Classical Zinc Finger Research Articles

ZNF692 promotes the migration and response to immunotherapy of clear cell renal cell carcinoma cells by targeting metabolic pathway

Clear cell renal cell carcinoma (ccRCC), with high mortality and poor prognosis, is the most common type of renal malignancy. It is necessary to identify new biomarkers that can serve as indicators for the detection of ccRCC at its early stages. In this study, we analyzed the role of classical zinc finger protein 692 (ZNF692) in ccRCC using datasets from The Cancer Genome Atlas (TCGA) and Single Cell Portal and immunohistochemical (IHC) staining of a tissue-microarray, and analyzed the function of ZNF692 in ccRCC cells. The analyses indicated that ZNF692 was upregulated in ccRCC samples compared with normal or paracancerous control samples (P < 0.001) and that the expression of this gene was linked to poor overall survival (HR = 2.1, P < 0.0001). The knockdown of ZNF692 inhibited the proliferation and migration of ccRCC cells by target GTPase-activating protein (SH3 domain)-binding protein 2 (G3BP2), and transmembrane 9 superfamily member 2 (TM9SF2)). T, B, proximal, and collecting tubule cells are the dominant cell types in normal kidney tissue where ZNF692 is expressed. In addition, immune checkpoint blockade (ICB) therapy dramatically changed the expression patterns of ZNF692. Collectively, these data indicate that ZNF692 may serve as prognosis, and as a potential indicator of the response to ICB therapy, a possibility needs to be verified by a case‒control study.

Decoding the Parkinson's Symphony: PARIS, Maestro of Transcriptional Regulation and Metal Coordination for Dopamine Release.

Parkin interacting substrate (PARIS) is a pivotal transcriptional regulator in the brain that orchestrates the activity of various enzymes through its intricate interactions with biomolecules, including nucleic acids. Notably, the binding of PARIS to insulin response sequences (IRSs) triggers a cascade of events that results in the functional loss in the substantia nigra, which impairs dopamine release and, subsequently, exacerbates the relentless neurodegeneration. Here, we report the details of the interactions of PARIS with IRSs via classical zinc finger (ZF) domains in PARIS, namely, PARIS(ZF2-4). Our biophysical studies with purified PARIS(ZF2-4) elucidated the binding partner of PARIS, which generates specific interactions with the IRS1 (5'-TATTTTT, Kd = 38.9 ± 2.4 nM) that is positioned in the promoter region of peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α). Mutational and metal-substitution studies demonstrated that Zn(II)-PARIS(ZF2-4) could recognize its binding partner selectively. Overall, our work provides submolecular details regarding PARIS and shows that it is a transcriptional factor that regulates dopamine release. Thus, PARIS could be a crucial target for therapeutic applications.

Reactivity of NiII, PdII and PtII complexes bearing phosphine ligands towards ZnII displacement and hydrolysis in Cis2His2 and Cis3His zinc-fingers domains

A systematic study of the effect of phosphine and bis-phosphine ligands in the interaction of NiII, PdII, and PtII complexes with two classes of zinc fingers was performed. The Cys2His2, finger 3 of specific protein-1, and the Cys2HisCys C-terminal zinc finger of nucleocapsid protein 7 of the HIV-1 were used as models of the respective class. In general, phosphine ligands favor the metal binding to the peptide, although the bis-phosphine ligands produce more specific binding than the monodentate. In the case of nickel complexes, the interaction of NiII ions with the sequence SKH, present in Cys2His2, results in hydrolysis, contrasting to the preferred zinc ejection produced by the NiII complexes with chelating phosphines, producing Ni(bis-phosphine) fingers. In the absence of the SKH sequence, zinc ejection is observed with the formation of nickel fingers, with reactivity dependent on the phosphine. On the other hand, Pd(phosphines) produces Pd2 fingers in the case of triphenylphosphine with the phosphine coordinated as intermediate species. The bis-phosphine ligands produce very clean spectra and a stable signal Pd(bis-phosphine)finger. Interestingly, phosphines produce very reactive platinum complexes, which eject zinc and promote peptide hydrolysis. The results reported here are relevant to the understanding of the mechanism of these interactions and how to modulate metallocompounds for zinc finger interference.

