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Design of a zinc-finger hydrolase with a synthetic αββ protein.

ABSTRACT: Recent advances in protein design have opened avenues for the creation of artificial enzymes needed for biotechnological and pharmaceutical applications. However, designing efficient enzymes remains an unrealized ambition, as the design must incorporate a catalytic apparatus specific for the desired reaction. Here we present a de novo design approach to evolve a minimal carbonic anhydrase mimic. We followed a step-by-step design of first folding the main chain followed by sequence variation for substrate binding and catalysis. To optimize the fold, we designed an αββ protein based on a Zn-finger. We then inverse-designed the sequences to provide stability to the fold along with flexibility of linker regions to optimize Zn binding and substrate hydrolysis. The resultant peptides were synthesized and assessed for Zn and substrate binding affinity by fluorescence and ITC followed by evaluation of catalytic efficiency with UV-based enzyme kinetic assays. We were successful in mimicking carbonic anhydrase activity in a peptide of twenty two residues, using p-nitrophenyl acetate as a CO2 surrogate. Although our design had modest activity, being a simple structure is an advantage for further improvement in efficiency. Our approach opens a way forward to evolving an efficient biocatalyst for any industrial reaction of interest.

SUBMITTER: Srivastava KR

PROVIDER: S-EPMC4015931 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Synthetic zinc finger nuclease design and rapid assembly.

Project description:Engineered zinc finger nucleases (ZFNs) are a tool for genome manipulation that are of great interest to scientists in many fields. To meet the needs of researchers wishing to employ ZFNs, an inexpensive, rapid assembly procedure would be beneficial to laboratories that do not have access to the proprietary reagents often required for ZFN production. Using freely available sequence data derived from the Zinc Finger Targeter database, we developed a protocol for synthesis and directed insertion of user-defined ZFNs into a versatile plasmid expression system. This oligonucleotide-based isothermal DNA assembly protocol was used to determine whether we could generate functional nucleases capable of endogenous gene editing. We targeted the human α-l-iduronidase (IDUA) gene on chromosome 4, mutations of which result in the severe lysosomal storage disease mucopolysaccharidosis type I. In approximately 1 week we were able to design, assemble, and test six IDUA-specific ZFNs. In a single-stranded annealing assay five of the six candidates we tested performed at a level comparable to or surpassing previously reported ZFNs. One of the five subsequently showed nuclease activity at the endogenous genomic IDUA locus. To our knowledge, this is the first demonstration of in silico-designed, oligonucleotide-assembled, synthetic ZFNs, requiring no specialized templates or reagents that are capable of endogenous human gene target site activity. This method, termed CoDA-syn (context-dependent assembly-synthetic), should facilitate a more widespread use of ZFNs in the research community.

| S-EPMC4076977 | biostudies-literature

RNA features governing zinc finger antiviral protein sensitivity enable attenuated virus design

Project description:Synonymous recoding of viral genome can attenuate their replication, but can have pleiotropic effects, with multiple mechanisms contributing to attenuation. We set out to design recoded viral genomes whose attenuation was specific and conditional. The zinc finger antiviral protein (ZAP) recognizes CpG dinucleotides and targets CpG-rich RNAs for depletion, but RNA features such as CpG numbers, spacing and surrounding nucleotide composition that enable specific modulation by ZAP are undescribed. Using synonymously mutated HIV-1 genomes, we define several sequence features that govern ZAP sensitivity and stable attenuation. Using features defined using HIV-1, we then designed a mutant enterovirus A71 genome whose attenuation was also stable and strictly ZAP-dependent, both in cell culture and in mice. This conditionally attenuated enterovirus A71 elicited neutralizing antibodies that were protective against wild-type enterovirus 71 infection and disease. Elucidation of the determinants of ZAP sensitivity can thus enable the rational design of conditionally attenuated viral vaccines.

2022-07-26 | GSE208611 | GEO

Modular Synthetic Inverters from Zinc Finger Proteins and Small RNAs.

Project description:Synthetic zinc finger proteins (ZFPs) can be created to target promoter DNA sequences, repressing transcription. The binding of small RNA (sRNA) to ZFP mRNA creates an ultrasensitive response to generate higher effective Hill coefficients. Here we combined three "off the shelf" ZFPs and three sRNAs to create new modular inverters in E. coli and quantify their behavior using induction fold. We found a general ordering of the effects of the ZFPs and sRNAs on induction fold that mostly held true when combining these parts. We then attempted to construct a ring oscillator using our new inverters. Our chosen parts performed insufficiently to create oscillations, but we include future directions for improvement upon our work presented here.

| S-EPMC4757538 | biostudies-literature

Design of a zinc finger protein binding a sequence upstream of the A20 gene.

