Inhibitory sites in enzymes: Zinc removal and reactivation by thionein

Thionein (T) has not been isolated previously from biological material. However, it is generated transiently in situ by removal of zinc from metallothionein under oxidoreductive conditions, particularly in the presence of selenium compounds. T very rapidly activates a group of enzymes in which zinc is bound at an inhibitory site. The reaction is selective, as is apparent from the fact that T does not remove zinc from the catalytic sites of zinc metalloenzymes. T instantaneously reverses the zinc inhibition with a stoichiometry commensurate with its known capacity to bind seven zinc atoms in the form of clusters in metallothionein. The zinc inhibition is much more pronounced than was previously reported, with dissociation constants in the low nanomolar range. Thus, T is an effective, endogenous chelating agent, suggesting the existence of a hitherto unknown and unrecognized biological regulatory system. T removes the metal from an inhibitory zinc-specific enzymatic site with a resultant marked increase of activity. The potential significance of this system is supported by the demonstration of its operations in enzymes involved in glycolysis and signal transduction.

Overexpression of a Novel Arabidopsis Gene Related to Putative Zinc-Transporter Genes from Animals Can Lead to Enhanced Zinc Resistance and Accumulation

We describe the isolation of an Arabidopsis gene that is closely related to the animal ZnT genes (Zn transporter). The protein encoded by the ZAT (Zn transporter of Arabidopsis thaliana) gene has 398 amino acid residues and is predicted to have six membrane-spanning domains. To obtain evidence for the postulated function of the Arabidopsis gene, transgenic plants with the ZAT coding sequence under control of the cauliflower mosaic virus 35S promoter were analyzed. Plants obtained with ZAT in the sense orientation exhibited enhanced Zn resistance and strongly increased Zn content in the roots under high Zn exposure. Antisense mRNA-producing plants were viable, with a wild-type level of Zn resistance and content, like plants expressing a truncated coding sequence lacking the C-terminal cytoplasmic domain of the protein. The availability of ZAT can lead to a better understanding of the mechanism of Zn homeostasis and resistance in plants.

Identification of the zinc-dependent endothelial cell binding protein for high molecular weight kininogen and factor XII: identity with the receptor that binds to the globular "heads" of C1q (gC1q-R).

High molecular weight kininogen (HK) and factor XII are known to bind to human umbilical vein endothelial cells (HUVEC) in a zinc-dependent and saturable manner indicating that HUVEC express specific binding site(s) for those proteins. However, identification and immunochemical characterization of the putative receptor site(s) has not been previously accomplished. In this report, we have identified a cell surface glycoprotein that is a likely candidate for the HK binding site on HUVECs. When solubilized HUVEC membranes were subjected to an HK-affinity column in the presence or absence of 50 microM ZnCl2 and the bound membrane proteins eluted, a single major protein peak was obtained only in the presence of zinc. SDS/PAGE analysis and silver staining of the protein peak revealed this protein to be 33 kDa and partial sequence analysis matched the NH2 terminus of gC1q-R, a membrane glycoprotein that binds to the globular "heads" of C1q. Two other minor proteins of approximately 70 kDa and 45 kDa were also obtained. Upon analysis by Western blotting, the 33-kDa band was found to react with several monoclonal antibodies (mAbs) recognizing different epitopes on gC1q-R. Ligand and dot blot analyses revealed zinc-dependent binding of biotinylated HK as well as biotinylated factor XII to the isolated 33-kDa HUVEC molecule as well as recombinant gC1q-R. In addition, binding of 125I-HK to HUVEC cells was inhibited by selected monoclonal anti-gC1q-R antibodies. C1q, however, did not inhibit 125I-HK binding to HUVEC nor did those monoclonals known to inhibit C1q binding to gC1q-R. Taken together, the data suggest that HK (and factor XII) bind to HUVECs via a 33-kDa cell surface glycoprotein that appears to be identical to gC1q-R but interact with a site on gC1q-R distinct from that which binds C1q.

