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J H Kägi

Publications and source records attributed to J H Kägi.

At least 19 recordsLinked to original sources
PubMed1998

Modulation of DNA binding of a tramtrack zinc finger peptide by the metallothionein-thionein conjugate pair.

The ability of metallothionein (MT) to modulate DNA binding by a two-finger peptide of Tramtrack (TTK), a CCHH zinc transcription factor, was investigated using metal-bound and metal-deficient forms of rabbit MT-2 and the TTK peptide. Thionein inhibited DNA binding by zinc-bound TTK, and Zn-MT restored DNA-binding by zinc-deficient apo-TTK. "Free" zinc at low concentrations was as effective as Zn-MT in restoring DNA binding by apopeptide but was inhibitory at concentrations equal to zinc bound to 2 mol eq and higher of Zn-MT. Substitution of cadmium for zinc reduced the affinity of the peptide for its DNA binding site. This effect was reversed by incubation with Zn-MT. The circular dichroic spectra of the TTK peptide indicated that zinc removal resulted in loss of alpha-helical structures, which are sites of DNA contact points. Reconstitution with cadmium resulted in stoichiometric substitution of 2 mol of Cd/mol of peptide but not recovery of alpha-helical structures. Incubation of Cd-TTK with Zn-MT restored the secondary structure expected for zinc-bound TTK. The ability of Zn-MT and thionein to restore or inhibit DNA-binding by TTK was associated with effects on the metallation status of the peptide and related alterations in its secondary structure.

Animals↗
PubMed1997

Metallothionein accretion in human hepatic cells is linked to cellular proliferation.

The basal amounts of metallothionein (MT) and its rates of biosynthesis were compared in resting and proliferating Chang liver (CCl-13) cells. In resting cells the total amounts of the detectable isoforms MT-2 and MT-1e were approx. 1.6x10(6) and 4x10(5) molecules per cell respectively. In exponentially growing cultures the cellular contents of both isoforms increased co-ordinately approx. 4-fold and decreased again to the initial values within 48 h after entering density-mediated growth arrest. As documented for MT-2 its transient accretion was attributable to a 10-fold rise in the rate of biosynthesis of this protein during the growth phase. Measurements of the relative amounts of MT-2 mRNA indicated the occurrence of a more than 50% increase within the first 12 h after subculturing of the cells, followed by a return to basal levels thereafter. These results denote a direct link between the programming of MT synthesis and proliferation and thus attest to a central housekeeping function of the MTs.

Blotting, Northern↗
PubMed1996

Separation and characterization of the metal-thiolate-cluster domains of recombinant sea urchin metallothionein.

Partial metal depletion and 113Cd-NMR studies have suggested that the recombinant Cd-containing metallothionein of the sea urchin Strongylocentrotus purpuratus (Cd7-MTA) binds its metal ions in a four-metal (Cd4Cys11) and a three-metal (Cd3Cys9) cluster associated with the N-terminal and C-terminal halves of the protein, respectively [Wang, Y., Mackay, E.A., Zerbe, O., Hess, D., Hunziker, P.E., Vasak, M. & Kägi, J.H.R. (1995) Biochemistry 34,7460-7467]. This partitioning has now been confirmed by bisecting native Cd7-MTA with subtilisin into products bearing only a single metal-thiolate cluster. Their separation by reverse-phase HPLC and on-line electrospray mass spectrometry in combination with sequence analysis revealed selective cleavage of the protein into a set of N-terminal polypeptides containing 37-39 residues with four Cd ions and a set of C-terminal polypeptides containing 24 and 25 residues with three Cd ions. Thus, sea urchin MTA like its mammalian counterparts is made up of two separate cluster-harboring domains. The fragmentation pattern indicated that the sites of cleavage are located in the peptide loop interspaced between the first two metal-bound cysteine residues of the C-terminal domain. Accordingly, with cleavage, one of the putative nine thiolate ligands of the three-metal cluster was lost to the N-terminal fragment. The coordinational consequences of this repartition were reflected in massive chiroptical changes accompanying the cleavage process. While the liberated N-terminal domain retained the CD profile of the four-metal cluster in the parent protein and thereby indicated preservation of its structure, the CD features attributable to the intact three-metal cluster were largely lost on cleavage. The vanished features bear strong resemblance to the large biphasic ellipticity signal at 250 nm which dominates the CD spectrum of native Cd7-MTA, and allow us thus to attribute this signal to excitonic coupling interactions of Cd-thiolate chromophores in the three-metal cluster.