Project description:Artificial transcription factors (ATFs) are composed of DNA-binding and functional domains. These domains can be fused together to create proteins that can bind a chosen DNA sequence. To construct a valid ATF, it is necessary to design suitable DNA-binding and functional domains. The Cys2-His2 zinc finger motif is the ideal structural scaffold on which to construct a sequence-specific protein. A20 is a cytoplasmic zinc finger protein that inhibits nuclear factor kappa-B activity and tumor necrosis factor (TNF)-mediated programmed cell death. A20 has been shown to prevent TNF-induced cytotoxicity in a variety of cell types including fibroblasts, B lymphocytes, WEHI 164 cells, NIH 3T3 cells and endothelial cells.In order to design a zinc finger protein (ZFP) structural domain that binds specific target sequences in the A20 gene promoter region, the structure and sequence composition of this promoter were analyzed by bioinformatics methods. The target sequences in the A20 promoter were submitted to the on-line ZF Tools server of the Barbas Laboratory, Scripps Research Institute (TSRI), to obtain a specific 18 bp target sequence and also the amino acid sequence of a ZFP that would bind to it. Sequence characterization and structural modeling of the predicted ZFP were performed by bioinformatics methods. The optimized DNA sequence of this artificial ZFP was recombined into the eukaryotic expression vector pIRES2-EGFP to construct pIRES2-EGFP/ZFP-flag recombinants, and the expression and biological activity of the ZFP were analyzed by RT-PCR, western blotting and EMSA, respectively. The ZFP was designed successfully and exhibited biological activity.It is feasible to design specific zinc finger proteins by bioinformatics methods.

| S-EPMC2278136 | biostudies-literature

Myocardial ischemic preconditioning upregulated protein 1(Mipu1):zinc finger protein 667 - a multifunctional KRAB/C2H2 zinc finger protein.

Project description:Myocardial ischemic preconditioning upregulated protein 1 (Mipu1) is a newly discovered upregulated gene produced in rats during the myocardial ischemic preconditioning process. Mipu1 cDNA contains a 1824-base pair open reading frame and encodes a 608 amino acid protein with an N-terminal Krüppel-associated box (KRAB) domain and classical zinc finger C2H2 motifs in the C-terminus. Mipu1 protein is located in the cell nucleus. Recent studies found that Mipu1 has a protective effect on the ischemia-reperfusion injury of heart, brain, and other organs. As a nuclear factor, Mipu1 may perform its protective function through directly transcribing and repressing the expression of proapoptotic genes to repress cell apoptosis. In addition, Mipu1 also plays an important role in regulating the gene expression of downstream inflammatory mediators by inhibiting the activation of activator protein-1 and serum response element.

| S-EPMC4288486 | biostudies-literature

Design and Development of Artificial Zinc Finger Transcription Factors and Zinc Finger Nucleases to the hTERT Locus.

Project description:The ability to direct human telomerase reverse transcriptase (hTERT) expression through either genetic control or tunable regulatory factors would advance not only our understanding of the transcriptional regulation of this gene, but also potentially produce new strategies for addressing telomerase-associated disease. In this work, we describe the engineering of artificial zinc finger transcription factors (ZFTFs) and ZF nucleases (ZFNs) to target sequences within the hTERT promoter and exon-1. We were able to identify several active ZFTFs that demonstrate a broadly tunable response when screened by a cell-based transcriptional reporter assay. Using the same DNA-binding domains, we generated ZFNs that were screened in combinatorial pairs in cell-based extrachromosomal single-strand annealing (SSA) assays and in gene-targeting assays using stably integrated constructs. Selected ZFN pairs were tested for the ability to induce sequence changes in a Cel1 assay and we observed frequencies of genomic modification up to 18.7% at the endogenous hTERT locus. These screening strategies have pinpointed several ZFN pairs that may be useful in gene editing of the hTERT locus. Our work provides a foundation for using engineered ZF proteins (ZFPs) for modulation of the hTERT locus.Molecular Therapy - Nucleic Acids (2013) 2, e87; doi:10.1038/mtna.2013.12; published online 23 April 2013.

| S-EPMC3650244 | biostudies-other

Design of a colicin E7 based chimeric zinc-finger nuclease.

Project description:Colicin E7 is a natural bacterial toxin. Its nuclease domain (NColE7) enters the target cell and kills it by digesting the nucleic acids. The HNH-motif as the catalytic centre of NColE7 at the C-terminus requires the positively charged N-terminal loop for the nuclease activity-offering opportunities for allosteric control in a NColE7-based artificial nuclease. Accordingly, four novel zinc finger nucleases were designed by computational methods exploiting the special structural features of NColE7. The constructed models were subjected to MD simulations. The comparison of structural stability and functional aspects showed that these models may function as safely controlled artificial nucleases. This study was complemented by random mutagenesis experiments identifying potentially important residues for NColE7 function outside the catalytic region.

| S-EPMC4104000 | biostudies-literature

Zinc-finger antiviral protein inhibits XMRV infection.