Recruitment of SMRT/N-CoR-mSin3A-HDAC-repressing complexes is not a general mechanism for BTB/POZ transcriptional repressors: The case of HIC-1 and γFBP-B

Hypermethylated in cancer (HIC-1), a new candidate tumor suppressor gene located in 17p13.3, encodes a protein with five C2H2 zinc fingers and an N-terminal broad complex, tramtrack, and bric à brac/poxviruses and zinc-finger (BTB/POZ) domain found in actin binding proteins or transcriptional regulators involved in chromatin modeling. In the human B cell lymphoma (BCL-6) and promyelocityc leukemia (PLZF) oncoproteins, this domain mediates transcriptional repression through its ability to recruit a silencing mediator of retinoid and thyroid hormone receptor (SMRT)/nuclear receptor corepressor (N-CoR)-mSin3A-histone deacetylase (HDAC) complex, a mechanism shared with numerous transcription factors. HIC-1 appears unique because it contains a 13-aa insertion acquired late in evolution, because it is not found in its avian homologue, γF1-binding protein isoform B (γFBP-B), a transcriptional repressor of the γF-crystallin gene. This insertion, located in a conserved region involved in the dimerization and scaffolding of the BTB/POZ domain, mainly affects slightly the ability of the HIC-1 and γFBP-B BTB/POZ domains to homo- and heterodimerize in vivo, as shown by mammalian two-hybrid experiments. Both the HIC-1 and γFBP-B BTB/POZ domains behave as autonomous transcriptional repression domains. However, in striking contrast with BCL-6 and PLZF, both HIC-1 and γFBP-B similarly fail to interact with members of the HDAC complexes (SMRT/N-CoR, mSin3A or HDAC-1) in vivo and in vitro. In addition, a general and specific inhibitor of HDACs, trichostatin A, did not alleviate the HIC-1- and γFBP-B-mediated transcriptional repression, as previously shown for BCL-6. Taken together, our studies show that the recruitment onto target promoters of an HDAC complex is not a general property of transcriptional repressors containing a conserved BTB/POZ domain.

Treble clef finger—a functionally diverse zinc-binding structural motif

Detection of similarity is particularly difficult for small proteins and thus connections between many of them remain unnoticed. Structure and sequence analysis of several metal-binding proteins reveals unexpected similarities in structural domains classified as different protein folds in SCOP and suggests unification of seven folds that belong to two protein classes. The common motif, termed treble clef finger in this study, forms the protein structural core and is 25–45 residues long. The treble clef motif is assembled around the central zinc ion and consists of a zinc knuckle, loop, β-hairpin and an α-helix. The knuckle and the first turn of the helix each incorporate two zinc ligands. Treble clef domains constitute the core of many structures such as ribosomal proteins L24E and S14, RING fingers, protein kinase cysteine-rich domains, nuclear receptor-like fingers, LIM domains, phosphatidylinositol-3-phosphate-binding domains and His-Me finger endonucleases. The treble clef finger is a uniquely versatile motif adaptable for various functions. This small domain with a 25 residue structural core can accommodate eight different metal-binding sites and can have many types of functions from binding of nucleic acids, proteins and small molecules, to catalysis of phosphodiester bond hydrolysis. Treble clef motifs are frequently incorporated in larger structures or occur in doublets. Present analysis suggests that the treble clef motif defines a distinct structural fold found in proteins with diverse functional properties and forms one of the major zinc finger groups.

Location and properties of metal-binding sites on the human prion protein

Although a functional role in copper binding has been suggested for the prion protein, evidence for binding at affinities characteristic of authentic metal-binding proteins has been lacking. By presentation of copper(II) ions in the presence of the weak chelator glycine, we have now characterized two high-affinity binding sites for divalent transition metals within the human prion protein. One is in the N-terminal octapeptide-repeat segment and has a Kd for copper(II) of 10−14 M, with other metals (Ni2+, Zn2+, and Mn2+) binding three or more orders of magnitude more weakly. However, NMR and fluorescence data reveal a previously unreported second site around histidines 96 and 111, a region of the molecule known to be crucial for prion propagation. The Kd for copper(II) at this site is 4 × 10−14 M, whereas nickel(II), zinc(II), and manganese(II) bind 6, 7, and 10 orders of magnitude more weakly, respectively, regardless of whether the protein is in its oxidized α-helical (α-PrP) or reduced β-sheet (β-PrP) conformation. A role for prion protein (PrP) in copper metabolism or transport seems likely and disturbance of this function may be involved in prion-related neurotoxicity.