Amino Acid Sequence↗
PubMed1995

Regulation of metallothionein gene expression in Cd- or Zn-adapted RK-13 cells.

We explored the molecular genetics underlying the massive induction of isoMTs by Zn2+ or Cd2+ in metal tolerant rabbit kidney (RK-13) sub-line cells, using band shift assays and Southern blotting analysis. In sub-line cells accommodated to intermediate metal concentrations (100 microM Zn2+; 1-20 microM Cd2+) evidence suggested that the increase in the capacity for isoMT synthesis is brought about by an increased binding activity of the nuclear transcription factors MTF-1 and Sp1. Using quantitative band shift analysis with a mouse MRE-d oligonucleotide probe, the binding of both transcription factors was found to be enhanced two to three times over the binding activity measured in the unexposed parental RK-13 cells. Their increase in binding activity is probably the cause of the overexpression of MT genes and the development of metal tolerance in these cells. In cells tolerant to the highest concentrations of metal the analysis of Southern blot signals revealed MT gene amplification to be the most probable cause of the increased MT production. Thus, in cells of sub-lines growing in the presence of 350 microM Zn2+, two of the isoMT genes were coordinately triplicated and in cells tolerant to 150 microM Cd2+ one isoMT gene was amplified two-fold.

Animals↗
PubMed1995

Characterization and sequential localization of the metal clusters in sea urchin metallothionein.

The mode of metal binding in sea urchin metallothionein (MT) was explored by electronic absorption, chiroptical, NMR, and mass spectroscopic methods. Recombinant sea urchin MT containing 7 equiv of the natural mixture of Cd isotopes was stripped of the metal by exposure to low pH and reconstituted with 113Cd (> 95% enriched). Comparison of the electronic spectroscopic and chiroptical features and the 113Cd NMR spectra of the reconstituted material with those of the native recombinant material indicated that the reconstituted material had regained the native conformation. The shoulder at 250 nm in the electronic absorption spectrum, the biphasic circular dichroism profile centered at 250 nm, and the chemical shift positions (605-695 ppm) of the seven 113Cd NMR resonances all strongly suggested that sea urchin MT like all other well characterized MTs contains clusters made up of tetrahedral Cd-thiolate units. The 113Cd chemical shift correlation spectrum of the reconstituted protein proved the existence of such metal clusters and allowed the unambiguous assignment of some of the metal connectivities. Homonuclear decoupling experiments in which Cd resonances were selectively saturated indicated moreover a partitioning of the metal complement into two separate clusters containing three and four Cd ions. The same proposition was supported by the selective reduction of three 113Cd resonances upon partial metal depletion following exposure of the protein to EDTA. Thus, the three-metal cluster is energetically less stable than the four-metal cluster.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Purification and characterisation of recombinant sea urchin metallothionein expressed in Escherichia coli.

Metallothioneins (MT) are metalloproteins expressed tissue specifically during the development of the sea urchin, Strongylocentrotus pururatus. To explore their structural and functional features and to compare them with those of the evolutionary distant mammalian MTs, one isoform (MTA) was obtained as the cadmium-containing form, from synthetic cDNA heterologously expressed in Escherichia coli. The purified protein was identified as the desired product by a combination of peptide-map analysis, amino acid sequence analysis and ion-spray mass spectroscopy. The existence of seven 113Cd NMR resonances revealed that the recombinant protein binds seven Cd ions/molecule. The position of the NMR resonances (605-695 ppm) and the electronic absorption features suggest that the sea urchin MTA, like the mammalian MTs, possesses tetrahedrally coordinated cadmium-thiolate clusters. With its large Stokes' radius, sea urchin MTA resembles the mammalian forms, suggesting a comparable elongated molecular shape. Measurements by spectrophotometric pH titration of cadmium binding by the recombinant protein suggest that it possesses two metal-thiolate clusters of distinctly different stability. At pH 7 the average apparent association constant for Cd2+ in the clusters is about 20-times weaker in sea urchin MTA than in rabbit MT-2.

