A persistent untranslated sequence within bacteriophage T4 DNA topoisomerase gene 60.

A 50-nucleotide untranslated region is shown to be present within the coding sequence of Escherichia coli bacteriophage T4 gene 60, which encodes one of the subunits for its type II DNA topoisomerase. This interruption is part of the transcribed messenger RNA and appears not to be removed before translation. Thus, the usual colinearity between messenger RNA and the encoded protein sequence apparently does not exist in this case. The interruption is bracketed by a direct repeat of five base pairs. A mechanism is proposed in which folding of the untranslated region brings together codons separated by the interruption so that the elongating ribosome may skip the 50 nucleotides during translation. The alternative possibility, that the protein is efficiently translated from a very minor and undetectable form of processed messenger RNA, seems unlikely, but has not been completely ruled out.

Crystal structure of Cd,Zn metallothionein.

The anomalous scattering data from five Cd in the native protein were used to determine the crystal structure of cadmium, zinc (Cd,Zn) metallothionein isoform II from rat liver. The structure of a 4-Cd cluster was solved by direct methods. A 2.3 A resolution electron density map was calculated by iterative single-wavelength anomalous scattering. The structure is folded into two domains. The amino terminal domain (beta) of residues 1 to 29 enfolds a three-metal cluster of one Cd and two Zn atoms coordinated by six terminal cysteine thiolate ligands and three bridging cysteine thiolates. The carboxyl terminal domain (alpha) of residues 30 to 61 enfolds a 4-Cd cluster coordinated by six terminal and five bridging cysteine thiolates. All seven metal sites have tetrahedral coordination geometry. The domains are roughly spherical, and the diameter is 15 to 20 A; there is limited contact between domains. The folding of alpha and beta is topologically similar but with opposite chirality. Redundant, short cysteine-containing sequences have similar roles in cluster formation in both alpha and beta.

Identification of SLF1 as a new copper homeostasis gene involved in copper sulfide mineralization in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, at least 12 genes are important for cells to propagate in medium containing elevated concentrations of copper salts (J. Welch, S. Fogel, C. Buchman, and M. Karin, EMBO J. 8:255-260, 1989). Complementation studies were carried out on a copper-sensitive mutation (cup14) from this group. A new yeast gene, designated SLF1, was identified as a multicopy suppressor of the cup14 mutation. Slf1 is important for the physiological process of copper sulfide (CuS) mineralization on the surface of cells cultured in medium containing copper salts. CuS mineralization causes the cells to turn brown. Disruption of SLF1, which is located close to the telomere region of chromosome IV, leads to limited copper sensitivity, and the resulting cells lack the normal brownish coloration when grown in CuSO4-containing medium. Overproduction of Slf1 in wild-type cells confers superresistance to CuSO4 and enhances the coloration of cells cultured in the presence of CuSO4. Upon addition of KCN to Cu-grown cells, the brownish coloration was bleached instantly, and copper ions were solubilized. These data are consistent with Slf1-dependent accumulation of CuS complexes on the cell surface. Disruption of SFL1 also results in loss of the ability of yeast cells to deplete Cu but not Cd ions from the growth medium, whereas overexpression enhances Ca depletion ability and the resulting deposition of CuS particles. It is proposed that Slfl participates in a copper homeostasis pathway, distinct from the Cup1 detoxification system, that leads to sulfide generation and CuS biomineralization on the cell surface. This process may coordinate with the Cup1 pathway at different copper concentrations to prevent copper-induced toxicity.

Copper-regulatory domain involved in gene expression.

Copper ion homeostasis in yeast is maintained through regulated expression of genes involved in copper ion uptake, Cu(I) sequestration, and defense against reactive oxygen intermediates. Positive and negative copper ion regulation is observed, and both effects are mediated by Cu(I)-sensing transcription factors. The mechanism of Cu(I) regulation is distinct for transcriptional activation versus transcriptional repression. Cu(I) activation of gene expression in S. cerevisiae and C. glabrata occurs through Cu-regulated DNA binding. The activation process involves Cu(I) cluster formation within the regulatory domain in Ace1 and Amt1. Cu(I) binding stabilizes a specific conformation capable of high-affinity interaction with specific DNA promoter sequences. Cu(I)-activated transcription factors are modular proteins in which the DNA-binding domain is distinct from the domain that mediates transcriptional activation. The all-or-nothing formation of the polycopper cluster permits a graded response of the cell to environmental copper. Cu(I) triggering may involve a metal exchange reaction converting Ace1 from a Zn(II)-specific conformer to a clustered Cu(I) conformer. The Cu(I) regulatory domain occurs in transcription factors from S. cerevisiae and C. glabrata. Sequence homologs are also known in Y. lipolytica and S. pombe, although no functional information is available for these candidate regulatory molecules. The presence of the Cu(I) regulatory domain in four distinct yeast strains suggests that this Cu-responsive domain may occur in other eukaryotes. Cu-mediated repression of gene expression in S. cerevisiae occurs through Cu(I) regulation of Mac1. Cu(I) binding to Mac1 appears to inhibit the transactivation domain. The Cu(I) specificity of this repression is likely to arise from formation of a polycopper thiolate cluster.

