Damage of zinc fingers in DNA repair proteins, a novel molecular mechanism in carcinogenesis

Zinc finger motifs participate in protein-nucleic acid and protein-protein interactions in many groups of proteins, including those involved in DNA repair. The Zn(II) ion, bonded tetrahedrally to cysteine thiolates and/or histidine imidazole groups, maintains the three-dimensional structure, crucial for the function of the domain. Zinc fingers can thus be compromised by a substitution of Zn(II) with another metal ion or by a release of Zn(II), due to the oxidation of thiolate donors. The latter may result from an action of redox-active metals or other oxidative agents. Studies in cell cultures and ex vivo demonstrated that soluble compounds of definite carcinogenic metals and metalloids, such as arsenic, cadmium and nickel, and putative carcinogens, including cobalt and lead, inhibit zinc finger containing DNA repair proteins. Further experiments demonstrated that these metals, as well as endogeneous oxidative substances, including hydrogen peroxide, nitrosoglutathione, and reducible selenium compounds damage or distort zinc finger domains. This reactivity can therefore be regarded as a novel molecular mechanism in carcinogenesis.     

The HIV-1 Rev binding family of proteins: the dog proteins as a study model

Various proteins that are required for the building of new complete human immunodeficiency type 1 virions (HIV-1) are coded by unspliced or partly spliced virus-derived mRNAs. HIV-1 has developed special strategies for moving these mRNAs to the cytoplasm to be translated. In the nucleus of the infected cell the virus-derived protein Regulator of expression of viral proteins (Rev) can bind both the viral intron-containing mRNAs and the cellular co-factor HIV-1 Rev binding protein (HRB) and this complex may be shuttled through the nuclear pores. HRB genes have been relatively well conserved during evolution, from Drosophila to humans. However, as a consequence of reading-frame shifts due to nt insertions/deletions, the protein products generated may differ considerably from the prototypal HRB protein, which comprises one Arf-GAP zinc finger domain, several Phenylalanine-Glycine (FG) motifs and four Asparagine-Proline-Phenylalanine (NPF) motifs. This variability is best exemplified by four HRB proteins of the dog, which are discussed here in more detail. The hypothesis is advanced that atypical HRB proteins may not be able to bind Rev and possibly have other, still undetermined, functions. Since the cellular co-factor HRB is essential for viral replication and spread but is not required for cell viability and main bodily functions, it might be an attractive candidate for anti-HIV-1 drug targeting.     

Design of novel zinc finger proteins: towards artificial control of specific gene expression.

In this review, we summarize design strategies for generating proteins with desired sequences such as long contiguous base pairs and diverse sequence specificities based on the nature of Cys(2)-His(2) zinc finger proteins. Recent progress towards artificial DNA binding proteins has been achieved by structure-based design processes and by selection strategies. Indeed, a multi-zinc finger protein with an 18 (or 27)-base pair address, and new zinc finger proteins for diverse DNA target sites (TATA-box and p53 binding site) have been created successfully. Such novel zinc finger proteins will probably be useful tools in molecular biology and potentially in human medicine.     

Structural requirements for the neuroprotective effects of aspirin analogues against N-methyl-D-aspartate and zinc ion neurotoxicity

In order to elucidate the structural requirements for the dual neuroprotective activity of aspirin against N-methyl-D-aspartate (NMDA) and zinc ion neurotoxicity, various aspirin analogues and derivatives, modified at the carboxylic group, the acetyl group, and the chain length between the carboxylic acid moiety and phenyl ring, were synthesized. Replacement of the carboxylic acid group with alkyl groups (compounds 2c and 2d) resulted in a dramatic increase in neuroprotective activity against NMDA neurotoxicity, while reduction of the carboxylic acid group to the alcohol (compound 2g) completely abolished this activity. In contrast to NMDA neurotoxicity, compounds that are devoid of the carboxylic acid group did not show any activity against zinc ion neurotoxicity. Replacement of the acetyl group with a propionyl (compound 5a) or butyryl group (compound 5b) did not significantly change the activity against NMDA neurotoxicity, but replacement of the acetyl group with a propionyl group (compound 5a) resulted in a slight decrease in activity against zinc ion neurotoxicity. Compound 12, which has ethylene units between the carboxylic acid moiety and phenyl ring in the structure of aspirin, exhibited greater neuroprotective activity against NMDA neurotoxicity than the compared compounds (aspirin, compound 9 and compound 17), which have different chain lengths. A similar trend was also observed in the neuroprotective activity against zinc ion neurotoxicity. These results indicate that the carboxylic acid group in aspirin is not indispensable for the inhibitory effect against NMDA neurotoxicity, but is essential for the inhibitory effect against zinc ion neurotoxicity. The acetyl group and ethylene unit's distance are favourable for the inhibitory effect against NMDA neurotoxicity as well as zinc ion neurotoxicity.     

Functional analysis of zinc finger proteins that bind to the silencer element in the glutathione transferase P gene

Glutathione transferase P (GST-P) gene expression is repressed in normal rats but markedly promoted during the early stage of chemical hepatocarcinogenesis. We have previously identified a silencer region in this gene promoter. The silencer is composed of several cis-elements to which at least three proteins (Silencer factor-A, -B, and -C: SF-A, SF-B, and SF-C) are known to bind. We cloned and characterized the nuclear factor 1 family and the CCAAT/enhancer-binding protein family as SF-A and SF-B, respectively. Recently, zinc finger proteins as candidates for SF-C, which binds to GST-P silencer 2 (GPS2), were isolated. These proteins include four Krüppel-like proteins (BTEB2, EZF, LKLF, and TIEG1) and other factors containing multiple zinc finger motifs (TFIIIA and MZFP). In the present study, we found that the zinc finger proteins showed the same DNA-binding affinities to GPS2. Moreover, transfection analyses revealed that BTEB2, EZF, and TIEGI repressed the GST-P promoter activity. Therefore, these three factors might contribute to the repression of the GST-P gene expression in normal rat liver.     

