Role of proteolysis in copper homoeostasis.

The cop operon of Enterococcus hirae controls cytoplasmic copper levels. It encodes two copper ATPases, a repressor, and the CopZ metallochaperone. Transcription of these genes is induced by copper. However, at higher copper concentrations, CopZ is degraded by a copper-activated proteolytic activity. This specific proteolysis of CopZ can also be demonstrated in vitro with E. hirae extracts. Growth of the cells in copper increases the copper-inducible proteolytic activity in extracts. Zymography reveals the presence of a copper-dependent protease in crude cell lysates. Copper-stimulated proteolysis of CopZ appears to play an important role in copper homoeostasis by E. hirae.

Tetrathiomolybdate inhibition of the Enterococcus hirae CopB copper ATPase.

Tetrathiomolybdate (TTM) avidly interacts with copper and has recently been employed to reduce excess copper in patients with Wilson disease. We found that TTM inhibits the purified Enterococcus hirae CopB copper ATPase with an IC(50) of 34 nM. Dithiomolybdate and trithiomolybdate, which commonly contaminate TTM, inhibited the copper ATPases with similar potency. Inhibition could be reversed by copper or silver, suggesting inhibition by substrate binding. These findings for the first time allowed an estimate of the high affinity of CopB for copper and silver. TTM is a new tool for the study of copper ATPases.

Bcl-x(L) antisense oligonucleotides induce apoptosis and increase sensitivity of pancreatic cancer cells to gemcitabine.

Pancreatic cancer is one of the leading causes of cancer-related death in Western countries. Bcl-x(L) is an anti-apoptotic factor of the Bcl-2 family, which is overexpressed in pancreatic cancer and its presence correlates with shorter patient survival. In this study, sequence-specific antisense oligonucleotides targeting the coding region of Bcl-x(L) were designed to examine whether apoptosis could be induced and chemosensitivity could be increased in pancreatic cancer cells. Five pancreatic cancer cell lines, Panc-1, MIA-PaCa-2, Capan-1, ASPC-1 and T3M4, were treated with Bcl-x(L) sense or antisense oligonucleotides and gemcitabine and the cell viability was examined by the SRB method. Apoptosis was determined using DAPI staining. In all examined pancreatic cancer cells, Bcl-x(L) expression was reduced after transfection of the antisense oligonucleotides. Cell death analysis using DAPI staining revealed that antisense, but not sense oligonucleotides caused apoptotic cell death. Furthermore, Bcl-x(L) antisense oligonucleotides enhanced the cytotoxic effects of gemcitabine in pancreatic cancer cells. Our results indicate that Bcl-x(L) antisense oligonucleotides effectively inhibited pancreatic cancer cell growth and caused apoptosis by reducing Bcl-x(L) protein levels. Bcl-x(L) antisense oligonucleotides also increased the chemosensitivity of pancreatic cancer cells, suggesting that Bcl-x(L) antisense therapy might be a potential future approach in this disease.

Interaction of the CopZ copper chaperone with the CopA copper ATPase of Enterococcus hirae assessed by surface plasmon resonance.

Intracellular copper routing in Enterococcus hirae can be accomplished by the CopZ metallochaperone. Using surface plasmon resonance analysis, we show here that CopZ interacts with the CopA copper ATPase. The binding affinity of CopZ for CopA was increased in the presence of copper, due to a 15-fold lower dissociation rate constant. Mutating the N-terminal copper binding motif of CopA from CxxC to SxxS abolished this copper-induced effect. Moreover, CopZ failed to show an interaction with an unrelated copper binding protein used as a control. These results show that (i) the CopA copper ATPase specifically interacts with the CopZ chaperone, (ii) this interaction is based on protein-protein interaction, and (iii) surface plasmon resonance is a novel tool for quantitative analysis of metallochaperone-target interactions.

Copper-induced proteolysis of the CopZ copper chaperone of Enterococcus hirae.

The cop operon is a key element of copper homeostasis in Enterococcus hirae. It encodes two copper ATPases, CopA and CopB, the CopY repressor, and the CopZ metallochaperone. It was previously shown that the transcription of the operon is induced by copper. The concomitant increase in the levels of Cop proteins, particularly the CopB copper export ATPase, allows uncompromised growth of E. hirae in up to 5 mm ambient copper. We here show by Western blotting that the steady-state level of CopZ was increased only up to 0.5 mm copper. At higher copper concentrations, the level of CopZ was decreased and became undetectable at 5 mm media copper. When CopZ was overexpressed from a plasmid, the cells exhibited increased sensitivity to copper and oxidative stress, suggesting that high CopZ expression could become toxic to cells. In wild-type cells, the level of mRNA transcripts from the cop operon remained high in up to 5 mm copper, suggesting that CopZ was proteolyzed. Cell extracts were found to contain a copper-activated proteolytic activity that degraded CopZ in vitro. In this assay, Cu-CopZ was more susceptible to degradation than apo-CopZ. The growth of E. hirae in copper increased the copper-inducible proteolytic activity in extracts. Zymographic studies showed the presence of a copper-dependent protease in crude cell lysates. Thus, copper-stimulated proteolysis plays an important role in the regulation of copper homeostasis in E. hirae.

