The EVER Proteins as a Natural Barrier against Papillomaviruses: a New Insight into the Pathogenesis of Human Papillomavirus Infections

Summary: Infections by human papillomaviruses (HPVs) are the most frequently occurring sexually transmitted diseases. The crucial role of genital oncogenic HPV in cervical carcinoma development is now well established. In contrast, the role of cutaneous HPV in skin cancer development remains a matter of debate. Cutaneous beta-HPV strains show an amazing ubiquity. The fact that a few oncogenic genotypes cause cancers in patients suffering from epidermodysplasia verruciformis is in sharp contrast to the unapparent course of infection in the general population. Our recent investigations revealed that a natural barrier exists in humans, which protects them against infection with these papillomaviruses. A central role in the function of this HPV-specific barrier is played by a complex of the zinc-transporting proteins EVER1, EVER2, and ZnT-1, which maintain cellular zinc homeostasis. Apparently, the deregulation of the cellular zinc balance emerges as an important step in the life cycles not only of cutaneous but also of genital HPVs, although the latter viruses have developed a mechanism by which they can break the barrier and impose a zinc imbalance. Herein, we present a previously unpublished list of the cellular partners of EVER proteins, which points to future directions concerning investigations of the mechanisms of action of the EVER/ZnT-1 complex. We also present a general overview of the pathogenesis of HPV infections, taking into account the latest discoveries regarding the role of cellular zinc homeostasis in the HPV life cycle. We propose a potential model for the mechanism of function of the anti-HPV barrier.     

Enterovirus 71 adsorption on metal ion-composite chitosan beads

In this study, we developed composite chitosan beads combining various metal ions, including Ni(2+), Cu(2+), Zn(2+), and Fe(2+), for direct adsorption of enterovirus 71 (EV71). The metal-ion species had significant effects on the adsorption capacity of beads. Among these metal ion-composite chitosan beads, Ni(2+)-chitosan beads exhibited the best adsorption capacity of EV71. Using a concentration of 0.01-M Ni(2+) was found to best provide for bead formation and EV71 adsorption. The adsorption of EV71 for Ni(2+)-chitosan beads at neutral or alkaline pH was favored. Under a competitive condition with albumin proteins, Ni(2+)-chitosan beads exhibited significant capacity of EV71 adsorption in culture media. The adsorption of EV71 on the Ni(2+)-chitosan beads was attributed to the strong binding between Ni(2+) ions chelated to the surface amino acid of EV71 capsids and Ni(2+) ions chelated on the chitosan materials. Moreover, the adsorbed EV71 retained its antigenicity and infectivity after desorption. The Ni(2+)-chitosan beads exhibit a promising application to EV71 adsorption and removal.     

Crystal structure of Helicobacter pylori neutrophil-activating protein with a di-nuclear ferroxidase center in a zinc or cadmium-bound form

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a Dps-like iron storage protein forming a dodecameric shell, and promotes adhesion of neutrophils to endothelial cells. The crystal structure of HP-NAP in a Zn(2+)- or Cd(2+)-bound form reveals the binding of two zinc or two cadmium ions and their bridged water molecule at the ferroxidase center (FOC). The two zinc ions are coordinated in a tetrahedral manner to the conserved residues among HP-NAP and Dps proteins. The two cadmium ions are coordinated in a trigonal-bipyramidal and distorted octahedral manner. In both structures, the second ion is more weakly coordinated than the first. Another zinc ion is found inside of the negatively-charged threefold-related pore, which is suitable for metal ions to pass through.     

The XLR gene product defines a novel set of proteins stabilized in the nucleus by zinc ions

The major product of the XLR (X-chromosomal, lymphocyte-regulated) locus is found to be a 30-kD nuclear protein with a relatively short (t1/2 approximately equal to 2 h) half-life. Together with its stage- and tissue-specific pattern of expression, this suggests a role for this protein in the regulation of differentiation in T and B lymphocytes. Interestingly, the XLR protein almost completely leaches out of the nucleus after lysis of cells in low salt buffer, but is stabilized in that location by metal cations, particularly Zn++. This stabilization is reversible by chelating agents (o-phenanthroline, EDTA) which also release a number of other polypeptides in addition to XLR. These results suggest that XLR represents a novel class of nuclear proteins, and that cations such as zinc may play a role in the localization of these proteins in the nucleus.     

