Association of erythroid transcription factors: complexes involving the LIM protein RBTN2 and the zinc-finger protein GATA1.

The RBTN2 LIM-domain protein, originally identified as an oncogenic protein in human T-cell leukemia, is essential for erythropoiesis. A possible role for RBTN2 in transcription during erythropoiesis has been investigated. Direct interaction of the RBTN2 protein was observed in vivo and in vitro with the GATA1 or -2 zinc-finger transcription factors, as well as with the basic helix-loop-helix protein TAL1. By using mammalian two-hybrid analysis, complexes involving RBTN2, TAL1, and GATA1, together with E47, the basic helix-loop-helix heterodimerization partner of TAL1, could be demonstrated. Thus, a molecular link exists between three proteins crucial for erythropoiesis, and the data suggest that variations in amounts of complexes involving RBTN2, TAL1, and GATA1 could be important for erythroid differentiation.

Subunit-destabilizing mutations in Drosophila copper/zinc superoxide dismutase: neuropathology and a model of dimer dysequilibrium.

Mutations in Cu/Zn superoxide dismutase (SOD), a hallmark of familial amyotrophic lateral sclerosis (FALS) in humans, are shown here to confer striking neuropathology in Drosophila. Heterozygotes with one wild-type and one deleted SOD allele retain the expected 50% of normal activity for this dimeric enzyme. However, heterozygotes with one wild-type and one missense SOD allele show lesser SOD activities, ranging from 37% for a heterozygote carrying a missense mutation predicted from structural models to destabilize the dimer interface, to an average of 13% for several heterozygotes carrying missense mutations predicted to destabilize the subunit fold. Genetic and biochemical evidence suggests a model of dimer dysequilibrium whereby SOD activity in missense heterozygotes is reduced through entrapment of wild-type subunits into unstable or enzymatically inactive heterodimers. This dramatic impairment of the activity of wild-type subunits in vivo has implications for our understanding of FALS and for possible therapeutic strategies.

A chloroplast processing enzyme involved in precursor maturation shares a zinc-binding motif with a recently recognized family of metalloendopeptidases.

Nuclear-encoded proteins targeted to the chloroplast are typically synthesized with N-terminal transit peptides which are proteolytically removed upon import. Structurally related proteins of 145 and 143 kDa copurify with a soluble chloroplast processing enzyme (CPE) that cleaves the precursor for the major light-harvesting chlorophyll a/b binding protein and have been implicated in the maturation of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and acyl carrier protein. The 145- and 143-kDa proteins have not been found as a heterodimer and thus may represent functionally independent isoforms encoded by separate genes. Here we describe the primary structure of a 140-kDa polypeptide encoded by cDNAs isolated by using antibodies raised against the 145/143-kDa doublet. The 140-kDa polypeptide contains a transit peptide, and strikingly, a His-Xaa-Xaa-Glu-His zinc-binding motif that is conserved in a recently recognized family of metalloendopeptidases, which includes Escherichia coli protease III, insulin-degrading enzyme, and subunit beta of the mitochondrial processing peptidase. Identity of 25-30%, concentrated near the N terminus of the 140-kDa polypeptide, is found with these proteases. Expression of CPE in leaves is not light dependent. Indeed, transcripts are present in dark-grown plants, and the 145/143-kDa doublet and proteolytic activity are both found in etioplasts, as well as in root plastids. Thus, CPE appears to be a necessary component of the import machinery in photosynthetic and nonphotosynthetic tissues, and it may function as a general stromal processing peptidase in plastids.

Functional domain analysis of glass, a zinc-finger-containing transcription factor in Drosophila.

The glass gene is required for proper photo-receptor differentiation during development of the Drosophila eye glass codes for a DNA-binding protein containing five zinc fingers that we show is a transcriptional activator. A comparison of the sequences of the glass genes from two species of Drosophila and a detailed functional domain analysis of the Drosophila melanogaster glass gene reveal that both the DNA-binding domain and the transcriptional-activation domain are highly conserved between the two species. Analysis of the DNA-binding domain of glass indicates that the three carboxyl-terminal zinc fingers alone are necessary and sufficient for DNA binding. We also show that a deletion mutant of glass containing only the DNA-binding domain can behave in a dominant-negative manner both in vivo and in a cell culture assay that measures transcriptional activation.

Structure-assisted redesign of a protein-zinc-binding site with femtomolar affinity.