Expression of the human Menkes ATPase in Xenopus laevis oocytes.

Menkes disease is an X-linked disorder of copper metabolism that is usually fatal. The affected gene has recently been cloned and encodes one of the two human copper ATPases. If the Menkes ATPase is defective, copper is trapped in the intestinal mucosa, leading to systemic copper deficiency. In order to study copper transport by this ATPase and the effects of disease mutations on its function, we developed a Xenopus laevis oocyte expression system. Wild-type Menkes ATPase cDNA and a fusion of this gene with the green fluorescent protein (GFP) gene was transcribed in vitro and the mRNA injected into oocytes. Expression in oocytes was analyzed by Western blotting and fluorescence microscopy. The Menkes ATPase-GFP chimera appeared to localize primarily to the plasma membrane as assessed by confocal microscopy. This system should thus provide an interesting new tool to study the function of the Menkes ATPase.

Structure-function analysis of purified Enterococcus hirae CopB copper ATPase: effect of Menkes/Wilson disease mutation homologues.

The Enterococcus hirae CopB ATPase (EC 3.6.1.3) confers copper resistance to the organism by expelling excess copper. Two related human ATPase genes, ATP7A (EC 3.6.1.36) and ATP7B (EC 3.6.1.36), have been cloned as the loci of mutations causing Menkes and Wilson diseases, diseases of copper metabolism. Many mutations in these genes have been identified in patients. Since it has not yet been possible to purify the human copper ATPases, it has proved difficult to test the impact of mutations on ATPase function. Some mutations occur in highly conserved sequence motifs, suggesting that their effect on function can be tested with a homologous enzyme. Here, we used the E. hirae CopB ATPase to investigate the impact of such mutations on enzyme function in vivo and in vitro. The Menkes disease mutation of Cys-1000-->Arg, changing the conserved Cys-Pro-Cys ('CPC') motif, was mimicked in CopB. The corresponding Cys-396-->Ser CopB ATPase was unable to restore copper resistance in a CopB knock-out mutant in vivo. The purified mutant ATPase still formed an acylphosphate intermediate, but possessed no detectable ATP hydrolytic activity. The most frequent Wilson disease mutation, His-1069-->Gln, was introduced into CopB as His-480-->Gln (H480Q). This mutant CopB also failed to confer copper resistance to a CopB knock-out strain. Purified H480Q CopB formed an acylphosphate intermediate and retained a small, but significant, ATPase activity. Our results reveal that Cys-396 and His-480 of CopB are key residues for ATPase function, and similar roles are suggested for Cys-1000 and His-1069 of Menkes and Wilson ATPases respectively.

Development and characterization of an animal model of carnitine deficiency.

Mammals cover their carnitine needs by diet and biosynthesis. The last step of carnitine biosynthesis is the conversion of butyrobetaine to carnitine by butyrobetaine hydroxylase. We investigated the effect of N-trimethyl-hydrazine-3-propionate (THP), a butyrobetaine analogue, on butyrobetaine hydroxylase kinetics, and carnitine biosynthesis and body homeostasis in rats fed a casein-based or a vegetarian diet. The K(m )of butyrobetaine hydroxylase purified from rat liver was 41 +/- 9 micromol x L(-1) for butyrobetaine and 37 +/- 5 micromol x L(-1) for THP, and THP was a competitive inhibitor of butyrobetaine hydroxylase (K(i) 16 +/- 2 micromol x L(-1)). In rats fed a vegetarian diet, renal excretion of total carnitine was increased by THP (20 mg.100 g(-1) x day(-1) for three weeks), averaging 96 +/- 36 and 5.3 +/- 1.2 micromol x day(-1) in THP-treated and control rats, respectively. After three weeks of treatment, the total carnitine plasma concentration (8.8 +/- 2.1 versus 52.8 +/- 11.4 micromol x L(-1)) and tissue levels were decreased in THP-treated rats (liver 0.19 +/- 0.03 versus 0.59 +/- 0.08 and muscle 0.24 +/- 0.04 versus 1.07 +/- 0.13 micromol x g(-1)). Carnitine biosynthesis was blocked in THP-treated rats (-0.22 +/- 0.13 versus 0.57 +/- 0.21 micromol x 100 g(-1) x day(-1)). Similar results were obtained in rats treated with the casein-based diet. THP inhibited carnitine transport by rat renal brush-border membrane vesicles competitively (K(i) 41 +/- 3 micromol x L(-1)). Palmitate metabolism in vivo was impaired in THP-treated rats and the livers showed mixed steatosis. Steady-state mRNA levels of the carnitine transporter rat OCTN2 were increased in THP-treated rats in skeletal muscle and small intestine. In conclusion, THP inhibits butyrobetaine hydroxylase competitively, blocks carnitine biosynthesis in vivo and interacts competitively with renal carnitine reabsorption. THP-treated rats develop systemic carnitine deficiency over three weeks and can therefore serve as an animal model for human carnitine deficiency.