Noncooperative metalation of metallothionein 1a and its isolated domains with zinc

Mammalian metallothioneins (MTs) are a family of small cysteine-rich proteins capable of binding 7 Zn(2+) or Cd(2+) ions into two distinct domains: an N-terminal β-domain that binds 3 Zn(2+) or Cd(2+) and a C-terminal α-domain that binds 4 Zn(2+) or Cd(2+). MT has been implicated in a number of physiological functions, including metal ion homeostasis, toxic metal detoxification, and as a protective agent against oxidative stress. Conventionally, MT has been understood to coordinate metal ions in a cooperative fashion. Under this mechanism of metalation, the only species of biological relevance would be the metal-free (apo-) form of the protein and the fully metalated (holo-) form of the protein. However, an increasing body of evidence suggests that metalation occurs in a noncooperative manner. If this latter mechanism is correct, then partially metalated forms of the protein will be stable and able to take part in cellular chemistry. We report in this paper conclusive evidence that shows that biologically essential zinc binds to MT in a noncooperative manner. In addition, we report for the first time the stability of a Zn(5)-MT species. The implications of these findings are discussed in terms of the mechanism of metalation.     

A transporter in the endoplasmic reticulum of Schizosaccharomyces pombe cells mediates zinc storage and differentially affects transition metal tolerance

The cation diffusion facilitator (CDF) family represents a class of ubiquitous metal transporters. Inactivation of a CDF in Schizosaccharomyces pombe, Zhf, causes drastically different effects on the tolerance toward various metals. A deletion mutant is Zn(2+)/Co(2+)-hypersensitive yet displays significantly enhanced Cd(2+) and Ni(2+) tolerance. Accumulation of zinc, cobalt, and cadmium is reduced in mutant cells. Non-vacuolar zinc content, as measured by analytical electron microscopy, is lower in zhf(-) cells compared with wild-type cells in the presence of elevated Zn(2+) concentrations. The protective effect against cadmium toxicity is independent of the phytochelatin detoxification pathway. Phytochelatin synthase-deficient cells show extremely enhanced (about 200-fold) cadmium tolerance when zhf is disrupted. Immunogold labeling indicates endoplasmic reticulum (ER) localization of Zhf. Electron spectroscopic imaging shows that accumulation of zinc coincides with Zhf localization, demonstrating a major role of the ER for metal storage and the involvement of Zhf in cellular zinc homeostasis. Also, these observations indicate that Cd(2+) ions exert their toxic effects on cellular metabolism in the ER rather than in the cytosol.     

Brain S100A5 is a novel calcium-, zinc-, and copper ion-binding protein of the EF-hand superfamily

S100A5 is a novel member of the EF-hand superfamily of calcium-binding proteins that is poorly characterized at the protein level. Immunohistochemical analysis demonstrates that it is expressed in very restricted regions of the adult brain. Here we characterized the human recombinant S100A5, especially its interaction with Ca(2+), Zn(2+), and Cu(2+). Flow dialysis revealed that the homodimeric S100A5 binds four Ca(2+) ions with strong positive cooperativity and an affinity 20-100-fold higher than the other S100 proteins studied under identical conditions. S100A5 also binds two Zn(2+) ions and four Cu(2+) ions per dimer. Cu(2+) binding strongly impairs the binding of Ca(2+); however, none of these ions change the alpha-helical-rich secondary structure. After covalent labeling of an exposed thiol with 2-(4'-(iodoacetamide)anilino)-naphthalene-6-sulfonic acid, binding of Cu(2+), but not of Ca(2+) or Zn(2+), strongly decreased its fluorescence. In light of the three-dimensional structure of S100 proteins, our data suggest that in each subunit the single Zn(2+) site is located at the opposite side of the EF-hands. The two Cu(2+)-binding sites likely share ligands of the EF-hands. The potential role of S100A5 in copper homeostasis is discussed.     

Inhibitory mechanism of store-operated Ca2+ channels by zinc

Capacitative calcium influx plays an important role in shaping the Ca(2+) response of various tissues and cell types. Inhibition by heavy metals is a hallmark of store-operated calcium channel (SOCC) activity. Paradoxically, although zinc is the only potentially physiological relevant ion, it is the least investigated in terms of inhibitory mechanism. In the present study, we characterize the inhibitory mechanism of the SOCC by Zn(2+) in the human salivary cell line, HSY, and rat salivary submandibular ducts and acini by monitoring SOCC activity using fluorescence imaging. Analysis of Zn(2+) inhibition indicated that Zn(2+) acts as a competitive inhibitor of Ca(2+) influx but does not permeate through the SOCC, suggesting that Zn(2+) interacts with an extracellular site of SOCC. Application of the reducing agents, dithiothreitol (DTT) and beta-mercaptoethanol, totally eliminated Zn(2+) and Cd(2+) inhibition of SOCC, suggesting that cysteines are part of the Zn(2+) and Cd(2+) binding site. Interestingly, reducing conditions failed to eliminate the inhibition of SOCC by La(3+) and Gd(3+), indicating that the Zn(2+) and lanthanides binding sites are distinct. Finally, we show that changes in redox potential and Zn(2+) are regulating, via SOCC activity, the agonist-induced Ca(2+) response in salivary ducts. The presence of a specific Zn(2+) site, responsive to physiological Zn(2+) and redox potential, may not only be instrumental for future structural studies of various SOCC candidates but may also reveal novel physiological aspects of the interaction between zinc, redox potential, and cellular Ca(2+) homeostasis.     