Long-term turnover of cadmium metallothionein in liver and kidney following a single low dose of cadmium in rats.

Rats were injected subcutaneously on two consecutive days with CdCl2, and sampled animals, killed at monthly intervals from 1 to 6 months thereafter, exhibited the presence of Cd,Zn-thionein in both the liver and kidney. At 6 months, hepatic thionein was present as the two major polymorphic forms previously demonstrated in short term Cd-injection studies. [35S]cysteine incorporation studies showed that both polymorphic forms of thionein underwent continual turnover at similar rates throughout th study. The slow hepatic and renal turnover of Cd, therefore, was not due to a highly stable form of Cd-thionein, but apparently due to an inefficient mechanism for excretion of Cd from these tissues. The Cd/Zn ratio of hepatic thionein remained relatively constant, suggesting that continual thionein induction results in a long-term hepatic trapping of Zn by thionein, but the ratio of renal thionein showed a marked increase during the course of the study.

Metal-ion regulation of gene expression in yeast.

Metal-responsive transcription factors exist in yeast to modulate expression of genes that encode proteins involved in cellular uptake of copper, iron and zinc ions. These signal transduction pathways function in the cellular regulation of the intracellular concentration of free metal ions. A second component of metal homeostasis is the regulation of metal-ion binding through protein-mediated metallation. Copper-specific chaperones exist in yeast that route copper ions to the site of biosynthesis of copper-metalloenzymes.

Structure of the cysteine-rich intestinal protein, CRIP.

LIM domains are Zn-binding arrays found in a number of proteins involved in the control of cell differentiation, including several developmentally regulated transcription factors and a human proto-oncogene product. The rat cysteine-rich intestinal protein, CRIP, is a 76-residue polypeptide which contains a LIM motif. The solution structure of CRIP has been determined by homonuclear and 1H-15N heteronuclear correlated nuclear magnetic resonance spectroscopy. Structures with individual distance violations of < or = 0.03 angstrom and penalties (squared sum of distance violations) of < or = 0.06 angstrom2 were generated with a total of 500 nuclear Overhauser effect (NOE)-derived distance restraints (averaging 15.6 restraints per refined residue). Superposition of backbone heavy atoms of ordered residues relative to mean atom positions is achieved with pairwise rms deviations of 0.54(+/-0.14) angstrom. As observed previously for a peptide with the sequence of the C-terminal LIM domain from the avian cysteine-rich protein, CRP (cCRP-LIM2), CRIP binds two equivalents of zinc, forming N-terminal CCHC (Cys3, Cys6, His24, Cys27) and C-terminal CCCC (Cys30, Cys33, Cys51, Cys55) modules. The CCHC and CCCC modules in CRIP contain two orthogonally-arrayed antiparallel beta-sheets. The C-terminal end of the CCHC module contains a tight turn and the C terminus of the CCCC module forms an alpha-helix. The modules pack via hydrophobic interactions, forming a compact structure that is similar to that observed for cCRP-LIM2. The most significant differences between the structures occur at the CCHC module-CCCC module interface, which results in a difference in the relative orientations of the modules, and at the C terminus where the alpha-helix appears to be packed more tightly against the preceding antiparallel beta-sheet. The greater abundance of NOE information obtained for CRIP relative to cCRP-LIM2, combined with the analysis of J-coupling and proton chemical shift data, have allowed a more detailed evaluation of the molecular level interactions that stabilize the fold of the LIM motif.

Characterization of a human chorionic gonadotropin-like protein from Candida albicans.

