial purification of active delta and epsilon subunits of the membrane ATPase from escherichia coli.

We have partially purified active delta and epsilon subunits of the E. coli membrane-bound Mg2+-ATPase (ECF1). Treating purified ECF1 with 50% pyridine precipitates the major subunits (alpha, beta, and gamma) of the enzyme, but the two minor subunits (delta and epsilon), which are present in relatively small amounts, remain in solution. The delta and epsilon subunits were then resolved from one another by anion exchange chromatography. The partially purified epsilon strongly inhibits the hydrolytic activity of ECF1. The epsilon fraction inhibits both the highly purified five-subunit ATPase and the enzyme deficient in the delta subunit. The latter result indicates that the delta subunit is not required for inhibition by epsilon. By contrast, two-subunit enzyme, consisting chiefly of the alpha and beta subunits, was insensitive to the ATPase inhibitor, suggesting that the gamma subunit may be required for inhibition by epsilon. The partially purified delta subunit restored the capacity of ATPase deficient in delta to recombine with ATPase-depleted membranes and to reconstitute ATP-dependent transhydrogenase. Previously we reported (Biochem, Biophys. Res. Commun. 62:764 [1975]) that a fraction containing both the delta and epsilon subunits of ECF1 restored the capacity of ATPase missing delta to recombine with depleted membranes and to function as a coupling factor in oxidative phosphorylation and for the energized transhydrogenase. These reconstitution experiments using isolated subunits provide rather substantial evidence that the delta subunit is essential for attaching the ATPase to the membrane and that the epsilon subunit has a regulatory function as an inhibitor of the ATPase activity of ECF1.     

Matrix isolation investigation of the complexes of acetonitrile and hydrogen cyanide with SiF4 and GeF4

1:1 molecular complexes of acetonitrile and hydrogen cyanide with silicon and germanium tetrafluorides have been isolated and studied in argon matrices at 14 K. The infrafed spectra of these complexes indicate that the acid and base subunits retain their structural integrity, but are perturbed in the complexes. The spectra indicate that all of the complexes are bound through the nitrogen of the nitrile group to the silicon or germanium atom; in this capacity, HCN is serving as a Lewis base, rather than as a Bronsted acid, as is more commonly the case. In the GeF₄ · HCN complex, for example, The C---H stretching mode was shifted 15 cm⁻¹ to lower energy, the C---N stretch roughly 40 cm⁻¹ to higher energy, and the bending mode approximately 30 cm^t- to higher energy. In addition, a number of perturbed modes of the acid subunit were observed; their locations are in good agreement with previous studies of GeF₄ complexes.     

PP2A in the regulation of cell motility and invasion

Cell motility is a very critical phenomenon that plays an important role in the development of eukaryotic organisms. One of the well studied cell motility phenomena is chemotaxis, which is described as a directional movement of cell in response to changes in external chemotactic gradient. Numerous studies conducted both in unicellular organism and in mammalian cells have demonstrated the importance of phosphatidylionositol-3 kinase (PI3K) in this process. In addition, it is now well established that although PI3K plays an activation role in chemotaxis, the role of phosphatases is also critical to maintain this dynamic cyclical process. Protein phosphatase 2A (PP2A) is a major serine/threonine phosphatase that is a key player in regulating PI3K signaling. PP2A is abundantly and ubiquitously expressed and has been highly conserved during the evolution of eukaryotes. PP2A is composed of three protein subunits, A, B, and C. Subunit 'A' is a 60-65 kDa structural component, 'C' is a 36-38 kDa catalytic subunit, and 'B' is a 54-130 kDa regulatory subunit. The core complex of PP2A is comprised of the A and C subunits, which are tightly associated and this dimeric core complexes with the regulatory B subunit. The B subunit determines the substrate specificity as well as the spatial and temporal functions of PP2A. PP2A plays an important role in regulating multiple signal transduction pathways, including cell-cycle regulation, cell-growth and development, cytoskeleton dynamics, and cell motility. This review focuses on the role of PP2A in regulating motility of normal and transformed cells.