PubMed Health⌕ Search

Biomedical subjects

C T Dameron

Publications and source records attributed to C T Dameron.

At least 19 recordsLinked to original sources
PubMed2000

Stoichiometry of complex formation between Copper(I) and the N-terminal domain of the Menkes protein.

The inherent cellular toxicity of copper ions demands that their concentration be carefully controlled. The cellular location of the Menkes ATPase, a key element in the control of intracellular copper, is regulated by the intracellular copper concentration through the N-terminus of the enzyme, comprising 6 homologous subdomains or modules, each approximately 70 residues in length and containing a -Cys-X-X-Cys- motif. Based on the proposal that binding of copper to these modules regulates the Menkes ATPase cellular location by promoting changes in the tertiary structure of the enzyme, we have expressed the entire N-terminal domain (MNKr) and the second metal-binding module (MNKr2) of the Menkes protein in E. coli and purified them to homogeneity. Ultraviolet-visible, luminescence, and X-ray absorption spectroscopy show that copper and silver bind to the single module, MNKr2, with a stoichiometry of one metal ion per module. However, the array of six modules, MNKr, binds Cu(I) to produce a homogeneous conformer with 4 mol equiv of metal ion. The metal ions are bound in an environment that is shielded from solvent molecules. We suggest a model of the Menkes protein in which the Cu(I) binding induces tertiary changes in the organization of the six metal-binding domains.

Adenosine Triphosphatases↗
PubMed2000

Kinetics of DNA alkylation, depurination and hydrolysis of anti diol epoxide of benzo(a)pyrene and the effect of cadmium on DNA alkylation.

Anti benzo[a]pyrene diol epoxide (BPDE) alkylates guanines of DNA at N7 in the major groove and at the exocyclic amino group in the minor groove. In this report we investigated the rates of BPDE hydrolysis, DNA alkylation and subsequent depurination of BPDE-adducted pBR322 DNA fragment using polyacrylamide gel electrophoresis. Preincubation studies showed that it hydrolyzed completely in triethanolamine buffer in <2 min. The depurination kinetics showed that a fraction of the N7 alkylated guanine depurinated rapidly; however a significant amount of N7 guanine alkylation remained stable to spontaneous depurination over a 4-h period. Similar results were obtained for the hydrolysis and alkylation rates of syn isomer but it required nearly 500 times more concentration to induce similar levels of N7 guanine alkylation. Cadmium ion strongly inhibited the N7 guanine alkylation of both isomers. But the minor groove alkylation was not affected as demonstrated by postlabeling assay which confirmed the presence of heat-and cadmium-stable minor groove adducts in BPDE-treated calf thymus DNA. Based on these and our earlier findings, we propose a mechanism for the synergistic effect of cadmium in chemically induced carcinogenesis.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed2000

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.

Animals↗
PubMed1999

Characterisation of copper-binding to the second sub-domain of the Menkes protein ATPase (MNKr2).

The Menkes ATPase (MNK) has an essential role in the translocation of copper across cellular membranes. In a complementary manner, the intracellular concentration of copper regulates the activity and cellular location of the ATPase through its six homologous amino-terminal domains. The roles of the six amino-terminal domains in the activation and cellular trafficking processes are unknown. Understanding the role of these domains relies on the development of an understanding of their metal-binding properties and structural properties. The second conserved sub-domain of MNK was over-expressed, purified and its copper-binding properties characterised. Reconstitution studies demonstrate that copper binds to MNKr2 as Cu(I) with a stoichiometry of one copper per domain. This is the first direct evidence of copper-binding to the MNK amino-terminal repeats. Circular dichroism studies suggest that the binding or loss of copper to MNKr2 does not cause substantial changes to the secondary structure of the protein.

Adenosine Triphosphatases↗
PubMed1999

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.

Amino Acid Sequence↗
PubMed1999

X-ray absorption spectroscopy of cadmium phytochelatin and model systems.

Higher plants, algae and some yeasts respond to potentially toxic heavy metals such as cadmium by synthesizing phytochelatins and related cysteine-rich polypeptides. We have used X-ray absorption spectroscopy to study the nature of cadmium binding in such peptides isolated from maize (Zea mays) exposed to low levels of cadmium, and in two synthetic cadmium-peptide complexes, Cd-(gamma-Glu-Cys)3Gly and Cd-(alpha-Glu-Cys)3Gly. We have used the synthetic ions [Cd(SPh)4]2-, [Cd4(SPh)10]2- and [S4Cd10(SPh)16]4-as crystallographically defined models for the cadmium site. The Cd K-edge extended X-ray absorption fine structure (EXAFS) data, together with the Cd K, LI, LII and LIII near-edge spectra, reveal a predominantly tetrahedral coordination of cadmium by sulfur in both the phytochelatin and synthetic peptide complexes. In particular, the Cd LIII-edge lacks a peak at 3534.9 e V which was found to be prominent for oxygen- or nitrogen-coordinated species. The Cd-S distance in the phytochelatin complex is 2.54 A. The Cd K-edge EXAFS does not show any isolated, well-defined Cd-Cd interactions; however, contrary to the conclusion of previous work, their absence is not necessarily indicative of isolated cadmium-thiolate ligation. Evidence from other studies suggests that high static disorder, combined with a large vibrational component, serve to effectively wash out this contribution to the EXAFS. The sulfur K-edge, moreover, shows a low-energy feature both in the phytochelatin and in the synthetic cadmium-peptide complexes which is consistent with sulfide bound in a cluster with cadmium as found for [S4Cd10(SPh)16]4-. This feature strongly suggests the presence of a polynuclear cadmium cluster in maize phytochelatin.

