Elisabeth Mintz

Hepatocyte Targeting and Intracellular Copper Chelation by a Thiol-Containing Glycocyclopeptide

Metal overload plays an important role in several diseases or intoxications, like in Wilsons disease, a major genetic disorder of copper metabolism in humans. To efficiently and selectively decrease copper concentration in the liver that is highly damaged, chelators should be targeted at the hepatocytes. In the present work, we synthesized a molecule able to both lower intracellular copper, namely Cu(I), and target hepatocytes, combining within the same structure a chelating unit and a carbohydrate recognition element. A cyclodecapeptide scaffold displaying a controlled conformation with two independent faces was chosen to introduce both units. One face displays a cluster of carbohydrates to ensure an efficient recognition of the asialoglycoprotein receptors, expressed on the surface of hepatocytes. The second face is devoted to metal ion complexation thanks to the thiolate functions of two cysteine side-chains. To obtain a chelator that is active only once inside the cells, the two thiol functions were oxidized in a disulfide bridge to afford the glycopeptide P(3). Two simple cyclodecapeptides modeling the reduced and complexing form of P(3) in cells proved a high affinity for Cu(I) and a high selectivity with respect to Zn(II). As expected, P(3) becomes an efficient Cu(I) chelator in the presence of glutathione that mimics the intracellular reducing environment. Finally, cellular uptake and ability to lower intracellular copper were demonstrated in hepatic cell lines, in particular in WIF-B9, making P(3) a good candidate to fight copper overload in the liver.

Zinc and copper toxicity in host defense against pathogens: Mycobacterium tuberculosis as a model example of an emerging paradigm

Microbial killing inside macrophages andother phagocytes involves a variety ofmechanisms, including, for instance, acid-ification of the phagocytosis vacuole—orphagosome—and the production of toxicoxygen and nitrogen radicals (Flannaganet al., 2009). In addition, the immunemodulation of nutrients available formicrobial development in infected cellsand tissues is a re-emerging conceptreferred to as “nutritional immunity”(Weinberg, 1975). This concept mostlydeveloped from knowledge of the intra-cellular microbial starvation mechanisminvolving phagosomal iron and man-ganese depletion through the metaltransporter NRAMP (Hood and Skaar,2012). Growing evidence suggests thatimmune defense against microorganismsalsoinvolvesmicrobialkillingbytransitionmetals, such as zinc and copper, presentin excess in the microbial environment,and a set of recent reports showed thatseveral bacterial pathogens, such as thetuberculosis (TB) bacillus,

Dissecting the role of the N‐terminal metal‐binding domains in activating the yeast copper ATPase in vivo

In yeast, copper delivery to the trans‐Golgi network involves interactions between the metallo‐chaperone Atx1 and the N‐terminus of Ccc2, the P‐type ATPase responsible for copper transport across trans‐Golgi network membranes. Disruption of the Atx1–Ccc2 route leads to cell growth arrest in a copper‐and‐iron‐limited medium, a phenotype allowing complementation studies. Coexpression of Atx1 and Ccc2 mutants in an atx1Δccc2Δ strain allowed us to study in vivo Atx1–Ccc2 and intra‐Ccc2 domain–domain interactions, leading to active copper transfer into the trans‐Golgi network. The Ccc2 N‐terminus encloses two copper‐binding domains, M1 and M2. We show that in vivo Atx1–M1 or Atx1–M2 interactions activate Ccc2. M1 or M2, expressed in place of the metallo‐chaperone Atx1, were not as efficient as Atx1 in delivering copper to the Ccc2 N‐terminus. However, when the Ccc2 N‐terminus was truncated, these independent metal‐binding domains behaved like functional metallo‐chaperones in delivering copper to another copper‐binding site in Ccc2 whose identity is still unknown. Therefore, we provide evidence of a dual role for the Ccc2 N‐terminus, namely to receive copper from Atx1 and to convey copper to another domain of Ccc2, thereby activating the ATPase. At variance with their prokaryotic homologues, Atx1 did not activate the Ccc2‐derived ATPase lacking its N‐terminus.

Interplay between glutathione, Atx1 and copper: X-ray absorption spectroscopy determination of Cu(I) environment in an Atx1 dimer.