Non-Conserved Amino Acid Residues Modulate the Thermodynamics of Zn(II) Binding to Classical ββα Zinc Finger Domains

Classical zinc fingers domains (ZFs) bind Zn(II) ion by a pair of cysteine and histidine residues to adopt a characteristic and stable ββα fold containing a small hydrophobic core. As a component of transcription factors, they recognize specific DNA sequences to transcript particular genes. The loss of Zn(II) disrupts the unique structure and function of the whole protein. It has been shown that the saturation of ZFs under cellular conditions is strictly related to their affinity for Zn(II). High affinity warrants their constant saturation, while medium affinity results in their transient structurization depending on cellular zinc availability. Therefore, there must be factors hidden in the sequence and structure of ZFs that impact Zn(II)-to-protein affinities to control their function. Using molecular dynamics simulations and experimental spectroscopic and calorimetric approaches, we showed that particular non-conserved residues derived from ZF sequences impact hydrogen bond formation. Our in silico and in vitro studies show that non-conserved residues can alter metal-coupled folding mechanisms and overall ZF stability. Furthermore, we show that Zn(II) binding to ZFs can also be entropically driven. This preference does not correlate either with Zn(II) binding site or with the extent of the secondary structure but is strictly related to a reservoir of interactions within the second coordination shell, which may loosen or tighten up the structure. Our findings shed new light on how the functionality of ZFs is modulated by non-coordinating residues diversity under cellular conditions. Moreover, they can be helpful for systematic backbone alteration of native ZF ββα scaffold to create artificial foldamers and proteins with improved stability.

Transcription Factors in Aureobasidium spp.: Classification, Regulation and a Newly Built Database.

Transcription factors (TFs) can regulate the synthesis of secondary metabolites through different metabolic pathways in Aureobasidium spp. In this study, a set of 16 superfamilies, 45 PFAM families of TFs with the DNA-binding domains, seven zinc finger families and eight categories of the C2H2 TFs have been identified in Aureobasidium spp. Among all the identified TFs, four superfamilies and six PFAM families are the fungal-specific types in this lineage. The Zn2Cys6 and fungal-specific domain regulators are found to be overwhelmingly predominated, while the C2H2 zinc finger class comprises a smaller regulator class. Since there are currently no databases that allow for easy exploration of the TFs in Aureobasidium spp., based on over 50 references and 2405 homologous TFs, the first TFs pipeline—the Aureobasidium Transcription Factor Database (ATFDB)—has been developed to accelerate the identification of metabolic regulation in various Aureobasidium species. It would be useful to investigate the mechanisms behind the wide adaptability and metabolite diversity of Aureobasidium spp.

The Current Status of SSRP1 in Cancer: Tribulation and Road Ahead.

Methods We search PubMed and Web of Sciences with keywords “SSRP1” and “Cancer.” Only English literature was included, and conference papers and abstract were all excluded. Results Transcription factors are classified into three groups based on their DNA binding motifs: simple helix-loop-helix (bHLH), classical zinc fingers (ZF-TFs), and homeodomains. The tumor-suppressive miR-497 (microRNA-497) acted as an undesirable regulator of SSRP1 upregulation, which led to tumor growth. The siRNA (small interfering RNA) knockdown of SSRP1 hindered cell proliferation along with incursion and glioma cell migration. Through the AKT (also known as protein kinase B) signaling pathway, SSRP1 silencing affected cancer apoptosis and cell proliferation. Conclusion The MAPK (mitogen-activated protein kinase) signaling pathway's phosphorylation was suppressed when SSRP1 was depleted. The effect of curaxins on p53 and NF-B (nuclear factor-κB), and their toxicity to cancer cells, is attributable to the FACT (facilitates chromatin transcription) complex's chromatin trapping.

Overexpression of Krueppel like factor 3 promotes subcutaneous adipocytes differentiation in goat Capra hircus.