Cadmium↗
PubMed1999

Molecular mechanisms of copper metabolism and the role of the Menkes disease protein.

Menkes disease is an X-linked, recessive disorder of copper metabolism that occurs in approximately 1 in 200,000 live births. The condition is characterized by skeletal abnormalities, severe mental retardation, neurologic degeneration, and patient mortality in early childhood. The symptoms of Menkes disease result from a deficiency of serum copper and copper-dependent enzymes. A candidate gene for the disease has been isolated and designated MNK. The MNK gene codes for a P-type cation transporting ATPase, based on homology to known P-type ATPases and in vitro experimentation. cDNA clones of MNK in Menkes patients show diminished or absented hybridization in northern blot experiments. The Menkes protein functions to export excess intracellular copper and activates upon Cu(I) binding to the six metal-binding repeats in the amino-terminal domain. The loss of Menkes protein activity blocks the export of dietary copper from the gastrointestinal tract and causes the copper deficiency associated with Menkes disease. Each of the Menkes protein amino-terminal repeats contains a conserved -X-Met-X-Cys-X-X-Cys- motif (where X is any amino acid). These metal-binding repeats are conserved in other cation exporting ATPases involved in metal metabolism and in proteins involved in cellular defense against heavy metals in both prokaryotes and eukaryotes. An overview of copper metabolism in humans and a discussion of our understanding of the molecular basis of cellular copper homeostasis is presented. This forms the basis for a discussion of Menkes disease and the protein deficit in this disease.

Adenosine Triphosphatases↗
PubMed1999

Copper chaperones: function, structure and copper-binding properties.

Copper is an absolute requirement for living systems and the intracellular trafficking of this metal to copper-dependent proteins is fundamental to normal cellular metabolism. The copper chaperones perform the dual functions of trafficking and the prevention of cytoplasmic exposure to copper ions in transit. Only a small number of copper chaperones have been identified at this time but their conservation across plant, bacterial and animal species suggests that the majority of living systems utilise these proteins for copper routing. The available data suggest that each copper-dependent protein in the cell is served by a specific copper chaperone. Although copper chaperones cannot be substituted for one another in a given cell type, copper chaperones that deliver to the same protein in different cell types appear to be functionally equivalent. The majority of the copper chaperones identified thus far have an "open-faced beta-sandwich" global fold with a conserved MXCXXC metal-binding motif. Specificity for a given copper-dependent protein appears to be mediated by the residues surrounding the copper-binding motif. Copper binds to such proteins as Cu(I) in a trigonal complex with three sulfur ligands. Only the copper chaperone specific for cytochrome-c-oxidase, Cox17, deviates from this design.

Amino Acid Sequence↗
PubMed1998

Cadmium inhibits BPDE alkylation of DNA in the major groove but not in the minor groove.

Cadmium, a constituent of tobacco, has the potential to act in synergy with other carcinogens in tobacco smoke. Working on the hypothesis that cadmium interactions with DNA enhances the mutagenic lesions induced by tobacco carcinogens, we investigated the site and sequence selectivity of DNA binding by cadmium using DNA reactive chemical probes. Our results show that this divalent cation binds to N7 guanines with a great preference for those occurring in runs of G's. Further, cadmium considerably diminishes N7 guanine alkylation by the tobacco carcinogen metabolite BPDE; however, the biologically relevant guanine alkylation in the minor groove by BPDE was not affected. The relevance of our findings to cadmium's role in the tobacco carcinogenesis is discussed.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1998

Mechanisms for protection against copper toxicity.

Essential transition metals such as copper, molybdenum, and zinc and nonessential metals like cadmium, mercury, and lead can be toxic at the cellular, tissue, and organ levels when present in excess. To avoid metal-induced toxicity most organisms use a redundant combination of metal-regulated import inhibition, sequestration, and enhanced export mechanisms. Combinations of these mechanisms are used to form detoxification pathways controlled through metal-binding proteins at transcriptional, translational, or enzymatic levels. In mammalian pathways copper is partially detoxified by sequestration in the metal-binding metallothioneins or export via the copper-translocating ATPases. Copper regulation of these two mechanisms is afforded by specific conformational changes induced in regulatory proteins on metal binding.