Project description:BACKGROUND: The zinc-finger antiviral protein (ZAP) is a host factor that specifically inhibits the replication of certain viruses, including Moloney murine leukemia virus (MoMLV), HIV-1, and certain alphaviruses and filoviruses. ZAP binds to specific viral mRNAs and recruits cellular mRNA degradation machinery to degrade the target RNA. The common features of ZAP-responsive RNA sequences remain elusive and thus whether a virus is susceptible to ZAP can only be determined experimentally. Xenotropic murine leukemia virus-related virus (XMRV) is a recently identified ?-retrovirus that was originally thought to be involved in prostate cancer and chronic fatigue syndrome but recently proved to be a laboratory artefact. Nonetheless, XMRV as a new retrovirus has been extensively studied. Since XMRV and MoMLV share only 67.9% sequence identity in the 3'UTRs, which is the target sequence of ZAP in MoMLV, whether XMRV is susceptible to ZAP remains to be determined. FINDINGS: We constructed an XMRV-luc vector, in which the coding sequences of Gag-Pol and part of Env were replaced with luciferase-coding sequence. Overexpression of ZAP potently inhibited the expression of XMRV-luc in a ZAP expression-level-dependent manner, while downregulation of endogenous ZAP rendered cells more sensitive to infection. Furthermore, ZAP inhibited the spreading of replication-competent XMRV. Consistent with the previously reported mechanisms by which ZAP inhibits viral infection, ZAP significantly inhibited the accumulation of XMRV-luc mRNA in the cytoplasm. The ZAP-responsive element in XMRV mRNA was mapped to the 3'UTR. CONCLUSIONS: ZAP inhibits XMRV replication by preventing the accumulation of viral mRNA in the cytoplasm. Documentation of ZAP inhibiting XMRV helps to broaden the spectrum of ZAP's antiviral activity. Comparison of the target sequences of ZAP in XMRV and MoMLV helps to better understand the features of ZAP-responsive elements.

| S-EPMC3376128 | biostudies-literature

Analysis of the interaction between Zinc finger protein 179 (Znf179) and promyelocytic leukemia zinc finger (Plzf).

Project description:BackgroundZinc finger protein 179 (Znf179), also known as ring finger protein 112 (Rnf112), is a member of the RING finger protein family and plays an important role in neuronal differentiation. To investigate novel mechanisms of Znf179 regulation and function, we performed a yeast two-hybrid screen to identify Znf179-interacting proteins.ResultsUsing a yeast two-hybrid screen, we have identified promyelocytic leukemia zinc finger (Plzf) as a specific interacting protein of Znf179. Further analysis showed that the region containing the first two zinc fingers of Plzf is critical for its interaction with Znf179. Although the transcriptional regulatory activity of Plzf was not affected by Znf179 in the Gal4-dependent transcription assay system, the cellular localization of Znf179 was changed from cytoplasm to nucleus when Plzf was co-expressed. We also found that Znf179 interacted with Plzf and regulated Plzf protein expression.ConclusionsOur results showed that Znf179 interacted with Plzf, resulting in its translocation from cytoplasm to the nucleus and increase of Plzf protein abundance. Although the precise nature and role of the Znf179-Plzf interaction remain to be elucidated, both of these two genes are involved in the regulation of neurogenesis. Our finding provides further research direction for studying the molecular functions of Znf179.

| S-EPMC3878200 | biostudies-literature

Protein delivery using Cys2-His2 zinc-finger domains.

Project description:The development of new methods for delivering proteins into cells is a central challenge for advancing both basic research and therapeutic applications. We previously reported that zinc-finger nuclease proteins are intrinsically cell-permeable due to the cell-penetrating activity of the Cys2-His2 zinc-finger domain. Here, we demonstrate that genetically fused zinc-finger motifs can transport proteins and enzymes into a wide range of primary and transformed mammalian cell types. We show that zinc-finger domains mediate protein uptake at efficiencies that exceed conventional protein transduction systems and do so without compromising enzyme activity. In addition, we demonstrate that zinc-finger proteins enter cells primarily through macropinocytosis and facilitate high levels of cytosolic delivery. These findings establish zinc-finger proteins as not only useful tools for targeted genome engineering but also effective reagents for protein delivery.

| S-EPMC4519095 | biostudies-literature

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