Purification and functional analysis of the copper ATPase CopA of Enterococcus hirae.

The Enterococcus hirae ATPase CopA is a member of the recently discovered heavy metal ATPases and shares 43% sequence identity with the human Menkes and Wilson copper ATPases. To study CopA biochemically, it was overexpressed in E. coli with an N-terminal histidine tag and purified to homogeneity by nickel affinity chromatography. The purified CopA catalyzed ATP hydrolysis with a V(max) of 0.15 micromol/min/mg and a K(m) for ATP of 0.2 mM and had an optimum pH of 6.25. The activity was 3- to 4-fold stimulated by reconstitution into proteoliposomes. The enzyme formed an acylphosphate intermediate. Its kinetics of formation and the effects of inhibitors and metal ions upon it support a function of CopA in copper transport. Purification and functional reconstitution of CopA provides the basis to study copper transport in vitro.

Epidermal growth factor is decreased in liver of rats with biliary cirrhosis but does not act as paracrine growth factor immediately after hepatectomy.

BACKGROUND/AIMS: Epidermal growth factor, a potent mitogen for hepatocytes and cholangiocytes, is thought to act as an immediate-early gene after partial hepatectomy. Since regeneration is impaired in cirrhosis, we explored the expression of epidermal growth factor in cirrhotic rat liver immediately after partial hepatectomy. METHODS: Cirrhosis was induced by bile duct ligation (n=21); sham-operated animals served as controls (n=21). Twenty-five days after initial surgery animals were subjected to 70% partial hepatectomy or sham operation; the liver was sampled before surgery and 20, 40 and 90 min thereafter. Epidermal growth factor mRNA levels were assessed by quantitative reverse transcription polymerase chain reaction. Protein expression was estimated by immunohistochemistry using a polyclonal antibody against epidermal growth factor. RESULTS: Before hepatectomy, epidermal growth factor mRNA averaged 70.3+/-39.9 pg/microg of total RNA in controls; this was markedly decreased to 21.9+/-12.7 pg/microg RNA in bile duct ligation (p<0.01). Epidermal growth factor mRNA did not increase after partial hepatectomy in either group, with the exception of sham-operated controls. Immunohistochemistry revealed that partial hepatectomy had no effect on epidermal growth factor expression. Hepatocytes showed uniformly cytosolic epidermal growth factor in controls, while in bile duct ligation immunostaining was faint or absent. Cholangiocytes exhibited a strong cytosolic staining in all experimental groups. CONCLUSIONS: The present study shows that epidermal growth factor is reduced in the cirrhotic liver. This could contribute to the loss of parenchymal liver tissue observed in cirrhosis. The lack of up-regulation after PH sheds doubt on the role of epidermal growth factor as an immediate-early gene in hepatic regeneration. Further, we demonstrate that epidermal growth factor accumulates in cholangiocytes. This observation is strong evidence for involvement of the mitogen epidermal growth factor in the proliferation of bile ducts during cirrhogenesis.

Intracellular copper routing: the role of copper chaperones.

Copper is required by all living systems. Cells have a variety of mechanisms to deal with this essential, yet toxic trace element. A recently discovered facet of homeostatic mechanisms is the protein-mediated, intracellular delivery of copper to target proteins. This routing is accomplished by a novel class of proteins, the 'copper chaperones'. They are a family of conserved proteins present in prokaryotes and eukaryotes, which suggests that copper chaperones are used throughout nature for intracellular copper routing.

NMR structure and metal interactions of the CopZ copper chaperone.