Previous research reported that KLF3 plays different roles in the regulation of adipose deposition across species. However, the exact function of KLF3 in goat subcutaneous adipocyte remains unknown. Here, the goat KLF3 gene was firstly cloned and showed that the mRNA sequence of the goat KLF3 gene was 1,264bp (GenBank accession number: KU041753.1) and its coding sequence was 1,037bp, encoding 345 amino acids with three classic zinc finger domains of KLFs family at its C-terminus. The alignment of the amino acid sequence of KLF3 among various species demonstrated that goat had the highest homology to that of sheep, presenting 99.4% similarity, while the homology similarity to that of mice presented only 93.62% in contrast. Furthermore, KLF3 had highest mRNA level in fat tissue and lowest level in the heart in comparison. Additionally, the mRNA level of KLF3 gradually tended to increase during adipogenesis. Interestingly, overexpression of KLF3 increased lipid accumulation. In line with this, the gain-of-function of KLF3 dramatically elevated the mRNA levels of TG synthetic genes and adipogenic maker genes (p<.01) . Moreover, overexpression of KLF3 upregulated all the potential target genes, except for C/EBPα. These results suggested that KLF3 is a positive regulator for subcutaneous adipocyte differentiation in goats.

Zinc Fingers.

Zinc finger (ZF) domains, that represent the majority of the DNA-binding motifs in eukaryotes, are involved in several processes ranging from RNA packaging to transcriptional activation, regulation of apoptosis, protein folding and assembly, and lipid binding. While their amino acid composition varies from one domain to the other, a shared feature is the coordination of a zinc ion, with a structural role, by a different combination of cysteines and histidines. The classical zinc finger domain (also called Cys2His2) that represents the most common class, uses two cysteines and two histidines to coordinate the metal ion, and forms a compact ββα architecture consisting in a β-sheet and an α-helix. GAG-knuckle resembles the classical ZF, treble clef and zinc ribbon are also well represented in the human genome. Zinc fingers are also present in prokaryotes. The first prokaryotic ZF domain found in the transcriptional regulator Ros protein was identified in Agrobacterium tumefaciens. It shows a Cys2His2 metal ion coordination sphere and folds in a domain significantly larger than its eukaryotic counterpart arranged in a βββαα topology. Interestingly, this domain does not strictly require the metal ion coordination to achieve the functional fold. Here, we report what is known on the main classes of eukaryotic and prokarotic ZFs, focusing our attention to the role of the metal ion, the folding mechanism, and the DNA binding. The hypothesis of a horizontal gene transfer from prokaryotes to eukaryotes is also discussed.

Overcharging of the Zinc Ion in the Structure of the Zinc-Finger Protein Is Needed for DNA Binding Stability

Zinc-finger structure, in which a Zn2+ ion binds to four cysteines or histidines in a tetrahedral structure, is a very common motif of nucleic acid-binding proteins. The corresponding interaction model is present in 3% of the genes in the human genome. As a result, the zinc finger has been extremely useful in various therapeutic and research capacities and in biotechnology. In a stable configuration of the zinc finger, the cysteine amino acids are deprotonated and become negatively charged. Thus, the Zn2+ ion is overscreened by four cysteine charges (overcharged). Whether this overcharged configuration is also stable when such a negatively charged zinc finger binds to a negatively charged DNA molecule is unknown. We investigated how the deprotonated state of cysteine influences its structure, dynamics, and function in binding to DNA molecules by using an all-atom molecular dynamics simulation up to the microsecond range of an androgen receptor protein dimer. Our results showed that the deprotonated state of cysteine residues is essential for the mechanical stabilization of the functional, folded conformation. This state stabilizes not only the protein structure but also the protein-DNA binding complex. The differences in the structural and energetic properties of the two sequence-identical monomers are also investigated and show the strong influence of DNA on the structure of the zinc-finger protein dimer upon complexation. Our result can potentially lead to a better molecular understanding of one of the most common classes of zinc fingers.

