Engineering the zinc finger recombinase for use in targeted genomic editing

No abstract available.

Identificação e avaliação da expressão de genes pertencentes a família CCCH zinc finger em duas cultivares de soja sob deficit hídrico.

2016

Involvement of the N-finger in the self-association of GATA-1

Zinc fingers are recognized as small protein domains that bind to specific DNA sequences. Recently however, zinc fingers from a number of proteins, in particular the GATA family of transcription factors, have also been implicated in specific protein-protein interactions. The erythroid protein GATA-1 contains two zinc fingers: the C-finger, which is sufficient for sequence-specific DNA-binding, and the N-finger, which appears both to modulate DNA-binding and to interact with other transcription factors. We have expressed and purified the N-finger domain and investigated its involvement in the self-association of GATA-1. We demonstrate that this domain does not homodimerize but instead makes intermolecular contacts with the C-finger, suggesting that GATA dimers are maintained by reciprocal N-finger-C-finger contacts. Deletion analysis identifies a 25-residue region, C-terminal to the core N-finger domain, that is sufficient for interaction with intact GATA-1. A similar subdomain exists C-terminal to the C-finger, and we show that self-association is substantially reduced when both subdomains are disrupted by mutation. Moreover, mutations that impair GATA-1 self-association also interfere with its ability to activate transcription in transfection studies.

Divergent functions of three Candida albicans zinc-cluster transcription factors (CTA4, ASG1 and CTF1) complementing pleiotropic drug resistance in Saccharomyces cerevisiae.

One of the mediators of pleiotropic drug resistance in Saccharomyces cerevisiae is the ABC-transporter gene PDR5. This gene is regulated by at least two transcription factors with Zn(2)-Cys(6) finger DNA-binding motifs, Pdr1p and Pdr3p. In this work, we searched for functional homologues of these transcription factors in Candida albicans. A C. albicans gene library was screened in a S. cerevisiae mutant lacking PDR1 and PDR3 and clones resistant to azole antifungals were isolated. From these clones, three genes responsible for azole resistance were identified. These genes (CTA4, ASG1 and CTF1) encode proteins with Zn(2)-Cys(6)-type zinc finger motifs in their N-terminal domains. The C. albicans genes expressed in S. cerevisiae could activate the transcription of a PDR5-lacZ reporter system and this reporter activity was PDRE-dependent. They could also confer resistance to azoles in a S. cerevisiae strain lacking PDR1, PDR3 and PDR5, suggesting that CTA4-, ASG1- and CTF1-dependent azole resistance can be caused by genes other than PDR5 in S. cerevisiae. Deletion of CTA4, ASG1 and CTF1 in C. albicans had no effect on fluconazole susceptibility and did not alter the expression of the ABC-transporter genes CDR1 and CDR2 or the major facilitator gene MDR1, which encode multidrug transporters known as mediators of azole resistance in C. albicans. However, additional phenotypic screening tests on the C. albicans mutants revealed that the presence of ASG1 was necessary to sustain growth on non-fermentative carbon sources (sodium acetate, acetic acid, ethanol). In conclusion, C. albicans possesses functional homologues of the S. cerevisiae Pdr1p and Pdr3p transcription factors; however, their properties in C. albicans have been rewired to other functions.

In silico analysis identifies a C3HC4-RING finger domain of a putative E3 ubiquitin-protein ligase located at the C-terminus of a polyglutamine-containing protein

Almost identical polyglutamine-containing proteins with unknown structures have been found in human, mouse and rat genomes (GenBank AJ277365, AF525300, AY879229). We infer that an identical new gene (RING) finger domain of real interest is located in each C-terminal segment. A three-dimensional (3-D) model was generated by remote homology modeling and the functional implications are discussed. The model consists of 65 residues from terminal position 707 to 772 of the human protein with a total length of 796 residues. The 3-D model predicts a ubiquitin-protein ligase (E3) as a binding site for ubiquitin-conjugating enzyme (E2). Both enzymes are part of the ubiquitin pathway to label unwanted proteins for subsequent enzymatic degradation. The molecular contact specificities are suggested for both the substrate recognition and the residues at the possible E2-binding surface. The predicted structure, of a ubiquitin-protein ligase (E3, enzyme class number 6.3.2.19, CATH code 3.30.40.10.4) may contribute to explain the process of ubiquitination. The 3-D model supports the idea of a C3HC4-RING finger with a partially new pattern. The putative E2-binding site is formed by a shallow hydrophobic groove on the surface adjacent to the helix and one zinc finger (L722, C739, P740, P741, R744). Solvent-exposed hydrophobic amino acids lie around both zinc fingers (I717, L722, F738, or P765, L766, V767, V733, P734). The 3-D structure was deposited in the protein databank theoretical model repository (2B9G, RCSB Protein Data Bank, NJ).