We have inserted a fourth protein ligand into the zinc coordination polyhedron of carbonic anhydrase II (CAII) that increases metal affinity 200-fold (Kd = 20 fM). The three-dimensional structures of threonine-199-->aspartate (T199D) and threonine-199-->glutamate (T199E) CAIIs, determined by x-ray crystallographic methods to resolutions of 2.35 Angstrum and 2.2 Angstrum, respectively, reveal a tetrahedral metal-binding site consisting of H94, H96, H119, and the engineered carboxylate side chain, which displaces zinc-bound hydroxide. Although the stereochemistry of neither engineered carboxylate-zinc interaction is comparable to that found in naturally occurring protein zinc-binding sites, protein-zinc affinity is enhanced in T199E CAII demonstrating that ligand-metal separation is a significant determinant of carboxylate-zinc affinity. In contrast, the three-dimensional structure of threonine-199-->histidine (T199H) CAII, determined to 2.25-Angstrum resolution, indicates that the engineered imidazole side chain rotates away from the metal and does not coordinate to zinc; this results in a weaker zinc-binding site. All three of these substitutions nearly obliterate CO2 hydrase activity, consistent with the role of zinc-bound hydroxide as catalytic nucleophile. The engineering of an additional protein ligand represents a general approach for increasing protein-metal affinity if the side chain can adopt a reasonable conformation and achieve inner-sphere zinc coordination. Moreover, this structure-assisted design approach may be effective in the development of high-sensitivity metal ion biosensors.

The retinoblastoma protein binds to RIZ, a zinc-finger protein that shares an epitope with the adenovirus E1A protein.

The retinoblastoma protein (Rb) is a target of viral oncoproteins. To explore the hypothesis that viral proteins may be structural mimics of cellular proteins, we have searched cDNA libraries for Rb-binding proteins. We report here the cloning of a cDNA for the protein RIZ from rat and human cells. RIZ is a 250-kDa nuclear protein containing eight zinc-finger motifs. It contains an Rb-binding motif that shares an antigenic epitope with the C terminus of E1A. A domain is conserved between RIZ and the PRDI-BF1/Blimp-1 differentiation factor. Other motifs of RIZ include putative GTPase and SH3 (src homology domain 3) domains. RIZ is preferentially expressed in both adult and embryonic rat neuroendocrine tissues. It is also expressed in human retinoblastoma cells and at low levels in all other human cell lines examined. While the function of RIZ is not yet clear, its structure and pattern of expression suggest a role for RIZ in transcriptional regulation during neuronal differentiation and pathogenesis of retinoblastoma.

Quantum Dot-Sensitized Solar Cells Based on Directly Adsorbed Zinc Copper Indium Sulfide Colloids

Heavy metal-based quantum dots (QDs) have demonstrated to behave as efficient sensitizers in QD-sensitized solar cells (QDSSCs), as attested by the countless works and encouraging efficiencies reported so far. However, their intrinsic toxicity has arisen as a major issue for the prospects of commercialization. Here, we examine the potential of environmentally friendly zinc copper indium sulfide (ZCIS) QDs for the fabrication of liquid-junction QDSSCs by means of photoelectrochemical measurements. A straightforward approach to directly adsorb ZCIS QDs on TiO2 from a colloidal dispersion is presented. Incident photon-to-current efficiency (IPCE) spectra of sensitized photoanodes show a marked dependence on the adsorption time, with longer times leading to poorer performances. Cyclic voltammograms point to a blockage of the channels of the mesoporous TiO2 film by the agglomeration of QDs as the main reason for the decrease in efficiency. Photoanodes were also submitted to the ZnS treatment. Its effects on electron recombination with the electrolyte are analyzed through electrochemical impedance spectroscopy and photopotential measurements. The corresponding results bring out the role of the ZnS coating as a barrier layer preventing electron leakage toward the electrolyte, as argued in other QD-sensitized systems. The beneficial effect of the ZnS coating is ultimately reflected on the power conversion efficiency of complete devices, reaching values of 2 %. In a more general vein, through these findings, we aim to call the attention to the potentiality of this quaternary alloy, virtually unexplored as a light harvester for sensitized devices.

Study of zinc protein ligands in a halophilic enzyme

Glucose dehydrogenase (EC 1.1.1.47) from the halophilic Archaeon Haloferax mediterranei belongs to the medium-chain alcohol dehydrogenase superfamily and requires a zinc ion for catalysis. The zinc ion is coordinated by a histidine, a water molecule and two other ligands from the protein or the substrate, which vary during the catalytic cycle of the enzyme. In many enzymes of this superfamily one of the zinc ligands is commonly cysteine, which is replaced by an aspartate residue at position 38 in the halophilic enzyme. This change has been only observed in glucose dehydrogenases from extremely halophilic microorganisms belonging to the Archaea Domain. This paper describes biochemical studies and structural comparisons to analyze the role of sequence differences between thermophilic and halophilic glucose dehydrogenases which contain a zinc ion within the protein surrounded by three ligands. Whilst the catalytic activity of the D38C GlcDH mutant is reduced, its thermal stability is enhanced, consistent with the greater structural similarity between this mutant and the homologous thermophilic enzyme from Thermoplasma acidophilum.