Expression and analysis of zinc finger family gene in Lenzites gibbosa

Zinc finger transcription factors play significant roles in the growth and development of plant and animal, but their function remains obscure in fungi. Lenzites gibbosa mycelia were extracted and sequenced by transcriptome analysis after growing on sawdust at different times to support mycelial growth of L. gibbosa in a nutrient matrix. Data bases used for analysis were the Kyoto encyclopedia of genes and genomes (KEGG) annotation, the cluster of orthologous groups of proteins (COG) and gene ontology (GO) annotation. Zinc finger class genes related to the growth and development of L. gibbosa were screened. GO annotation and enrichment analysis of differentially expressed genes were carried out. A total of 114.55 Gb Clean Data were obtained from the L. gibbosa transcriptome. The average Clean Data in each sample was 6.16 Gb. The relative efficiency of reads between each sample and the reference genome was 88.5% to 91.4%. The COG analysis showed that most zinc finger protein genes were related to replication, recombination and repair function. GO enrichment analysis showed that the expressed genes involved in cellular process, cell part and binding. We identified seventy-two expressed genes including seven up-regulated genes and sixty-five down-regulated genes by applying DESeq2 data analysis software. By comparing the significantly expressed genes with KEGG database, 66 annotated sequences were obtained, and 35 primary metabolic pathways were annotated. Pathway enrichment analysis showed that differentially expressed genes were significantly enriched in protein processing in endoplasmic reticulum and ubiquitin-mediated proteolysis pathways. Gene_11750 and gene_5266 are highly correlated with the growth and development of L. gibbosa and are closely related to protein processing in endoplasmic reticulum and ubiquitin-mediated proteolysis pathway. According to gene functional analysis, seven important differentially expressed genes related to the growth and development of L. gibbosa were identified.

Model peptide for anti-sigma factor domain HHCC zinc fingers: high reactivity toward 1O2 leads to domain unfolding.

All organisms have to cope with the deleterious effects of reactive oxygen species. Some of them are able to mount a transcriptional response to various oxidative stresses, which involves sensor proteins capable of assessing the redox status of the cell or to detect reactive oxygen species. In this article, we describe the design, synthesis and characterization of Zn·LASD(HHCC), a model for the Zn(Cys)2(His)2 zinc finger site of ChrR, a sensor protein involved in the bacterial defence against singlet oxygen that belongs to the family of zinc-binding anti-sigma factors possessing a characteristic H/C-X24/25-H-X3-C-X2-C motif. The 46-amino acid model peptide LASD(HHCC) was synthetized by solid phase peptide synthesis and its Zn2+-binding properties were investigated using electronic absorption, circular dichroism and NMR. LASD(HHCC) forms a 1 : 1 complex with Zn2+, namely Zn·LASD(HHCC), that adopts a well-defined conformation with the Zn2+ ion capping a 3-helix core that reproduces almost perfectly the fold of the ChrR in the vicinity of its zinc site. H2O2 reacts with Zn·LASD(HHCC) to yield a disulfide with a second order rate constant of 0.030 ± 0.002 M-1 s-1. Zn·LASD(HHCC) reacts rapidly with singlet oxygen to yield sulfinates and sulfonates. A lower limit of the chemical reaction rate constant between Zn·LASD(HHCC) and 1O2 was determined to be 3.9 × 106 M-1 s-1. Therefore, the Zn(Cys)2(His)2 site of Zn·LASD(HHCC) appears to be at least 5 times more reactive toward these two oxidants than that of a classical ββα zinc finger. Consequences for the activation mechanism of ChrR are discussed.

Ni(II), Hg(II), and Pb(II) Coordination in the Prokaryotic Zinc-Finger Ros87.

Zinc ion binding is a principal event in the achievement of the correct fold in classical zinc finger domains since the motif is largely unfolded in the absence of metal. In the case of a prokaryotic zinc finger, the larger βββαα domain contributes to the folding mechanism with a larger hydrophobic core. For these reasons, following the great amount of attention devoted to unveiling the effect of xenobiotic metal ion replacement in zinc fingers and in zinc-containing proteins in general, the prokaryotic zinc finger domain appears to be an interesting model for studying metal ion interaction with metalloproteins. Here, we explore the binding of Ni(II), Hg(II), and Pb(II) to Ros87, the DNA binding domain of the prokaryotic zinc finger protein Ros. We measured Ros87-metal ion dissociation constants and monitored the effects on the structure and function of the domain. Interestingly, we found that the protein folds in the presence of Ni(II) with important structural perturbations, while in the presence of Pb(II) and Hg(II) it does not appear to be significantly folded. Accordingly, an overall strong reduction in the DNA binding capability is observed for all of the examined proteins. Our data integrate and complement the information collected in the past few years concerning the functional and structural effects of metal ion substitution in classical zinc fingers in order to contribute to a better comprehension of the toxicity of these metals in biological systems.