Effect of zinc binding and precipitation on structures of recombinant human growth hormone and nerve growth factor

Metal-induced precipitation of protein therapeutics is being used and further developed as a processing step in protein formulation and may have utility in protein purification and bulk storage. In such processes, it is imperative that native protein structure is maintained and the metal complexation is reversible. In the current study, we investigated the effects of zinc-induced precipitation on recombinant human growth hormone (rhGH) and recombinant human nerve growth factor (rhNGF). On the addition of ethylenediaminetetraacetic acid (EDTA), the precipitates were dissolved, yielding complete recovery of native protein in both cases. Both proteins have specific metal binding sites and require specific molar ratios of zinc to protein to initiate precipitation (zinc:rhGH > 2:1; zinc:rhNGF > 18:1). Furthermore, the secondary structures of both proteins were unperturbed in soluble zinc complexes and zinc-induced precipitates, as measured by infrared and circular dichroism spectroscopies. The soluble zinc complex of rhGH had minor tertiary structural alterations, whereas zinc binding did not alter the tertiary structure of rhNGF. These studies indicated that metal-induced precipitation provides a method to maintain proteins in their native state in precipitates, which may be useful for purification, storage, and formulation.     

Co(II) and Cd(II) substitute for Zn(II) in the zinc finger derived from the DNA repair protein XPA, demonstrating a variety of potential mechanisms of toxicity

XPA is one of the key members of the protein complex of the nucleotide excision repair (NER) pathway of DNA repair. The CCCC zinc finger domain of XPA is involved in the interactions with other NER proteins. To study the possible molecular mechanisms of XPA inhibition, we previously investigated Zn(II) and Ni(II) interactions with the synthetic 37 amino acid peptide (XPAzf), AcDYVICEECGKEFMDSYLMNHFDLPTCDNCRDADDKHKam, representing the XPA zinc finger sequence (Bal, W., Schwerdtle, T., and Hartwig, A. (2003) Mechanism of nickel assault on the zinc finger of DNA repair protein XPA. Chem. Res. Toxicol. 16, 242-248). In this work, we extended these studies on other carcinogenic metal ions, Co(II) and Cd(II). The binding constants and complex geometries were determined using UV-vis and CD spectroscopies, and oxidative damage to XPAzf was studied with HPLC. The conditional binding constants determined for Co(II) and Cd(II) in 50 mM phosphate buffer, pH 7.4, are 10(7.4)+/-(0.4) and 10(12.8)+/-(0.5), respectively, yielding binding constant ratios Zn(II)/Co(II) of 100 and Zn(II)/ Cd(II) of 0.001, which are the lowest values reported for zinc fingers so far. The Co(II) ion forms a tetrahedral complex with the sulfurs of XPAzf, which is isostructural with the native zinc finger. The Cd(II) complex is somewhat less structured. The oxidation of Zn(II)-saturated XPAzf by H2O2 is accelerated in the presence of Co(II), but the concentration profile of this effect indicates the formation of an active Co(II) complex external to the metal-sulfur center. The Cd(II)-saturated XPAzf is very resistant to oxidation by H2O2. Overall, our results indicate that XPAzf can undergo Co(II) and Cd(II) assault under specific conditions.     

Zinc ion dependent B-cell epitope, associated with primary Sjogren's syndrome, resides within the putative zinc finger domain of Ro60kD autoantigen: physical and immunologic properties

The Ro/La ribonucleoprotein (RNP) complex is composed of the proteins Ro60kD, Ro52kD, and La48kD that are in association with one small cytoplasmic RNA (YRNA). Specific protein-RNA and protein-protein interactions are thought to occur through the RNP and zinc-finger secondary structure elements of the Ro60kD protein. The aim of our study was to investigate the antigenic properties of the zinc finger domain of the Ro60KD autoantigen and its contribution to the formation of Ro/La RNP complex. It was found that the peptide VSLVCEKLCNEKLLKKARIHPFHILIA (Zif-1), which corresponds to the natural sequence of the zinc finger domain (301-327), and the peptide C(Acm)NEKLLKKARIC(Acm), analogous to the intermediate loop 310-319 (Zif-3) of the same domain of Ro60KD, are recognized by the majority of anti-Ro/SSA and anti-La/SSB positive sera (82.6% and 77.1%, respectively) in the absence of zinc ions. The same sera failed to react with Zif-1 peptide in the presence of Zn2+. In contrast, the addition of zinc ions was necessary for the binding of Zif-1 to recombinant Ro52KD as shown by direct binding experiments of the recombinant protein with synthetic peptides. Our data suggest the zinc finger domain of Ro60kD contains a B-cell epitope with high specificity for primary Sjogren's syndrome. Furthermore, depending on the presence of zinc ions, the zinc finger domain of the Ro60KD protein can exist in two different conformational states favoring either an interaction with the Ro52KD protein or binding with autoantibodies.