Adenosine Triphosphatases↗
PubMed1997

Biochemical characterization of procoagulant albumin.

Procoagulant albumin (Pro-Alb) is an anionic form of albumin isolated from normal human plasma that regulates vascular endothelial cell hemostatic properties, including induction of tissue factor activity. We investigated the biochemical modification of Pro-Alb that was associated with procoagulant-inducing activity. Tryptic digestion of Pro-Alb identified greatest bioactivity in the carboxy-terminus of the molecule, a region associated with lipid binding sites. Activated charcoal treatment and phopholipase C digestion reduced the procoagulant-inducing activity of Pro-Alb, and Pro-Alb contained 2.3-fold more phosphorus than inactive albumin. We conclude that modification of albumin by phospholipid imparts tissue factor-inducing activity to Pro-Alb.

Amino Acid Sequence↗
PubMed1995

Distinct regions of Cu(I).ACE1 contact two spatially resolved DNA major groove sites.

The interaction between the Cu(I).ACE1 (CuACE1) transcription factor and its DNA binding site in the yeast metallothionein gene was studied by systematically altering the DNA sequence through base substitution, modification, and deletions as well as by altering the protein structure through chemical modification. We show here that CuACE1 is comprised of two distinct domains that contact DNA through minor groove interactions located between two major groove interaction sites. The minor groove interactions are shown to be critical for formation of a stable CuACE1.DNA complex. The NH2-terminal segment of ACE1 is shown to contact the 5'-most distal major groove site.

Base Sequence↗
PubMed1994

The metallothionein structural motif in gene expression.

Metalloregulation in eukaryotic organisms is poorly understood. Only a limited number of physiological processes are currently known to be regulated by metal ions. Copper salts stimulate transcription of MT and SOD genes in fungi and repress expression of cytochrome c6 in algae. The Cu-activation of gene expression in fungi is mediated by a Cu1+ specific sensor protein. The mechanism of Cu-activation of the sensor molecule, ACE1 (and probably AMT1), appears to be the formation of a CuS polymetallic cluster as the structural core of the proteins. Structural similarities between ACE1, AMT1 and metallothionein suggest that the MT motif is a good structural model to explain the metal-specific activation and specificity of the two signal transducing proteins. Metal ion specificity is achieved by the propensity of proteins with a MT motif to form multinuclear CuS centers. Coordination inorganic chemistry appears to be the driving force for Cu1+ metalloregulation in biology. The metalloregulation of transcriptional activation of mammalian MT genes will be intriguing as metal ion selectivity is not as apparent. The MT motif is not expected to be a highly redundant structural theme. The intriguing observation by Uchida et al. (154) that the growth inhibitory factor (MTIII) deficient in the brains of Alzheimer's patients is homologous to metallothioneins raises the likelihood that coordination chemistry will be critical in stabilizing the bioactive form of MTIII. The motif may be observed in yet to be identified metalloregulatory proteins that regulate other processes in a Cu- or Zn-specific manner. Formation of metal:thiolate polymetallic clusters allows a significant volume of the protein structure to be altered, so metal-induced structural dynamics are possible. CuS polynuclear clusters may be more general than the MT motif in biology. The only molecules currently known to form CuS polynuclear clusters include MT, ACE1 and the Cu(gamma EC)nG complexes, although AMT1 and MTIII are expected to be the next members in the list. Three other Cys-rich proteins, papilloma viral E7 and LIM motif-containing CRP and CRIP, isolated as Zn2+ proteins exhibit facile metal exchange in vitro with Cu1+. The resulting Cu1+ proteins show optical properties similar to CuMTs (202,203). Although CuS clusters may form in E7 and LIM-containing proteins, it is premature to ascribe any biological significance to the Cu1+ conformers.

Amino Acid Sequence↗
PubMed1993

Distinct metal binding configurations in ACE1.