X-ray absorption techniques have been used to characterise the primary coordination sphere of Cu(I) bound to glutathionate (GS−), to Atx1 and in Cu2I(GS−)2(Atx1)2, a complex recently proposed as the major form of Atx1 in the cytosol. In each complex, Cu(I) was shown to be triply coordinated. When only glutathione is provided, each Cu(I) is triply coordinated by sulphur atoms in the binuclear complex CuI2(GS−)5, involving bridging and terminal thiolates. In the presence of Atx1 and excess of glutathione, under conditions where CuI2(GS−)2(Atx1)2 is formed, each Cu(I) is triply coordinated by sulphur atoms. Given these constraints, there are two different ways for Cu(I) to bridge the Atx1 dimer: either both Cu(I) ions contribute to bridging the dimer, or only one Cu(I) ion is responsible for bridging, the other one being coordinated to two glutathione molecules. These two models are discussed as regards Cu(I) transfer to Ccc2a.

The cadmium transport sites of CadA, the Cd2+-ATPase from Listeria monocytogenes.

CadA, the Cd2+-ATPase from Listeria monocytogenes, belongs to the Zn2+/Cd2+/Pb2+-ATPase bacterial subfamily of P1B-ATPases that ensure detoxification of the bacteria. Whereas it is the major determinant of Listeria resistance to Cd2+, CadA expressed in Saccharomyces cerevisiae severely decreases yeast tolerance to Cd2+ (Wu, C. C., Bal, N., Pérard, J., Lowe, J., Boscheron, C., Mintz, E., and Catty, P. (2004) Biochem. Biophys. Res. Commun. 324, 1034–1040). This phenotype, which reflects in vivo Cd2+-transport activity, was used to select from 33 point mutations, shared out among the eight transmembrane (TM) segments of CadA, those that affect the activity of the protein. Six mutations affecting CadA were found: M149A in TM3; E164A in TM4; C354A, P355A, and C356A in TM6; and D692A in TM8. Functional studies of the six mutants produced in Sf9 cells revealed that Cys354 and Cys356 in TM6 as well as Asp692 in TM8 and Met149 in TM3 could participate at the Cd2+-binding site(s). In the canonical Cys-Pro-Cys motif of P1B-ATPases, the two cysteines act at distinct steps in the transport mechanism, Cys354 being directly involved in Cd2+ binding, while Cys356 seems to be required for Cd2+ occlusion. This confirms an earlier observation that the two equivalent Cys of Ccc2, the yeast Cu+-ATPase, also act at different steps. In TM4, Glu164, which is conserved among P1B-ATPases, may be required for Cd2+ release. Finally, analysis of the role of Cd2+ in the phosphorylation from ATP and from Pi of the mutants suggests that two Cd2+ ions are involved in the reaction cycle of CadA.

Cyclic AMP-dependent protein kinase controls energy interconversion during the catalytic cycle of the yeast copper-ATPase

The pathogenesis of human Menkes and Wilson diseases depends on alterations in copper transport. Some reports suggest that intracellular traffic of copper might be regulated by kinase‐mediated phosphorylation. However, there is no evidence showing the influence of kinase‐related processes in coupled ATP hydrolysis/copper transport cycles. Here, we show that cyclic AMP‐dependent protein kinase (PKA) regulates Ccc2p, the yeast Cu(I)‐ATPase, with PKA‐mediated phosphorylation of a conserved serine (Ser258) being crucial for catalysis. Long‐range intramolecular communication between Ser258 and Asp627 (at the catalytic site) modulates the key pumping event: the conversion of the high‐energy to the low‐energy phosphorylated intermediate associated with copper release.

Metal homeostasis disruption and mitochondrial dysfunction in hepatocytes exposed to sub-toxic doses of zinc oxide nanoparticles

Increased production and use of zinc oxide nanoparticles (ZnO-NPs) in consumer products has prompted the scientific community to investigate their potential toxicity, and understand their impact on the environment and organisms. Molecular mechanisms involved in ZnO-NP toxicity are still under debate and focus essentially on high dose expositions. In our study, we chose to evaluate the effect of sub-toxic doses of ZnO-NPs on human hepatocytes (HepG2) with a focus on metal homeostasis and redox balance disruptions. We showed massive dissolution of ZnO-NPs outside the cell, transport and accumulation of zinc ions inside the cell but no evidence of nanoparticle entry, even when analysed by high resolution TEM microscopy coupled with EDX. Gene expression analysis highlighted zinc homeostasis disruptions as shown by metallothionein 1X and zinc transporter 1 and 2 (ZnT1, ZnT2) over-expression. Major oxidative stress response genes, such as superoxide dismutase 1, 2 and catalase were not induced. Phase 2 enzymes in term of antioxidant response, such as heme oxygenase 1 (HMOX1) and the regulating subunit of the glutamate-cysteine ligase (GCLM) were slightly upregulated, but these observations may be linked solely to metal homeostasis disruptions, as these actors are involved in both metal and ROS responses. Finally, we observed abnormal mitochondria morphologies and autophagy vesicles in response to ZnO-NPs, indicating a potential role of mitochondria in storing and protecting cells from zinc excess but ultimately causing cell death at higher doses.