A recently discovered family of proteins that function as copper chaperones route copper to proteins that either require it for their function or are involved in its transport. In Enterococcus hirae the copper chaperone function is performed by the 8-kDa protein CopZ. This paper describes the NMR structure of apo-CopZ, obtained using uniformly (15)N-labeled CopZ overexpressed in Escherichia coli and NMR studies of the impact of Cu(I) binding on the CopZ structure. The protein has a betaalphabetabetaalphabeta fold, where the four beta-strands form an antiparallel twisted beta-sheet, and the two helices are located on the same side of the beta-sheet. A sequence motif GMXCXXC in the loop between the first beta-strand and the first alpha-helix contains the primary ligands, which bind copper(I). Binding of copper(I) caused major structural changes in this molecular region, as manifested by the fact that most NMR signals of the loop and the N-terminal part of the first helix were broadened beyond detection. This effect was strictly localized, because the remainder of the apo-CopZ structure was maintained after addition of Cu(I). NMR relaxation data showed a decreased correlation time of overall molecular tumbling for Cu(I)-CopZ when compared with apo-CopZ, indicating aggregation of Cu(I)-CopZ. The structure of CopZ is the first three-dimensional structure of a cupro-protein for which the metal ion is an exchangeable substrate rather than an integral part of the structure. Implications of the present structural work for the in vivo function of CopZ are discussed, whereby it is of special interest that the distribution of charged residues on the CopZ surface is highly uneven and suggests preferred recognition sites for other proteins that might be involved in copper transfer.

Effects of promoter mutations on the in vivo regulation of the cop operon of Enterococcus hirae by copper(I) and copper(II).

The cop operon of Enterococcus hirae encodes a repressor, CopY, a copper chaperone, CopZ, and two copper ATPases, CopA and CopB. Regulation of the cop operon is bi-phasic, with copper addition as well as copper chelation leading to induction. Using a plasmid-borne system with a reporter gene, induction of wild-type and mutant cop promoters by high and low copper conditions was investigated. Only mutations that impaired the interaction of CopY with both DNA binding sites had a marked effect on regulation, leading to hyperinduction by copper(I) or copper(II). Chelation of copper(II), but not copper(I), also induced the operon, but induction by copper chelation was not significantly affected by the mutations. E. hirae mutants with reduced extracellular copper reductase activity exhibited the same induction kinetics as wild-type cells. These results show that copper addition and copper chelation induce the cop operon by different routes.

The Enterococcus hirae copper chaperone CopZ delivers copper(I) to the CopY repressor.

Expression of the cop operon which effects copper homeostasis in Enterococcus hirae is controlled by the copper responsive repressor CopY. Purified Zn(II)CopY binds to a synthetic cop promoter fragment in vitro. Here we show that the 8 kDa protein CopZ acts as a copper chaperone by specifically delivering copper(I) to Zn(II)CopY and releasing CopY from the DNA. As shown by gel filtration and luminescence spectroscopy, two copper(I) are thereby quantitatively transferred from Cu(I)CopZ to Zn(II)CopY, with displacement of the zinc(II) and transfer of copper from a non-luminescent, exposed, binding site in CopZ to a luminescent, solvent shielded, binding site in CopY.

[How (deficient) copper causes illness]. / Wie (kein) Kupfer krank macht.

Copper is an essential cofactor in all cells. However, it remains largely unknown how cells deal with this element, which is essential yet toxic. Through the study of microbial model systems on the one hand, and the investigation of inherited diseases in copper metabolism on the other, important insights into the way cells deal with copper can be gained. Two key new elements of copper metabolism have emerged from these studies: ATP-driven copper pumps and intracellular copper transport proteins, the copper chaperones.

False positive staining in the TUNEL assay to detect apoptosis in liver and intestine is caused by endogenous nucleases and inhibited by diethyl pyrocarbonate.

BACKGROUND: The terminal transferase uridyl nick end labelling (TUNEL) assay allows the easy demonstration of cell death as a result of apoptosis. However, when this assay is applied to liver tissue, the number of TUNEL positive cells is dependent on the time of incubation with proteinase K. AIM: To test whether false positive results are the result of the release of endogenous endonucleases by proteinase K and can be abolished by pretreatment with diethyl pyrocarbonate (DEPC). METHODS: Involution of hyperplastic ductules in bile duct ligated rats after biliary decompression by Roux-en-Y anastomosis and acute CCl4 intoxication were studied as models of apoptosis and necrosis, respectively. A standard TUNEL assay was applied to formalin fixed tissue sections mounted with cement. To inhibit putative endogenous endonucleases, tissue slides were pre-incubated with DEPC. RESULTS: In the standard TUNEL assay, the number of positive nuclei was highly dependent upon the length of time that sections were incubated with proteinase K. After pretreatment with DEPC, only cells that also exhibited morphological features of apoptosis stained positive. DEPC pretreatment abolished false positive staining in CCl4 induced hepatocyte necrosis and blocked interference by endogenous alkaline phosphatase in intestine. The method of gluing the tissue section to the glass slide was found to be of utmost importance because the effect of DEPC was abolished on silanised slides. CONCLUSIONS: False positive staining in the TUNEL assay in the liver is caused by the release of endogenous endonucleases as a result of proteinase treatment. This can be abolished by pretreatment of tissue slides with DEPC.