High intracellular Zn<Superscript>2+</Superscript> ions modulate the VHR,ZAP-70 and ERK activities of LNCaP prostate cancer cells

Malignant prostate tissues have markedly reduced zinc (Zn(2+)) contents in comparison to non-malignant tissues. In this study, we restored a high intracellular Zn(2+) level to LNCaP prostate cancer cells by culturing the cells in a growth medium supplemented with a supraphysiological concentration of Zn(2+) (10 mug/ml) over 5 weeks. The intracellular Zn(2+) level increased in the Zn(2+)-treated cells, and there was a marked increase in the presence of zincosomes, a Zn(2+)-specific intracellular organelle. The proliferation rate of the Zn(2+)-treated cells was markedly reduced. There was also a significant increase (36.6% +/- 6.4%) in the total tyrosine phosphorylated proteins. Vaccinia H1-related (VHR) phosphatase, zeta chain-associated protein-70 (ZAP-70) kinase and phosphorylated extracellular signal-regulated protein kinase 1 and 2 (p-ERK 1 and 2) were also present in higher abundance. Treatment with TPEN, which chelates Zn(2+), reduced the abundance of VHR phosphatase and ZAP-70 kinase, but increased the abundance of p-ERK 1. However, the TPEN treatment restored the Zn(2+)-treated LNCaP cell proliferation to a rate comparable to that of the non Zn(2+)-treated cells. These results highlight the importance of a high intracellular Zn(2+) content and the VHR/ZAP-70-associated pathways in the modulation of LNCaP prostate cancer cell growth.     

Zinc influx and physiological consequences in the beta-insulinoma cell line, Min6

In the mammalian pancreas, high concentrations of Zn(2+) are co-secreted with insulin, which may then permeate via abundant L-type Ca(2+) channels (LTCC) present on the beta-cells. Neither the mechanisms utilized by these cells to lower cytosolic Zn(2+) nor the implications of increased intracellular Zn(2+) on beta-cell survival are well understood. To address this, we employed cell imaging of Zn(2+) and Ca(2+) in the beta-insulinoma cell line, Min6. Depolarization induced an intense zinc influx that was blocked by nifedipine and verapamil, indicating that Zn(2+) permeates via the LTCC. Both Ca(2+) and Zn(2+) permeated concomitantly, yet while Ca(2+) was subsequently removed from the cytosol, Zn(2+) was retained in the cells. Fluorescent staining of vesicular Zn(2+) using ZP1 demonstrated that Zn(2+) could be slowly sequestered following a brief exposure to low concentration of Zn(2+). In contrast, cells were unable to sequester Zn(2+) following application of high concentrations, which was followed by massive cell death. Our results demonstrate homeostatic crosstalk between the plasma membrane and intracellular zinc transporters and suggest that attenuating zinc influx may enhance beta-cell survival.     

A role for ZnT-1 in regulating cellular cation influx

The ZnTs are a growing family of proteins involved in lowering or sequestration of cellular zinc. Using fluorescent measurements of zinc transport we have addressed the mechanism of action of the most ubiquitously expressed member of this family, ZnT-1. This protein has been shown to lower levels of intracellular zinc though the mechanism has remained elusive. The rate of zinc efflux in HEK293 cells expressing ZnT-1 was not accelerated in comparison to control cells, suggesting that ZnT-1 may be involved in regulating influx rather than efflux of zinc. Co-expression of the L-type calcium channel, a major route for zinc influx, and ZnT-1 resulted in a 3-fold reduction in the rate of zinc influx in HEK293 and PC-12 cells, indicating that ZnT-1 modulates zinc permeation through this channel. Immunoblot analysis indicates that ZnT-1 expression does not modulate LTCC expression. Our findings therefore indicate that ZnT-1 modulates the permeation of cations through LTCC, thereby, regulating cation homeostasis through this pathway. Furthermore, ZnT-1 may play a role in cellular ion homeostasis and thereby confer protection against pathophysiological events linked to cellular Ca(2+) or Zn(2+) permeation and cell death.     