A cobalt complex that selectively disrupts the structure and function of zinc fingers

Zinc finger domains are structures that mediate sequence recognition for a large number of DNA-binding proteins. These domains consist of sequences of amino acids containing cysteine and histidine residues tetrahedrally coordinated to a zinc ion. In this report, we present a means to selectively inhibit a zinc finger transcription factor with cobalt(III) Schiff-base complexes. 1H NMR spectroscopy confirmed that the structure of a zinc finger peptide is disrupted by axial ligation of the cobalt(III) complex to the nitrogen of the imidazole ring of a histidine residue. Fluorescence studies reveal that the zinc ion is displaced from the model zinc finger peptide in the presence of the cobalt complex. In addition, gel-shift and filter-binding assays reveal that cobalt complexes inhibit binding of a complete zinc finger protein, human transcription factor Sp1, to its consensus sequence. Finally, a DNA-coupled conjugate of the cobalt complexes selectively inhibited Sp1 in the presence of several other transcription factors.

Design of polyzinc finger peptides with structured linkers

Zinc finger domains are perhaps the most versatile of all known DNA binding domains. By fusing up to six zinc finger modules, which normally recognize up to 18 bp of DNA, designer transcription factors can be produced to target unique sequences within large genomes. However, not all continuous DNA sequences make good zinc finger binding sites. To avoid having to target unfavorable DNA sequences, we designed multizinc finger peptides with linkers capable of spanning long stretches of nonbound DNA. Two three-finger domains were fused by using either transcription factor IIIA for the Xenopus 5S RNA gene (TFIIIA) finger 4 or a non-sequence-specific zinc finger as a “structured” linker. Our gel-shift results demonstrate that these peptides are able to bind with picomolar affinities to target sequences containing 0–10 bp of nonbound DNA. Furthermore, these peptides display greater sequence selectivity and bind with higher affinity than similar six-finger peptides containing long, flexible linkers. These peptides are likely to be of use in understanding the behavior of polydactyl proteins in nature and in the targeting of human, animal, or plant genomes for numerous applications. We also suggest that in certain polydactyl peptides an individual finger can “flip” out of the major groove to allow its neighbors to bind shorter, nontarget DNA sequences.

Improved DNA binding specificity from polyzinc finger peptides by using strings of two-finger units

Multizinc finger peptides are likely to reach increased prominence in the search for the “ideal” designer transcription factor for in vivo applications such as gene therapy. However, for these treatments to be effective and safe, the peptides must bind with high affinity and, more importantly, with great specificity. Our previous research has shown that zinc finger arrays can be made to bind 18 bp of DNA with picomolar affinity, but also has suggested that arrays of fingers also may bind tightly to related sequences. This work addresses the question of zinc finger DNA binding specificity. We show that by changing the way in which zinc finger arrays are constructed—by linking three two-finger domains rather than two three-finger units—far greater target specificity can be achieved through increased discrimination against mutated or closely related sequences. These new peptides have the added capability of being able to span two short gaps of unbound DNA, although still binding with picomolar affinity to their target sites. We believe that this new method of constructing zinc finger arrays will offer greater efficacy in the fields of gene therapy and in the production of transgenic organisms than previously reported zinc finger arrays.

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.

Exploring the DNA-binding specificities of zinc fingers with DNA microarrays

A key step in the regulation of networks that control gene expression is the sequence-specific binding of transcription factors to their DNA recognition sites. A more complete understanding of these DNA–protein interactions will permit a more comprehensive and quantitative mapping of the regulatory pathways within cells, as well as a deeper understanding of the potential functions of individual genes regulated by newly identified DNA-binding sites. Here we describe a DNA microarray-based method to characterize sequence-specific DNA recognition by zinc-finger proteins. A phage display library, prepared by randomizing critical amino acid residues in the second of three fingers of the mouse Zif268 domain, provided a rich source of zinc-finger proteins with variant DNA-binding specificities. Microarrays containing all possible 3-bp binding sites for the variable zinc fingers permitted the quantitation of the binding site preferences of the entire library, pools of zinc fingers corresponding to different rounds of selection from this library, as well as individual Zif268 variants that were isolated from the library by using specific DNA sequences. The results demonstrate the feasibility of using DNA microarrays for genome-wide identification of putative transcription factor-binding sites.