Amino Acid Sequence↗
PubMed1993

Complete amino acid sequences of five dimeric and four monomeric forms of metallothionein from the edible mussel Mytilus edulis.

Cadmium-induced metallothioneins from the common sea mussel, Mytilus edulis, were shown to comprise of two groups of isoforms having apparent molecular masses of 10 kDa and 20 kDa. The 10-kDa group was resolved by anion-exchange chromatography into four fractions while the 20-kDa group was resolved into three fractions using this method. After metal removal and S-methylation of the cysteine residues using methyl-p-nitrobenzenesulphonate the complete amino acid sequences were determined. Five isoforms of the 20-kDa group were shown to possess monomeric units consisting of 71 amino acids. These proteins were distinct from the four 72-amino-acid proteins of the 10-kDa group. The FASTA algorithm has been used to compare the degree of similarity between the mussel metallothionein MT-10-IV isoform and other metallothioneins. The mussel MT-10-IV isoform exhibited substantial similarity to other molluscan metallothioneins. Moreover, the mussel metallothionein exhibited more similarity to vertebrate metallothioneins than to those of non-molluscan invertebrates, thus suggesting that the mussel metallothioneins are class I metallothioneins.

Amino Acid Sequence↗
PubMed1993

Purification and primary structure of snail metallothionein. Similarity of the N-terminal sequence with histones H4 and H2A.

A cadmium-binding metallothionein has been purified from metal-exposed Roman snails (Helix pomatia) using gel-permeation, ion-exchange and reverse-phase high-performance liquid chromatography. The S-methylated protein was digested with trypsin and the endoproteinases Asp-N, Glu-C and Arg-C. While most of the resulting peptides could be sequenced by Edman degradation, the intact protein, as well as the N-terminal peptide, proved to be blocked. Analysis by mass spectrometry showed that the N-terminal amino acid was an acetylated serine residue. Snail metallothionein, which is suggested to be involved in the detoxification of cadmium, contains 66 amino acid residues, 18 of which are cysteine residues arranged in seven Cys-Xaa-Cys motifs. The calculated molecular mass of the protein is 6.62 kDa. The primary structure of snail metallothionein reveals a clear relationship with molluscan and vertebrate metallothioneins, but lower similarity with metallothioneins of other invertebrate species. The N-terminal region of the isolated protein proved to be unique among the metallothionein sequences determined so far, showing high degrees of similarity with the N-terminal sequences of histones H2A and H4 which may be important for regulatory functions.

Amino Acid Sequence↗
PubMed1993

Cell- and inducer-specific accretion of human isometallothioneins.

The synthesis of metallothionein (MT) was investigated in three different human epithelial cell lines, each derived from one of the embryonic germ layers. The accretion of different isoforms of the protein was monitored using a sensitive neutral-pH h.p.l.c. method. Induction of MT synthesis by zinc ions and dexamethasone revealed differences between the three cell lines, both with respect to the number and the amounts of the different isoMTs formed. Dose-response experiments showed that an increase in dexamethasone concentration enhances MT accretion asymptotically to a limit, whereas within the concentration range explored zinc produces an exponential augmentation.

Cells, Cultured↗
PubMed1993

Induction of metallothionein synthesis by cadmium and zinc in cultured rabbit kidney cells (RK-13).