Studies from our laboratory have demonstrated that human CG (hCG), human LH, (hLH), and an hCG-like protein extracted from Xanthomonas maltophilia were able to induce Candida albicans transition from the blastospore to the germ tube stage. In the present study, we describe the characterization of an hCG-like material extracted from Candida albicans blastospores (CaCGLP), which is potent in inducing transition and presumably represents the endogenous transition-inducing substance. This material was extracted from Candida albicans blastospores with glacial acetic acid and purified by affinity chromatography using a polyclonal rabbit anti-hCG antibody. The product obtained is a 68-kilodalton single band protein, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Under reduced conditions a protein smear is seen. Amino acid analysis showed a predominance of glycine (22%), followed by serine (12%), and glutamate (12%). This protein reacted in the following hCG immunoassays: 1) a polyclonal rabbit anti-hCG equilibrium assay, 2) a carboxyl-tail hCG equilibrium assay, 3) two hCG equilibrium assays using monoclonal antibodies (CG no. 4 and CG no. 9), 4) a free alpha-subunit equilibrium assay using a monoclonal antibody, and 5) an ultrasensitive immunoradiometric assay for hCG which does not cross-react with hLH, nor the free beta-subunit of hCG. The CaCGLP showed no reaction in a specific hLH immunoradiometric assay. When CaCGLP was tested in the transition assay, in the presence of 4% rat serum, it was found that this protein was 100 times more potent than hCG in producing Candida albicans transition. We conclude that Candida albicans produces a protein that has certain tertiary structure similarities to hCG and that this material is able to induce germ tube formation. We postulate that CaCGLP has an autocrine/paracrine effect in Candida albicans as a transition factor to control its own pathogenicity.

Cloning system for Candida glabrata using elements from the metallothionein-IIa-encoding gene that confer autonomous replication.

The yeast Candida glabrata harbors two distinct gene families that encode metallothioneins (MTs). One of these loci, the MT-IIa locus, exhibits selective and tandem amplification in many wild type strains of C. glabrata. The present paper demonstrates that the amplified MT-IIa gene contains autonomously replicating sequences (ARS). These ARS elements have been used to construct vectors capable of replicating in C. glabrata. The ARS element(s) in the MT-IIa gene were localized to a 457-bp segment downstream from the MT-IIa coding sequence. Although plasmids containing this fragment transform C. glabrata with high frequency, the stability of the transformants and the copy number of the plasmid improve when the entire 1.25-kb MT-IIa gene is used. Transformation of C. glabrata with plasmids carrying the 2 microns circle ARS of Saccharomyces cerevisiae led to the formation of micro-colonies, indicating that the ARS elements of 2 microns plasmids replicate only to a limited extent in C. glabrata. Conversely, a C. glabrata plasmid carrying three copies of the MT-IIa gene was able to transform S. cerevisiae.

Disruption analysis of metallothionein-encoding genes in Candida glabrata.

Candida glabrata harbors multiple genes encoding metallothionein (MT). We have disrupted MT-IIa, an amplified locus, and MT-IIb, a single-copy gene, to determine the roles of various MT genes in CuSO4 resistance in C. glabrata. The concentration of CuSO4 required to inhibit the growth by 50% (IC50) of a C. glabrata strain harboring an amplified MT-IIa locus and a single-copy MT-IIb and MT-I genes was 7 mM in a synthetic complete medium. The IC50 decreased to approx. 1 mM when the amplified MT-IIa locus was deleted. The disruption of the MT-IIb gene decreased the IC50 further to 0.1 mM. The CuSO4 resistance in a strain lacking both of the MT-II genes was attributable to MT-I; no evidence was found for the production of (gamma EC)nG isopeptides. The comparison of the nucleotide sequence of MT-IIb to that of MT-IIa revealed the same coding sequence with differences in the 5' region. However, substantial differences were found in the 3' region. MT-IIb was expressed since we were able to purify the protein from the strain that had an intact MT-IIb gene, but a deleted MT-IIa gene. In addition, CuSO4 resistance was provided by MT-IIb. Northern analysis of the total RNA from varied C. glabrata strains indicated no significant changes in the expression of MT-I in the presence or absence of the MT-II genes.

A plant metallothionein produced in E. coli.

A metallothionein cDNA was generated from pea (Pisum sativum L.) roots, amplified by PCR and inserted into a plasmid for expression in E. coli. Purification of the resultant product generated 3 pools of cadmium-containing material after DEAE-cellulose chromatography. The amino acid composition of each was in excellent agreement with that predicted for pea metallothionein. A cadmium content of approximately 6 g.atoms per mole of protein was estimated. N-terminal sequence analysis revealed that the recombinant molecule had been proteolysed within the extended region linking the 2 cysteine-rich (putative) metal-binding regions. The significance of these findings in terms of the protein folding/targeting of the molecule are considered.

Calcium, magnesium and the conformation of parvalbumin during muscular activity.