Zinc- and iron-rubredoxins from Clostridium pasteurianum at atomic resolution: a high-precision model of a ZnS4 coordination unit in a protein.

The Zn(Scys)4 unit is present in numerous proteins, where it assumes structural, regulatory, or catalytic roles. The same coordination is found naturally around iron in rubredoxins, several structures of which have been refined at resolutions of, or near to, 1 A. The fold of the small protein rubredoxin around its metal ion is an excellent model for many zinc finger proteins. Zn-substituted rubredoxin and its Fe-containing counterpart were both obtained as the products of the expression in Escherichia coli of the rubredoxin-encoding gene from Clostridium pasteurianum. The structures of both proteins have been refined with an anisotropic model at atomic resolution (1.1 A, R = 8.3% for Fe-rubredoxin, and 1.2 A, R = 9.6% for Zn-rubredoxin) and are very similar. The most significant differences are increased lengths of the M-S bonds in Zn-rubredoxin (average length, 2.345 A) as compared with Fe-rubredoxin (average length, 2.262 A). An increase of the CA-CB-SG-M dihedral angles involving Cys-6 and Cys-39, the first cysteines of each of the Cys-Xaa-Xaa-Cys metal binding motifs, has been observed. Another consequence of the replacement of iron by zinc is that the region around residues 36-46 undergoes larger displacements than the remainder of the polypeptide chain. Despite these changes, the main features of the FeS4 site, namely a local 2-fold symmetry and the characteristic network of N-H...S hydrogen bonds, are conserved in the ZnS4 site. The Zn-substituted rubredoxin provides the first precise structure of a Zn(Scys)4 unit in a protein. The nearly identical fold of rubredoxin around iron or zinc suggests that at least in some of the sites where the metal has mainly a structural role-e.g., zinc fingers-the choice of the relevant metal may be directed by its cellular availability and mobilization processes rather than by its chemical nature.

The in vitro ejection of zinc from human immunodeficiency virus (HIV) type 1 nucleocapsid protein by disulfide benzamides with cellular anti-HIV activity.

Several disulfide benzamides have been shown to possess wide-spectrum antiretroviral activity in cell culture at low micromolar to submicromolar concentrations, inhibiting human immunodeficiency virus (HIV) type 1 (HIV-1) clinical and drug-resistant strains along with HIV-2 and simian immunodeficiency virus [Rice, W. G., Supko, J. G., Malspeis, L., Buckheit, R. W., Jr., Clanton, D., Bu, M., Graham, L., Schaeffer, C. A., Turpin, J. A., Domagala, J., Gogliotti, R., Bader, J. P., Halliday, S. M., Coren, L., Sowder, R. C., II, Arthur, L. O. & Henderson, L. E. (1995) Science 270, 1194-1197]. Rice and coworkers have proposed that the compounds act by "attacking" the two zinc fingers of HIV nucleocapsid protein. Shown here is evidence that low micromolar concentrations of the anti-HIV disulfide benzamides eject zinc from HIV nucleocapsid protein (NCp7) in vitro, as monitored by the zinc-specific fluorescent probe N-(6-methoxy-8-quinoyl)-p-toluenesulfonamide (TSQ). Structurally similar disulfide benzamides that do not inhibit HIV-1 in culture do not eject zinc, nor do analogs of the antiviral compounds with the disulfide replaced with a methylene sulfide. The kinetics of NCp7 zinc ejection by disulfide benzamides were found to be nonsaturable and biexponential, with the rate of ejection from the C-terminal zinc finger 7-fold faster than that from the N-terminal. The antiviral compounds were found to inhibit the zinc-dependent binding of NCp7 to HIV psi RNA, as studied by gel-shift assays, and the data correlated well with the zinc ejection data. Anti-HIV disulfide benzamides specifically eject NCp7 zinc and abolish the protein's ability to bind psi RNA in vitro, providing evidence for a possible antiretroviral mechanism of action of these compounds. Congeners of this class are under advanced preclinical evaluation as a potential chemotherapy for acquired immunodeficiency syndrome.