High resolution X-ray structures of different metal-substituted forms of phosphotriesterase from Pseudomonas diminuta

Phosphotriesterase, isolated from the soil-dwelling bacterium Pseudomonas diminuta, catalyzes the detoxification of organophosphate-based insecticides and chemical warfare agents. The enzyme has attracted significant research attention in light of its possible employment as a bioremediation tool. As naturally isolated, the enzyme is dimeric. Each subunit contains a binuclear zinc center that is situated at the C-terminal portion of a "TIM" barrel motif. The two zincs are separated by approximately 3.4 A and coordinated to the protein via the side chains of His 55, His 57, His 201, His 230, Asp 301, and a carboxylated Lys 169. Both Lys 169 and a water molecule (or hydroxide ion) serve to bridge the two zinc ions together. Interestingly, these metals can be replaced with cadmium or manganese ions without loss of enzymatic activity. Here we describe the three-dimensional structures of the Zn(2+)/Zn(2+)-, Zn(2+)/Cd(2+)-, Cd(2+)/Cd(2+)-, and Mn(2+)/Mn(2+)-substituted forms of phosphotriesterase determined and refined to a nominal resolution of 1.3 A. In each case, the more buried metal ion, referred to as the alpha-metal, is surrounded by ligands in a trigonal bipyramidal ligation sphere. For the more solvent-exposed or beta-metal ion, however, the observed coordination spheres are either octahedral (in the Cd(2+)/Cd(2+)-, Mn(2+)/Mn(2+)-, and the mixed Zn(2+)/Cd(2+)-species) or trigonal bipyramidal (in the Zn(2+)/Zn(2+)-protein). By measuring the anomalous X-ray data from crystals of the Zn(2+)/Cd(2+)-species, it has been possible to determine that the alpha-metal ion is zinc and the beta-site is occupied by cadmium.     

Metallothionein can function as a chaperone for zinc uptake transport into prostate and liver mitochondria

In mammalian cells the cytosolic concentration of free Zn(2+) ions is extremely low (nM-fM range) and unlikely to provide an adequate pool for the uptake and accumulation of zinc in mitochondria. We previously identified a mitochondrial uptake transport process that effectively transports zinc directly from low molecular weight zinc ligands independent of and in the absence of available free Zn(2+) ions. Since metallothionein (MT) is an important ligand form of cellular zinc, we determined if Zn(7)-MT was an effective chaperone and donor for delivery and uptake of zinc by prostate and liver mitochondria. The results reveal that both intact mitochondria and mitoplasts effectively accumulated zinc from Zn(7)-MT. The study confirms and extends our previous report that the putative zinc transporter is associated with the inner mitochondrial membrane and involves a direct exchange of zinc from the ligand to the transporter. The ventral prostate cells contain no detectable MT; so that ligands (such as citrate, aspartate) other than MT are zinc donors for mitochondrial zinc accumulation. However, in liver and perhaps other cells, Zn(7)-MT is probably important in the cytosolic trafficking of zinc to the mitochondria for the uptake of zinc into the mitochondrial matrix by the putative zinc uptake transporter.     

Development of novel Zn2+ loaded nanoparticles designed for cell-type targeted drug release in CNS neurons: in vitro evidences

Intact synaptic function and plasticity are fundamental prerequisites to a healthy brain. Therefore, synaptic proteins are one of the major targets for drugs used as neuro-chemical therapeutics. Unfortunately, the majority of drugs is not able to cross the blood-brain barrier (BBB) and is therefore distributed within the CNS parenchyma. Here, we report the development of novel biodegradable Nanoparticles (NPs), made of poly-lactide-co-glycolide (PLGA) conjugated with glycopeptides that are able to cross the BBB and deliver for example Zn(2+) ions. We also provide a thorough characterization of loaded and unloaded NPs for their stability, cellular uptake, release properties, toxicity, and impact on cell trafficking. Our data reveal that these NPs are biocompatible, and can be used to elevate intracellular levels of Zn(2+). Importantly, by engineering the surface of NPs with antibodies against NCAM1 and CD44, we were able to selectively target neurons or glial cells, respectively. Our results indicate that these biodegradable NPs provide a potential new venue for the delivery Zn(2+) to the CNS and thus a means to explore the influence of altered zinc levels linked to neuropsychological disorders such as depression.     