The conformation of perch parvalbumin in the Ca-, Mg- and metal-free state was studied by intrinsic fluorescence, trypsin susceptibility, thiol titration and circular dichroism. The data reveal that Ca-parvalbumin has a more compact structure than the metal-free protein, with a high alpha-helical content and a buried thiol. No difference in conformation could be detected between Mg- and Ca-parvalvumin, indicating that the Ca-Mg exchange that may take place during muscular activity is accompanied by little or no structural changes. Furthermore, recently published kinetic parameters can now be interpreted as meaning that, during the contraction-relaxation cycle, parvalbumin often stays in the Mg-form instead of switching to the Ca-form which is predominant in vitro.

Formation of the metal-thiolate clusters of rat liver metallothionein.

The isoforms of rat liver apo-metallothionein (MT) were reconstituted in vitro with Cd and Zn ions to study the order of binding of the seven metal sites. Reconstitution with seven Cd ions resulted in a metalloprotein similar to induced Cd,Zn-MT by the criteria of electrophoretic mobility, insensitivity to proteolysis by subtilisin and the pH-dependent release of Cd. Proteolytic digestion of MT reconstituted with sub-optimal quantities of Cd followed by separation of Cd-containing polypeptide fragments by electrophoresis and chromatography revealed metal ion binding initially occurs in cluster A. Upon saturation of the four sites in cluster A, binding occurs in the three metal center, cluster B. Samples reconstituted with one to four Cd or Zn ions per protein molecule, followed by digestion with subtilisin, yielded increasing amounts of a proteolytically stable polypeptide fragment identical with the alpha fragment domain encompassing the four metal center. Samples renatured with five to seven Cd ions per MT molecule showed decreasing quantities of alpha fragment and increasing amounts of nativelike MT. The binding process in each domain is cooperative. Reconstitution of apo-MT with two Cd ions followed by proteolysis yields a 50% recovery of saturated Cd4-alpha cluster. Likewise, when Cd5-renatured MT was digested with subtilisin, 30% of the molecules were identified as Cd7-MT with the remainder as Cd4-alpha fragment.

Crystals of cadmium, zinc metallothionein.

Single crystals have been grown of Cd,Zn metallothionein isoform II from rat liver. The space group is P41212(P43212) with unit cell dimensions a = b = 31.0 A and c = 120.0 A, and one molecule in the crystallographic asymmetric unit. The crystals are square bipyramids elongated on the tetragonal c-axis and are grown by repetitive seeding. The crystals are suitable for high resolution structure analysis. Assays of dissolved crystals show that the crystals have the same Cd and Zn content and amino acid composition as the native, as-isolated protein.

Uptake of cadmium-109, a metallothionein-binding radiometal, by tumors in mice as a function of the transformed phenotype.

Metallothionein (MT) is an intracellular protein that binds many metals with isotopes having imaging or radiotherapeutic potential. To determine whether uptake of radioisotopes that bind to MT is increased in tumors, we measured the uptake of cadmium-109 (Cd-109) in tumors and in normal tissues of mice. Tumors were grown in Balb/C mice from cultured Balb/3T3 cells transformed by the Moloney murine sarcoma virus (MMSV). Uptake of Cd-109 by MMSV tumors exceeded that by normal tissues examined, with the exception of liver and kidney (the organs known to be richest in metallothionein). The MMSV tumor:background ratios of activity were greater for Cd-109 than for gallium-67 for many of the normal tissues examined. The magnitude of uptake of Cd-109 by tumors from four related cell lines paralleled their degree of expression of two indices of the transformed, or malignant, phenotype. We conclude that metals that bind to MT may be useful for imaging or radiotherapy of cancer.

Enhanced neurotrophic activity in Alzheimer's disease cortex is not associated with down-regulation of metallothionein-III (GIF).

Alzheimer's disease (AD) is a chronic neurodegenerative disorder for which the pathogenic mechanisms are not well understood. Previous studies demonstrated that extracts prepared from AD brains could increase the survival of rat cortical neurons in vitro. Additional studies indicated that this enhanced neurotrophic activity of AD brain was due to a reduction of a growth inhibitory factor (GIF) that was subsequently shown to be a new member of the metallothionein (MT) gene family, and designated MT-III. The study presented here examined the association between neurotrophic activity and MT-III expression in frontal cortices from eight AD and five control brains, and further characterized the inhibitory activity of MT-III. On average, AD extracts stimulated the survival of approximately 2-fold more rat cortical neurons than control extracts, demonstrating that AD brain possesses elevated neurotrophic activity. When recombinant MTs were added to cultures grown in the presence of brain extract, MT-III but not MT-I had an inhibitory effect on neuron survival, confirming that MT-III is a specific inhibitory factor in this assay. However, in contrast to previous reports, neither MT-III mRNA nor MT-III protein levels were significantly decreased in the AD group. Therefore, the difference in neurotrophic activity between the AD and control brain samples examined in this study is probably not directly mediated by MT-III. These results suggest that MT-III down-regulation is not an important pathogenic event in some cases of AD.