The effects of increasing concentrations of Zn(II) and Cd(II) on the expression of the four isometallothioneins (isoMTs), namely MT-1a, MT-2a, MT-2d and MT-2e, in rabbit kidney cells (RK-13) and the development of cellular tolerance to these metal ions were studied. The results showed that, whereas in parental cells MT concentration was low and composed nearly exclusively of MT-2a and MT-1a, all four isoMTs increased massively in abundance when the cells were exposed to toxic concentrations of Zn(II) or Cd(II), the relative increase being largest in the two minor isoforms MT-2d and MT-2e. While the response of the four isoMTs to the challenge by Zn(II) or Cd(II) was qualitatively comparable, there were differences in sensitivity and delay time, Cd(II) being the more efficient inducer and much faster in eliciting the onset of isoMT synthesis. An even larger production of isoMTs resulted when RK-13 cells were cultured in the presence of a series of metal concentrations yielding sub-lines of increased metal tolerance. In this instance too, there were marked differences in the response to Cd(II) and Zn(II). Thus, in cells of sub-lines selected for tolerance to moderate concentrations of Cd(II) the kinetic analysis of isoMT accretion gave indications of a saturable induction process while no such evidence was forthcoming for Zn(II). In sub-line cells selected for tolerance to the highest concentrations of Cd(II) or Zn(II) isoMT formation was increased by another order of magnitude, reaching for some isoforms a 100- to 1000-fold augmentation over the amounts measured in cells of the unexposed parental cells. A potentiation of this magnitude goes beyond the range of ordinary regulation of gene expression. It is to be viewed instead as an enlargement of the capacity of isoMT synthesis acquired by a variety of mechanisms in the surviving cells.

Animals↗
PubMed1993

Resolution and quantification of four metallothionein isoforms from rabbit kidney cells.

Reversed-phase high-performance liquid chromatography coupled with an on-line integrating chromatographic data system was used to separate and quantify the four major isometallothioneins MT-1a, MT-2a, MT-2d, and MT-2e present in metallothionein samples from cultured rabbit kidney cells as prepared by gel-filtration and/or ion-exchange chromatography. The standard curves for each of these isoforms showed closely comparable linear correlations between the amount of protein applied to the column and their integrated absorbance peak area at 220 nm. The lower limit of quantification is 30 pmol, sufficient to assess basal isometallothionein concentrations and to follow their variation upon metal exposure.

Animals↗
PubMed1992

Comparison of the NMR solution structure and the x-ray crystal structure of rat metallothionein-2.

Metallothioneins are small cysteine-rich proteins capable of binding heavy metal ions such as Zn2+ and Cd2+. They are ubiquitous tissue components in higher organisms, which tentatively have been attributed both unspecific protective functions against toxic metal ions and highly specific roles in fundamental zinc-regulated cellular processes. In this paper a detailed comparison of the NMR solution structure [Schultze, P., Wörgötter, E., Braun, W., Wagner, G., Vasák, M., Kägi, J. H. R. & Wüthrich, K. (1988) J. Mol. Biol. 203, 251-268] and a recent x-ray crystal structure [Robbins, A. H., McRee, D. E., Williamson, M., Collett, S. A., Xoung, N. H., Furey, W. F., Wang, B. C. & Stout, C. D. (1991) J. Mol. Biol. 221, 1269-1293] of rat metallothionein-2 shows that the metallothionein structures in crystals and in solution have identical molecular architectures. The structures obtained with both techniques now present a reliable basis for discussions on structure-function correlations in this class of metalloproteins.

Amino Acid Sequence↗
PubMed1992

Spectral characteristics of cadmium-containing phytochelatin complexes isolated from Schizosaccharomyces pombe.