Metal selectivity determinants in a family of transition metal transporters

Metal tolerance proteins (MTPs) are plant members of the cation diffusion facilitator (CDF) transporter family involved in cellular metal homeostasis. Members of the CDF family are ubiquitously found in all living entities and show principal selectivity for Zn(2+), Mn(2+), and Fe(2+). Little is known regarding metal selectivity determinants of CDFs. We identified a novel cereal member of CDFs in barley, termed HvMTP1, that localizes to the vacuolar membrane. Unlike its close relative AtMTP1, which is highly selective for Zn(2+), HvMTP1 exhibits selectivity for both Zn(2+) and Co(2+) as assessed by its ability to suppress yeast mutant phenotypes for both metals. Expression of HvMTP1/AtMTP1 chimeras in yeast revealed a five-residue sequence within the AtMTP1 N-segment of the His-rich intracytoplasmic loop that confines specificity to Zn(2+). Furthermore, mutants of AtMTP1 generated through random mutagenesis revealed residues embedded within transmembrane domain 3 that additionally specify the high degree of Zn(2+) selectivity. We propose that the His-rich loop, which might play a role as a zinc chaperone, determines the identity of the metal ions that are transported. The residues within transmembrane domain 3 can also influence metal selectivity, possibly through conformational changes induced at the cation transport site located within the membrane or at the cytoplasmic C-terminal domain.     

Characterization of the PIB-Type ATPases present in Thermus thermophilus

P(IB)-type ATPases transport heavy metals (Cu(2+), Cu(+), Ag(+), Zn(2+), Cd(2+), Co(2+)) across biomembranes, playing a key role in homeostasis and in the mechanisms of biotolerance of these metals. Three genes coding for putative P(IB)-type ATPases are present in the genome of Thermus thermophilus (HB8 and HB27): the TTC1358, TTC1371, and TTC0354 genes; these genes are annotated, respectively, as two copper transporter (CopA and CopB) genes and a zinc-cadmium transporter (Zn(2+)/Cd(2+)-ATPase) gene. We cloned and expressed the three proteins with 8His tags using a T. thermophilus expression system. After purification, each of the proteins was shown to have phosphodiesterase activity at 65°C with ATP and p-nitrophenyl phosphate (pNPP) as substrates. CopA was found to have greater activity in the presence of Cu(+), while CopB was found to have greater activity in the presence of Cu(2+). The putative Zn(2+)/Cd(2+)-ATPase was truncated at the N terminus and was, surprisingly, activated in vitro by copper but not by zinc or cadmium. When expressed in Escherichia coli, however, the putative Zn(2+)/Cd(2+)-ATPase could be isolated as a full-length protein and the ATPase activity was increased by the addition of Zn(2+) and Cd(2+) as well as by Cu(+). Mutant strains in which each of the three P-type ATPases was deleted singly were constructed. In each case, the deletion increased the sensitivity of the strain to growth in the presence of copper in the medium, indicating that each of the three can pump copper out of the cells and play a role in copper detoxification.     

Thionein/metallothionein control Zn(II) availability and the activity of enzymes

Fundamental issues in zinc biology are how proteins control the concentrations of free Zn(II) ions and how tightly they interact with them. Since, basically, the Zn(II) stability constants of only two cytosolic zinc enzymes, carbonic anhydrase and superoxide dismutase, have been reported, the affinity for Zn(II) of another zinc enzyme, sorbitol dehydrogenase (SDH), was determined. Its log K is 11.2 +/- 0.1, which is similar to the log K values of carbonic anhydrase and superoxide dismutase despite considerable differences in the coordination environments of Zn(II) in these enzymes. Protein tyrosine phosphatase 1B (PTP 1B), on the other hand, is not classified as a zinc enzyme but is strongly inhibited by Zn(II), with log K = 7.8 +/- 0.1. In order to test whether or not metallothionein (MT) can serve as a source for Zn(II) ions, it was used to control free Zn(II) ion concentrations. MT makes Zn(II) available for both PTP 1B and the apoform of SDH. However, whether or not Zn(II) ions are indeed available for interaction with these enzymes depends on the thionein (T) to MT ratio and the redox poise. At ratios [T/(MT + T) = 0.08-0.31] prevailing in tissues and cells, picomolar concentrations of free Zn(II) are available from MT for reconstituting apoenzymes with Zn(II). Under conditions of decreased ratios, nanomolar concentrations of free Zn(II) become available and affect enzymes that are not zinc metalloenzymes. The match between the Zn(II) buffering capacity of MT and the Zn(II) affinity of proteins suggests a function of MT in controlling cellular Zn(II) availability.