Expression of KLF6-SV2 in colorectal cancer and its impact on proliferation and apoptosis.

Kruppel like factor 6 (KLF6), a member of KLF family, which has classic zinc finger structure, is broadly considered to have anticancer activity. The role of SV2 variant, one of KLF6 alternative splicing isoforms has not yet been definite in the colorectal cancer. This study aimed to detect the expression of the KLF6-SV2 in colorectal cancer and investigate its impact on cell proliferation and apoptosis. qRT-PCR was used to quantitatively determine KLF6-SV2 mRNA expression in colorectal cancer samples, corresponding normal tissue, normal colonic mucosal cell line FHC and seven colorectal cancer cell lines. SW480 and SW620 cell models with over-expressing KLF6-SV2 were constructed. Cell proliferation, cell cycle and apoptosis were measured respectively using MTT assay, DNA ploidy detection and Annexin V flow cytometry. Meanwhile, expression of p53, p21 and Bax were detected by qRT-PCR and western blot. The mRNA expression level of KLF6-SV2 in colorectal cancer tissues (0.783±0.409) was decreased than in corresponding normal tissues (1.086±0.449) (P<0.01), and expression in SW480 and SW620 were lower than in FHC, HCT116, LoVo, HT29, Caco-2 and RKO. In cell lines over-expressing KLF6-SV2, cell proliferation was markedly suppressed, cell cycle was blocked and cell apoptosis was significantly induced. Simultaneously, expression of p21 and Bax were remarkably up-regulated, while p53 remained unchanged. Decreased expression of KLF6-SV2 may be associated with the occurrence and development of colorectal cancer. KLF6-SV2 plays a role as tumor suppressor by efficiently blocking cell proliferation, arresting cell cycle and inducing apoptosis in colorectal cancer, which may be related to increased expression of p21 and Bax.

TvZNF1 is a C2H2 zinc finger protein of Trichomonas vaginalis.

The zinc fingers proteins (ZNF) are the largest family of DNA binding proteins and can act as transcriptional factors in eukaryotes. ZNF are implicated in activation in response to environmental stimulus by biometals such as Zn2+. Many of these proteins have the classical C2H2 zinc finger motifs (C2H2-ZNFm) of approximately 30 amino acids, where a Zn2+ ion is coordinated by two cysteine and two histidine residues. Trichomonas vaginalis is a protozoan parasite than responds to environmental changes including Zn2+. Until now has not been described any ZNF that could be involved in the regulation of genic expression of T. vaginalis. Here, we characterized in silico and experimentally an annoted ZNF (TvZNF1) from T. vaginalis and isolated the gene, tvznf1 encoding it. TvZNF1 have eight C2H2-ZNFm with residues that maybe involved in the structural stability of DNA binding motifs. In this work we confirmed the Zn2+ upregulation expression of tvznf1 gene. Recombinant TvZNF1 was able to bind to specific DNA sequences according to EMSA assay. Additionally, we demonstrated that recombinant TvZNF1 bind to MRE signature in vitro, which strongly suggests its role in transcriptional regulation, similar to the one observed for mammalian MTF-1. This result suggested a conserved mechanism of genic regulation mediated by ZNFs in T. vaginalis.

Miz-1 and Max compete to engage c-Myc: implication for the mechanism of inhibition of c-Myc transcriptional activity by Miz-1.

c-Myc is a basic helix-loop-helix leucine zipper (b-HLH-LZ) transcription factor deregulated in the majority of human cancers. As a heterodimer with Max, another b-HLH-LZ transcription factor, deregulated and persistent c-Myc accumulates at transcriptionally active promoters and enhancers and amplifies transcription. This leads to the so-called transcriptional addiction of tumor cells. Recent studies have showed that c-Myc transcriptional activities can be reversed by its association with Miz-1, a POZ transcription factor containing 13 classical zinc fingers. Although evidences have led to suggest that c-Myc interacts with both Miz-1 and Max to form a ternary repressive complex, earlier evidences also suggest that Miz-1 and Max may compete to engage c-Myc. In such a scenario, the Miz-1/c-Myc complex would be the entity responsible for the inhibition of c-Myc transcriptional amplification. Considering the implications of the Miz-1/c-Myc interaction, it is highly important to solve this duality. While two potential c-Myc interacting domains (hereafter termed MID) have been identified in Miz-1 by yeast two-hybrid, with the b-HLH-LZ as a bait, the biophysical characterization of these interactions has not been reported so far. Here, we report that the MID located between the 12th and 13th zinc finger of Miz-1 and the b-HLH-LZ of Max compete to form a complex with the b-HLH-LZ of c-Myc. Our results support the notion that the repressive action of Miz-1 on c-Myc does not rely on the formation of a ternary complex. The implications of these observations for the mechanism of inhibition of c-Myc transcriptional activity by Miz-1 are discussed. Proteins 2017; 85:199-206. © 2016 Wiley Periodicals, Inc.