NapA Na(+)/H(+) Antiporter as a Sodium Extrusion System Supplementary to the Vacuolar Na(+)-ATPase in Enterococcus hirae.

Enterococcus hirae has two sodium extrusion systems: the NapA Na(+)/H(+) antiporter and the vacuolar Na(+)-ATPase. We found that a NapA mutant, WD4, which is deficient in Na(+)/H(+) antiporter activity, grew well in the pH range of 6 to 10 up to 200 mM sodium. This was due to active, potential-independent sodium extrusion by the Na(+)-ATPase, which was induced under these conditions. The NapA Na(+)/H(+) antiporter is thus not a prerequisite for growth of E. hirae in the presence of sodium, but plays a supplementary role in sodium extrusion at acidic pH.

Topical application of synthetic pyrethroids to cattle as a source of persistent environmental contamination.

Following the application of permethrin or cyhalothrin to cattle for the control of ectoparasites, the occurrence and persistence of these chemicals was assessed on the animals and in their environment. The release of permethrin from ear tags containing 1 g of the drug on cattle was followed for 65 days and lead to concentrations of 5 to 35 micrograms of permethrin per gram of hair on the shoulders. On the flanks of the animals, the corresponding values were 10 times lower. Across the 1.5 acre pasture, high concentrations of permethrin were measured at various locations and long after treatment: 6 micrograms/g on bark of a birch after one week, 5 micrograms/g on a pole of the fence after two weeks, 1 microgram/g in grass from a resting site of the animal after six weeks, and 0.5 microgram/g in bark of a pine tree after three month and two weeks after the animals had left the pasture. In similar assays, cyhalothrin applied to milk cows as a pour-on preparation was monitored. One week following treatment with 0.2 g/animal, hair cut from the shoulders contained 5 micrograms/g of the insecticide, which disappeared with a half-life of 12 days. Dust collected two weeks after the pour-on treatment from the milk barn where the cows were milked twice daily contained 47 micrograms/g of cyhalothrin, which disappeared with a half-life of 44 days. These results show that synthetic pyrethroids used on farm animals can be the source of widespread and persistent contamination.

Hepatic mitochondrial proliferation in rats with secondary biliary cirrhosis: time course and mechanisms.

It is well known that the hepatic mitochondrial protein content is increased in rats 4 weeks after bile duct ligation. In the present study, we measured the time course of this increase and assessed the levels of selected mitochondrial messenger RNA (mRNA) species and the rate of mitochondrial protein synthesis by isolated mitochondria. Three days after surgery, the mitochondrial protein content was not significantly different between bile duct-ligated (BDL) and control rats, averaging 1,140 +/- 220 mg/liver in BDL and 1,260 +/- 50 mg/liver in sham-operated control rats. However, in comparison with control rats, it was increased in BDL rats by 35% at 7 days, by 81% at 14 days, and by 27% at 28 days after surgery. In vitro mitochondrial protein synthesis, which was assessed as the fractional incorporation of [35S]-methionine into mitochondrial protein, was not different between BDL and control rats at 3 days after surgery, but was decreased in BDL rats by 63% at 7 days, by 55% at 14 days, and by 36% at 28 days after surgery. Northern blot analysis revealed an increase in the mRNA levels of adenosine triphosphate (ATP)ase subunit 6 and apocytochrome b in BDL rats at day 7, but no significant differences between BDL and control rats in mitochondrial mRNA and ribosomal RNA species 14 and 28 days after surgery. These results show that the hepatic mitochondrial protein content rises early after surgery in BDL rats, but this rise cannot be ascribed to elevated rates of mitochondrial protein synthesis. Thus, increased synthesis of nuclearly encoded mitochondrial proteins and/or decreased degradation of mitochondrial proteins appear likely mechanisms that lead to the observed increase in the hepatic mitochondrial protein content in BDL rats.