Phytochelatins, heavy-metal-containing peptides with structures (gamma EC)nG, where n = 2-8, have been isolated from higher plants and the fission yeast Schizosaccharomyces pombe. The present work describes the isolation and characterization of several naturally occurring mixed complexes of these peptides from S. pombe exposed to 1 mM CdCl2. A lower-molecular-mass fraction from Sephadex G-50 chromatography yielded three distinct species on further fractionation. HPLC chromatography revealed the presence of peptides with n = 1-4 in varying amounts in these three complexes, referred to as complexes I, II and III. Stoichiometries are proposed for these complexes, based on [Cd], [SH], [S2-] and the amino acid content. Ultraviolet absorption and magnetic circular dichroism spectra of complexes II and III are similar, whereas the CD spectra of these two complexes are strikingly different. Compared to both complexes II and III, the CD bands of complex I are relatively weak. Ultraviolet absorption, CD and magnetic circular dichroism spectra provide a basis for the discussion of structural differences in these complexes.

Cadmium↗
PubMed1992

Comparison of the solution conformations of human [Zn7]-metallothionein-2 and [Cd7]-metallothionein-2 using nuclear magnetic resonance spectroscopy.

The solution structure of native human [Zn7]-metallothionein-2 has been compared with the previously determined structure of human [Cd7]-metallothionein-2. The comparison was based on complete sequence-specific 1H nuclear magnetic resonance assignments for human [Zn7]-metallothionein-2 obtained using the sequential assignment method. The secondary structure was found to be very similar in the [Zn7]- and [Cd7]- forms of the protein. Only seven amide protons in [Zn7]- metallothionein-2 were found to have exchange rates lower than approximately 0.2 min-1 at pH 7.0 and 10 degrees C, which corresponds closely to the results of amide proton exchange studies with the [Cd7]- form of the protein. Finally, the 1H-1H distance constraints determined from nuclear Overhauser enhancement spectroscopy for human [Zn7]-metallothionein-2 were checked for compatibility with the [Cd7]-metallothionein-2 structure. Overall, although no direct method is available for identifying the metal-polypeptide co-ordinative bonds in the Zn(2+)-containing protein, these measurements provided several independent lines of evidence showing that the [Zn7]- and [Cd7]- forms of human metallothionein-2 have the same molecular architecture.

Amides↗
PubMed1991

Zinc transfer from transcription factor IIIA fingers to thionein clusters.

The rapid induction of thionein (apometallothionein) by many endogenous stimuli such as steroid hormones, cytokines, and second messengers suggests that this cysteine-rich, metal binding protein participates in an as yet undefined role in cellular regulatory processes. This study demonstrates with DNA and RNA binding assays and in vitro transcription measurements that thionein suppresses the binding of the Xenopus laevis zinc finger transcription factor IIIA (TFIIIA) to 5S RNA and to the 5S RNA gene and abrogates the capacity of TFIIIA to initiate the RNA polymerase III-catalyzed synthesis of 5S RNA. The effect is reversed by the addition of zinc and is not observed in the TFIIIA-independent transcription of a tRNA gene by the same RNA polymerase. In view of the strong tendency of thionein to complex posttransition metals such as zinc, one effect of its enhanced synthesis in vivo could be to reduce the intracellular disposability of zinc and thus modulate the actions of zinc-dependent enzymes and proteins, most notably those of the zinc finger transcription factors.

Animals↗
PubMed1991

Thionein (apometallothionein) can modulate DNA binding and transcription activation by zinc finger containing factor Sp1.

A number of transcription factors contain so-called zinc finger domains for the interaction with their cognate DNA sequence. It has been shown that removal of the zinc ions complexed in these zinc fingers abrogates DNA binding and transcription activation. Therefore we wanted to test the hypothesis that the activity of transcription factors could be regulated by physiological chelators of zinc. A prominent candidate for such a chelator is the Cys-rich protein thionein (apometallothionein) that is inducible by heavy metal loads, and by other environmental stimuli. Here we show with DNA binding and in vitro transcription assays that thionein indeed can inactivate the zinc finger-containing Sp1 in a reversible manner. By contrast, transcription factor Oct-1, which binds DNA via a homeo-domain, i.e. a helix-turn-helix motif not involving zinc ions, is refractory to thionein action. We propose that modulation of intracellular thionein concentration is used for the coordinated regulation of a large subset of genes whose transcription depends on zinc finger proteins.

Animals↗