Structural Analyses of Zinc Finger Domains for Specific Interactions with DNA

Zinc finger proteins are among the most extensively applied metalloproteins in the field of biotechnology owing to their unique structural and functional aspects as transcriptional and translational regulators. The classical zinc fingers are the largest family of zinc proteins and they provide critical roles in physiological systems from prokaryotes to eukaryotes. Two cysteine and two histidine residues (Cys₂His₂) coordinate to the zinc ion for the structural functions to generate a ββα fold, and this secondary structure supports specific interactions with their binding partners, including DNA, RNA, lipids, proteins, and small molecules. In this account, the structural similarity and differences of well-known Cys₂His₂-type zinc fingers such as zinc interaction factor 268 (ZIF268), transcription factor IIIA (TFIIIA), GAGA, and Ros will be explained. These proteins perform their specific roles in species from archaea to eukaryotes and they show significant structural similarity; however, their aligned amino acids present low sequence homology. These zinc finger proteins have different numbers of domains for their structural roles to maintain biological progress through transcriptional regulations from exogenous stresses. The superimposed structures of these finger domains provide interesting details when these fingers are applied to specific gene binding and editing. The structural information in this study will aid in the selection of unique types of zinc finger applications in vivo and in vitro approaches, because biophysical backgrounds including complex structures and binding affinities aid in the protein design area.

The zinc fingers of YY1 bind single-stranded RNA with low sequence specificity.

Classical zinc fingers (ZFs) are traditionally considered to act as sequence-specific DNA-binding domains. More recently, classical ZFs have been recognised as potential RNA-binding modules, raising the intriguing possibility that classical-ZF transcription factors are involved in post-transcriptional gene regulation via direct RNA binding. To date, however, only one classical ZF-RNA complex, that involving TFIIIA, has been structurally characterised. Yin Yang-1 (YY1) is a multi-functional transcription factor involved in many regulatory processes, and binds DNA via four classical ZFs. Recent evidence suggests that YY1 also interacts with RNA, but the molecular nature of the interaction remains unknown. In the present work, we directly assess the ability of YY1 to bind RNA using in vitro assays. Systematic Evolution of Ligands by EXponential enrichment (SELEX) was used to identify preferred RNA sequences bound by the YY1 ZFs from a randomised library over multiple rounds of selection. However, a strong motif was not consistently recovered, suggesting that the RNA sequence selectivity of these domains is modest. YY1 ZF residues involved in binding to single-stranded RNA were identified by NMR spectroscopy and found to be largely distinct from the set of residues involved in DNA binding, suggesting that interactions between YY1 and ssRNA constitute a separate mode of nucleic acid binding. Our data are consistent with recent reports that YY1 can bind to RNA in a low-specificity, yet physiologically relevant manner.

Revisiting and re-engineering the classical zinc finger peptide: consensus peptide-1 (CP-1).