The ACE1 protein of Saccharomyces cerevisiae mediates the metal-induced expression of the CUP1 metallothionein (MT) genes. Curiously, ACE1 resembles the MT protein in the types of metal complexes that form. ACE1 binds Cd(II) and Cu(I) ions in distinct configurations, but only the Cu(I) conformer of ACE1 forms a high-affinity and specific complex with DNA. Cu(I) ions associated with ACE1 are known to assemble in a polymetallic CuI-thiolate cluster that resembles Cu-metallothionein in metal coordination properties [Dameron, C. T., Winge, D. R., George, G. N., Sansone, M., Hu, S., & Hamer, D. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 6127-6131]. In contrast to the Cu(I) nuclearity of 6-7 mol equiv in ACE1 and 7 mol equiv in yeast MT, divalent ions, including Cd(II), Zn(II) and Co(II), bind with a maximal stoichiometry of near 4 mol equiv in ACE1 and 4 mol equiv in yeast MT. Charge-transfer bands consistent with metal:thiolate coordination were observed in CdACE1 and CoACE1. Spectroscopic studies of CoIIACE1 and EXAFS analysis of CdIIACE1 revealed tetrahedral coordination geometry in these complexes. Similar tetrahedral coordination complexes were observed with Co(II) and Cd(II) complexes of MT from S. cerevisiae. Metal binding in ACE1 is clearly similar to that in MT, and therefore the MT-metal clusters appear to be a good structural model of the metal center of ACE1.

Bacteriophage T7↗
PubMed1992

Metal thiolate coordination in the E7 proteins of human papilloma virus 16 and cottontail rabbit papilloma virus as expressed in Escherichia coli.

The oncogenic E7 proteins of human papilloma virus (HPV 16) and of cottontail rabbit papilloma virus (CRPV) have been purified from an expression system in Escherichia coli. The proteins as purified from E. coli contain one tightly bound Zn(II) ion per molecule. The metal site shows facile exchange with either Cd(II) or Cu(I). The HPV 16 E7 maximally bound one Cd(II) or two Cu(I) ions, while the CRPV E7 bound two Cd(II) or three Cu(I) ions. The Cd(II) and Cu(I) E7 molecules exhibited optical transitions in the ultraviolet suggestive of metal:thiolate coordination. E7 proteins from HPV 16 and CRPV contain 7 and 8 cysteines/molecule, respectively. Reaction of the E7 proteins with the sulfhydryl reagent, dithiodipyridine, revealed that all the cysteinyl sulfurs are present in the reduced thiol state. Cu(I)-E7 molecules are luminescent with maximal emission at 570 nm. The observed emission at room temperature is indicative of metal coordination within a compact protein environment shielded from solvent interactions. The emission maxima occurs at the same wavelength (570 nm) as Cu(I)-cysteinyl sulfur clusters in Cu(I)-metallothioneins. The single Zn(II) atom in each protein can be removed from E7 in the presence of EDTA. The resulting apoE7 molecules remain soluble and can be partially reconstituted with Cd(II) to regain the ultraviolet charge transfer transitions.

Animals↗
PubMed1992

Sequestration of cadmium and copper by recombinant rainbow trout and human metallothioneins and by chimeric (mermaid and fishman) proteins with interchanged domains.

A family of synthetic genes was constructed encoding a rainbow trout metallothionein (MT), a human MT, and two chimeric molecules which contained respectively (i) the N-terminal (or head) domain of human MT followed by the C-terminal (or tail) domain of a fish MT (termed mermaid MT) and (ii) the head domain of fish MT fused with the tail domain of human MT (denoted fishman MT). These were expressed in Escherichia coli and the four recombinant proteins were purified to homogeneity therefrom. All four were found to bind 7 g atoms of Cd(II) per mol; at pH 7.0, but not at pH 8.6, four Cd(II) ions were sequestered preferentially in the tail (or alpha) domain. Reciprocally, copper was found to bind preferentially in the head (or beta) domain. The human and fishman MTs displayed a stoichiometry of 12 g atoms of Cu(I) per mol, while rainbow trout and mermaid MTs bound only 10. The significance of these findings is discussed in relation to the different positional organization of cysteine residues close to the N and C termini of mammalian and piscine metallothioneins.

Amino Acid Sequence↗
PubMed1991

A copper-thiolate polynuclear cluster in the ACE1 transcription factor.

ACE1 is the transcriptional activator of the metallothionein (CUP1 locus) gene in Saccharomyces cerevisiae. Previous data had implicated the N-terminal domain of ACE1 as responsible for the Cu-dependent specific DNA binding. An expression system in Escherichia coli was constructed to enable the isolation of an ACE1 domain containing the DNA and Cu-binding regions. Here we report the purification and characterization of the Cu-ACE1 truncated molecule. Spectroscopic techniques showed that ACE1 contains an unusual type of DNA binding structure that is based on a polynuclear Cu(I)-cysteinyl thiolate cluster. The cluster consists of six or seven Cu(I) ions coordinated to cysteinyl thiolates in a trigonal geometry distorted from planarity. The Cu(I)-cysteine cluster of Cu-ACE1 exhibits structural properties analogous to the Cu(I)-thiolate polynuclear cluster in yeast Cu-metallothionein itself, suggesting an unusual mechanism for the evolution of this regulatory factor. The Cu cluster organizes and stabilizes the conformation of the N-terminal domain of ACE1 for specific DNA binding.

Amino Acid Sequence↗