Protection against cadmium toxicity in yeast by alcohol dehydrogenase.

A cDNA expression library from Schizosaccharomyces pombe was transformed into Saccharomyces cerevisiae to screen for genes capable of conferring cadmium resistance to S. cerevisiae cells. The cDNA library was cloned into the S. cerevisiae expression vector pDB20 which is designed to express cDNAs via the constitutively-expressed promoter of the gene for alcohol dehydrogenase I (ADH1). Terminator and polyadenylation signals are also provided by the ADH1 gene. Cadmium resistant colonies were shown to arise by a recombination event leading to the exchange of the S. pombe DNA with the chromosomal ADH1 gene and a consequent dramatic increase in the ADH1 gene expression due to the high copy number of the plasmid. The overexpression of ADH1 effectively buffered the cells for cadmium ions by formation of Cd-ADH.

Structural and functional diversity of copper-metallothioneins from the American lobster Homarus americanus.

The role of copper metallothionein (CuMT) in copper metabolism and metalloenzyme activation is poorly understood. We have chosen marine crustaceans, in which a direct correlation exists between levels of Cu(I)MT and Cu(I)-hemocyanin during the molt cycle (Engel and Brouwer, Biol. Bull. 173, 239-251, 1987) as unique model systems to study the involvement of MTs in metalloprotein activation and degradation. We have isolated three low-molecular weight, cysteine-rich copper proteins from the American lobster Homarus americanus, which we designate as CuMT-1, CuMT-2, and CuMT-3, respectively. As a first attempt to fully characterize these proteins, we have determined the sequence of the first 56 amino acids of CuMT-1. The results show this protein to belong to the class I MTs, i.e., related in primary structure to equine renal MT. CuMT-1 cannot transfer its copper to copper-depleted apohemocyanin. CuMT-2 belongs to the same class of MTs as CuMT-1, but CuMT-3 does not. The latter can reactivate lobster hemocyanin containing reduced amounts of Cu(I). Spectroscopic studies show that Cu(I) transfer from CuMT-3 to apohemocyanin initially results in the formation of distorted binuclear-copper sites, which subsequently slowly return to their native stereochemical configuration. Finally, we present evidence that shows that the class I MTs in marine crustacea are involved in the sequestration of elevated levels of heavy-metal ions. These observations strongly suggest that the different forms of MT have different biological functions.

X-ray absorption studies of the copper-beta domain of rat liver metallothionein.

Rat liver metallothionein contains two domains, each of which enfolds a separate metal-thiolate cluster. The binding stoichiometry of these clusters depends on the particular metal ion bound. In the aminoterminal beta domain the cluster can accommodate either three Cd(II) ions or six Cu(I) ions. The Cd ions are known to be coordinated in a tetrahedral geometry. In order to better understand the binding of Cu ions in this domain, the Cu-beta domain fragment of metallothionein was prepared and investigated by x-ray absorption spectroscopy. Quantitative analysis of the EXAFS data indicates copper-sulfur distances of 2.25 +/- 0.03 A. The EXAFS amplitudes and distance results are most consistent with trigonal coordination. A trigonal biprism is proposed for the Cu6Cys9 complex in which Cu occupies each vertex and cysteinyl sulfur bridges at each of the nine edges.

Conversion in the peptides coating cadmium:sulfide crystallites in Candida glabrata.

Cultures of Candida glabrata treated with CdCl2 form intracellular Cd(II) complexes that evolve with the time of culturing. Initially, glutathione (gamma ECG) appears to be the major buffering component. One type of Cd(II)-glutathione complex exists as a cadmium:sulfide (CdS) crystallite coated with glutathione. A time dependent change in the coating of the CdS particles occurs with a decrease in the (gamma ECG) content and a corresponding increase in the abundance of (gamma EC)nG peptides with (gamma EC)2G becoming the predominant peptide. The des-Gly variant (gamma EC)2 appears in significant concentration only in late cultures. The evolution in isopeptide coating appears to be dependent on the sulfide content of the CdS particles. Cellular conditions that enhance the generation of sulfide ions facilitate the conversion from gamma ECG to (gamma EC)2G.