Zinc plays key structural and catalytic roles in biology. Structural zinc sites are often referred to as zinc finger (ZF) sites, and the classical ZF contains a Cys2His2 motif that is involved in coordinating Zn(II). An optimized Cys2His2 ZF, named consensus peptide 1 (CP-1), was identified more than 20 years ago using a limited set of sequenced proteins. We have reexamined the CP-1 sequence, using our current, much larger database of sequenced proteins that have been identified from high-throughput sequencing methods, and found the sequence to be largely unchanged. The CCHH ligand set of CP-1 was then altered to a CAHH motif to impart hydrolytic activity. This ligand set mimics the His2Cys ligand set of peptide deformylase (PDF), a hydrolytically active M(II)-centered (M = Zn or Fe) protein. The resultant peptide [CP-1(CAHH)] was evaluated for its ability to coordinate Zn(II) and Co(II) ions, adopt secondary structure, and promote hydrolysis. CP-1(CAHH) was found to coordinate Co(II) and Zn(II) and a pentacoordinate geometry for Co(II)-CP-1(CAHH) was implicated from UV-vis data. This suggests a His2Cys(H2O)2 environment at the metal center. The Zn(II)-bound CP-1(CAHH) was shown to adopt partial secondary structure by 1-D (1)H NMR spectroscopy. Both Zn(II)-CP-1(CAHH) and Co(II)-CP-1(CAHH) show good hydrolytic activity toward the test substrate 4-nitrophenyl acetate, exhibiting faster rates than most active synthetic Zn(II) complexes.

Oncoprotein ZNF322A transcriptionally deregulates alpha-adducin, cyclin D1 and p53 to promote tumor growth and metastasis in lung cancer.

ZNF322A encoding a classical Cys2His2 zinc finger transcription factor was previously revealed as a potential oncogene in lung cancer patients. However, the oncogenic role of ZNF322A and its underlying mechanism in lung tumorigenesis remain elusive. Here we show ZNF322A protein overexpression in 123 Asian and 74 Caucasian lung cancer patients. Multivariate Cox regression analysis indicated that ZNF322A was an independent risk factor for a poor outcome in lung cancer, corroborating the Kaplan–Meier results that patients with ZNF322A protein overexpression had significantly poorer overall survival than other patients. Overexpression of ZNF322A promoted cell proliferation and soft agar growth by prolonging cell cycle in S phase in multiple lung cell lines, including the immortalized lung cell BEAS-2B. In addition, ZNF322A overexpression enhanced cell migration and invasion, whereas knockdown of ZNF322A reduced cell growth, invasion and metastasis abilities in vitro and in vivo. Quantitative proteomic analysis revealed potential ZNF322A-regulated downstream targets, including alpha-adducin (ADD1), cyclin D1 (CCND1), and p53. Using luciferase promoter activity assay combined with site-directed mutagenesis and sequential chromatin immunoprecipitation-PCR assay, we found that ZNF322A could form a complex with c-Jun and cooperatively activate ADD1 and CCND1 but repress p53 gene transcription by recruiting differential chromatin modifiers, such as histone deacetylase 3, in an AP-1 element dependent manner. Reconstitution experiments indicated that CCND1 and p53 were important to ZNF322A-mediated promotion of cell proliferation, whereas ADD1 was necessary for ZNF322A-mediated cell migration and invasion. Our results provide compelling evidence that ZNF322A overexpression transcriptionally dysregulates genes involved in cell growth and motility therefore contributes to lung tumorigenesis and poor prognosis.

Reactivity of a Zn(Cys)2(His)2 Zinc Finger with Singlet Oxygen: Oxidation Directed toward Cysteines but not Histidines.

Singlet oxygen ((1)O2) is an important reactive oxygen species in biology that has deleterious effects. Proteins constitute the main target of (1)O2 in cells. Several organisms are able to mount a transcriptional defense against (1)O2. ChrR and MBS are two proteins with Zn(Cys)2(His)2 zinc finger sites that are involved in the regulation of the defense against (1)O2. In this article, we investigate the reactivity of Zn⋅CPF, a Zn(Cys)2(His)2 classical ββα zinc finger, with (1)O2. We show that Zn⋅CPF interacts with (1)O2 mainly by physical quenching using a combination of (1)O2 luminescence quenching and kinetic competition experiments. The chemical reaction, which accounts for 5% of the interaction, leads to oxidation of cysteines but not histidines. Primary photooxidation products, identified by HPLC and mass spectrometry, are sulfinate (75±5%) and disulfides (25±5%). The peptides that have a single cysteine thiolate oxidized into a sulfinate are still able to bind one equivalent Zn(2+) but with a dramatic reduction of the binding constant compared to Zn⋅CPF despite the preservation of the ββα fold, as shown by NMR and CD titrations. Finally, Zn⋅CPF is compared to Zn⋅LTC, a treble clef Zn(Cys)4 zinc finger, to gain further insight into the behavior of zinc fingers toward (1)O2.