Homozygous Inactivating Mutation In Nanos3 In Two Sisters With Primary Ovarian Insufficiency.

Despite the increasing understanding of female reproduction, the molecular diagnosis of primary ovarian insufficiency (POI) is seldom obtained. The RNA-binding protein NANOS3 poses as an interesting candidate gene for POI since members of the Nanos family have an evolutionarily conserved function in germ cell development and maintenance by repressing apoptosis. We performed mutational analysis of NANOS3 in a cohort of 85 Brazilian women with familial or isolated POI, presenting with primary or secondary amenorrhea, and in ethnically-matched control women. A homozygous p.Glu120Lys mutation in NANOS3 was identified in two sisters with primary amenorrhea. The substituted amino acid is located within the second C2HC motif in the conserved zinc finger domain of NANOS3 and in silico molecular modelling suggests destabilization of protein-RNA interaction. In vitro analyses of apoptosis through flow cytometry and confocal microscopy show that NANOS3 capacity to prevent apoptosis was impaired by this mutation. The identification of an inactivating missense mutation in NANOS3 suggests a mechanism for POI involving increased primordial germ cells (PGCs) apoptosis during embryonic cell migration and highlights the importance of NANOS proteins in human ovarian biology.

Gene expression profile of the plant pathogen Fusarium graminearum under the antagonistic effect of Pantoea agglomerans

The pathogenic fungus Fusarium graminearum is an ongoing threat to agriculture, causing losses in grain yield and quality in diverse crops. Substantial progress has been made in the identification of genes involved in the suppression of phytopathogens by antagonistic microorganisms; however, limited information regarding responses of plant pathogens to these biocontrol agents is available. Gene expression analysis was used to identify differentially expressed transcripts of the fungal plant pathogen F. graminearum under antagonistic effect of the bacterium Pantoea agglomerans. A macroarray was constructed, using 1014 transcripts from an F. graminearum cDNA library. Probes consisted of the cDNA of F. graminearum grown in the presence and in the absence of P. agglomerans. Twenty-nine genes were either up (19) or down (10) regulated during interaction with the antagonist bacterium. Genes encoding proteins associated with fungal defense and/or virulence or with nutritional and oxidative stress responses were induced. The repressed genes coded for a zinc finger protein associated with cell division, proteins containing cellular signaling domains, respiratory chain proteins, and chaperone-type proteins. These data give molecular and biochemical evidence of response of F. graminearum to an antagonist and could help develop effective biocontrol procedures for pathogenic plant fungi.

AtPIN: Arabidopsis thaliana Protein Interaction Network

Background: Protein-protein interactions (PPIs) constitute one of the most crucial conditions to sustain life in living organisms. To study PPI in Arabidopsis thaliana we have developed AtPIN, a database and web interface for searching and building interaction networks based on publicly available protein-protein interaction datasets. Description: All interactions were divided into experimentally demonstrated or predicted. The PPIs in the AtPIN database present a cellular compartment classification (C(3)) which divides the PPI into 4 classes according to its interaction evidence and subcellular localization. It has been shown in the literature that a pair of genuine interacting proteins are generally expected to have a common cellular role and proteins that have common interaction partners have a high chance of sharing a common function. In AtPIN, due to its integrative profile, the reliability index for a reported PPI can be postulated in terms of the proportion of interaction partners that two proteins have in common. For this, we implement the Functional Similarity Weight (FSW) calculation for all first level interactions present in AtPIN database. In order to identify target proteins of cytosolic glutamyl-tRNA synthetase (Cyt-gluRS) (AT5G26710) we combined two approaches, AtPIN search and yeast two-hybrid screening. Interestingly, the proteins glutamine synthetase (AT5G35630), a disease resistance protein (AT3G50950) and a zinc finger protein (AT5G24930), which has been predicted as target proteins for Cyt-gluRS by AtPIN, were also detected in the experimental screening. Conclusions: AtPIN is a friendly and easy-to-use tool that aggregates information on Arabidopsis thaliana PPIs, ontology, and sub-cellular localization, and might be a useful and reliable strategy to map protein-protein interactions in Arabidopsis. AtPIN can be accessed at http://bioinfo.esalq.usp.br/atpin.