Differential display of intestinal mRNAs regulated by dietary zinc.

Regulation of gene expression by zinc is well established, especially through the metal response elements of the metallothionein genes; however, most other aspects of the functions of zinc in gene expression remain unknown. We have looked for intestinal mRNAs that are regulated by dietary zinc status. Using the reverse transcriptase-PCR method of mRNA differential display, we compared intestinal mRNA from rats that were maintained for 18 days in one of three dietary groups: zinc-deficient, zinc-adequate, and pair-fed zinc-adequate. At the end of this period, total RNA was prepared from the intestine and analyzed by mRNA differential display. Under these conditions, only differentially displayed cDNA bands that varied in the zinc-deficient group, relative to the zinc-adequate groups, were selected. Utilizing two anchored oligo-dT3' PCR primers and a total of 27 arbitrary decamers as 5' PCR primers, our results yielded 47 differentially displayed cDNA bands from intestinal RNA. Thirty were increased in zinc deficiency, and 17 were decreased. Nineteen bands were subcloned and sequenced. Eleven of these were detectable on Northern blots, of which four were confirmed as regulated. Three of these have homology to known genes: cholecystokinin, uroguanylin, and ubiquinone oxidoreductase. The fourth is a novel sequence as it has no significant homology in GenBank. The remainder of those cloned included novel sequences, as well as matches to reported expressed sequence tags, and functionally identified genes. Further characterization of the regulated sequences identified here will show whether they are primary or secondary effects of zinc deficiency.

The single Cys2-His2 zinc finger domain of the GAGA protein flanked by basic residues is sufficient for high-affinity specific DNA binding.

Specific DNA binding to the core consensus site GAGAGAG has been shown with an 82-residue peptide (residues 310-391) taken from the Drosophila transcription factor GAGA. Using a series of deletion mutants, it was demonstrated that the minimal domain required for specific binding (residues 310-372) includes a single zinc finger of the Cys2-His2 family and a stretch of basic amino acids located on the N-terminal end of the zinc finger. In gel retardation assays, the specific binding seen with either the peptide or the whole protein is zinc dependent and corresponds to a dissociation constant of approximately 5 x 10(-9) M for the purified peptide. It has previously been thought that a single zinc finger of the Cys2-His2 family is incapable of specific, high-affinity binding to DNA. The combination of an N-terminal basic region with a single Cys2-His2 zinc finger in the GAGA protein can thus be viewed as a novel DNA binding domain. This raises the possibility that other proteins carrying only one Cys2-His2 finger are also capable of high-affinity specific binding to DNA.

The leukemia-associated-protein (LAP) domain, a cysteine-rich motif, is present in a wide range of proteins, including MLL, AF10, and MLLT6 proteins.

We have identified and further characterized a Caenorhabditis elegans gene, CEZF, that encodes a protein with substantial homology to the zinc finger and leucine zipper motifs of the human gene products AF10, MLLT6, and BR140. The first part of the zinc finger region of CEZF has strong similarity to the corresponding regions of AF10 (66%) and MLLT6 (64%) at the cDNA level. As this region is structurally different from previously described zinc finger motifs, sequence homology searches were done. Twenty-five other proteins with a similar motif were identified. Because the functional domain of this motif is potentially disrupted in leukemia-associated chromosomal translocations, we propose the name of leukemia-associated protein (LAP) finger. On the basis of these comparisons, the LAP domain consensus sequence is Cys1-Xaa1-2-Cys2-Xaa9-21-Cys3-Xaa2-4 -Cys4-Xaa4-5-His5-Xaa2-Cys6-Xaa12-46 - Cys7-Xaa2-Cys8, where subscripted numbers represent the number of amino acid residues. We review the evidence that this motif binds zinc, is the important DNA-binding domain in this group of regulatory proteins, and may be involved in leukemogenesis.