Ca2+/Calmodulin-dependent Protein Kinase II Is an Essential Mediator in the Coordinated Regulation of Electrocyte Ca2+-ATPase by Calmodulin and Protein Kinase A

The aim of this study was to investigate (a) whether Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) participates in the regulation of plasma membrane Ca2+-ATPase and (b) its possible cross-talk with other kinase-mediated modulatory pathways of the pump. Using isolated innervated membranes of the electrocytes from Electrophorus electricus L., we found that stimulation of endogenous protein kinase A (PKA) strongly phosphorylated membrane-bound CaM kinase II with simultaneous substantial activation of the Ca2+ pump (≈2-fold). The addition of cAMP (5-50 pm), forskolin (10 nm), or cholera toxin (10 or 100 nm) stimulated both CaM kinase II phosphorylation and Ca2+-ATPase activity, whereas these activation processes were cancelled by an inhibitor of the PKA α-catalytic subunit. When CaM kinase II was blocked by its specific inhibitor KN-93, the Ca2+-ATPase activity decreased to the levels measured in the absence of calmodulin; the unusually high Ca2+ affinity dropped 2-fold; and the PKA-mediated stimulation of Ca2+-ATPase was no longer seen. Hydroxylamine-resistant phosphorylation of the Ca2+-ATPase strongly increased when the PKA pathway was activated, and this phosphorylation was suppressed by inhibition of CaM kinase II. We conclude that CaM kinase II is an intermediate in a complex regulatory network of the electrocyte Ca2+ pump, which also involves calmodulin and PKA.

Cd2+- or Hg2+-binding proteins can replace the Cu+-chaperone Atx1 in delivering Cu+ to the secretory pathway in yeast

Copper delivery to Ccc2 – the Golgi Cu+‐ATPase – was investigated in vivo, replacing the Cu+‐chaperone Atx1 by various structural homologues in an atx1‐Δ yeast strain. Various proteins, displaying the same ferredoxin‐like fold and (M/L)(T/S)CXXC metal‐binding motif as Atx1 and known as Cu+‐, Cd2+‐ or Hg2+‐binding proteins were able to replace Atx1. Therefore, regardless of their original function, these proteins could all bind copper and transfer it to Ccc2, suggesting that Ccc2 is opportunistic and can interact with many different proteins to gain Cu+. The possible role of electrostatic potential surfaces in the docking of Ccc2 with these Atx1‐homologues is discussed.

Visualization, quantification and coordination of Ag+ ions released from silver nanoparticles in hepatocytes

Silver nanoparticles (AgNPs) can enter eukaryotic cells and exert toxic effects, most probably as a consequence of the release of Ag+ ions. Due to the elusive nature of Ag+ ionic species, quantitative information concerning AgNP intracellular dissolution is missing. By using a synchrotron nanoprobe, silver is visualized and quantified in hepatocytes (HepG2) exposed to AgNPs; the synergistic use of electron microscopy allows for the discrimination between nanoparticular and ionic forms of silver within a single cell. AgNPs are located in endocytosis vesicles, while the visualized Ag+ ions diffuse in the cell. The averaged NP dissolution rates, measured by X-ray absorption spectroscopy, highlight the faster dissolution of citrate-coated AgNPs with respect to the less toxic PVP-coated AgNPs; these results are confirmed at the single-cell level. The released Ag+ ions recombine with thiol-bearing biomolecules: the Ag-S distances measured in cellulo, and the quantitative evaluation of gene expression, provide independent evidence of the involvement of glutathione and metallothioneins in Ag+ binding. The combined use of cutting-edge imaging techniques, atomic spectroscopy and molecular biology brings insight into the fate of AgNPs in hepatocytes, and more generally into the physicochemical transformations of metallic nanoparticles in biological environments and the resulting disruption of metal homeostasis.