Zinc finger proteins are abundant in the human proteome and are responsible for a variety of functions. The domains that constitute zinc finger proteins are compact spherical structures, each ...comprising approximately 30 amino acid residues, but they also have precise molecular factor functions: zinc binding and DNA recognition. Due to the biological importance of zinc finger proteins and their unique structural and functional properties, many artificial zinc finger proteins have been created and are expected to improve their functions and biological applications. In this study, we review previous studies on the redesign and application of artificial zinc finger proteins, focusing on the experimental results obtained by our research group. In addition, we systematically review various design strategies used to construct artificial zinc finger proteins and discuss in detail their potential biological applications, including gene editing. This review will provide relevant information to researchers involved or interested in the field of artificial zinc finger proteins as a potential new treatment for various diseases.
Cytochrome c (cyt c) is a stable protein that functions in a monomeric state as an electron donor for cytochrome c oxidase. It is also released to the cytosol when permeabilization of the ...mitochondrial outer membrane occurs at the early stage of apoptosis. For nearly half a century, it has been known that cyt c forms polymers, but the polymerization mechanism remains unknown. We found that cyt c forms polymers by successive domain swapping, where the C-terminal helix is displaced from its original position in the monomer and Met-heme coordination is perturbed significantly. In the crystal structures of dimeric and trimeric cyt c, the C-terminal helices are replaced by the corresponding domain of other cyt c molecules and Met80 is dissociated from the heme. The solution structures of dimeric, trimeric, and tetrameric cyt c were linear based on small-angle X-ray scattering measurements, where the trimeric linear structure shifted toward the cyclic structure by addition of PEG and (NH₄)₂HPO₄. The absorption and CD spectra of high-order oligomers (∼40 mer) were similar to those of dimeric and trimeric cyt c but different from those of monomeric cyt c. For dimeric, trimeric, and tetrameric cyt c, the ΔH of the oligomer dissociation to monomers was estimated to be about -20 kcal/mol per protomer unit, where Met-heme coordination appears to contribute largely to ΔH. The present results suggest that cyt c polymerization occurs by successive domain swapping, which may be a common mechanism of protein polymerization.
The use of membrane-permeable peptides as carrier vectors for the intracellular delivery of various proteins and macromolecules for modifying cellular function is well documented. Arginine-rich ...peptides, including those derived from human immunodeficiency virus 1 Tat protein, are among the representative classes of these vectors. The internalization mechanism of these vector peptides and their protein conjugates was previously regarded as separate from endocytosis, but more recent reevaluations have concluded that endocytosis is involved in their internalization. In this report, we show that the uptake of octa-arginine (R8) peptide by HeLa cells was significantly suppressed by the macropinocytosis inhibitor ethylisopropylamiloride (EIPA) and the F-actin polymerization inhibitor cytochalasin D, suggesting a role for macropinocytosis in the uptake of the peptide. In agreement with this we observed that treatment of the cells with R8 peptide induced significant rearrangement of the actin cytoskeleton. The internalization efficiency and contribution of macropinocytosis were also observed to have a dependency on the chain length of the oligoarginine peptides. Uptake of penetratin, another representative peptide carrier, was less sensitive to EIPA and penetratin did not have such distinct effects on actin localization. The above observations suggest that penetratin and R8 peptides have distinct internalization mechanisms.
The design of DNA‐binding proteins for the specific control of the gene expression is one of the big challenges for several research laboratories in the post‐genomic era. An artificial transcription ...factor with the desired DNA binding specificity could work as a powerful tool and drug to regulate the target gene. The zinc‐finger proteins, which typically contain many fingers linked in a tandem fashion, are some of the most intensively studied DNA‐binding proteins. In particular, the Cys2His2‐type zinc finger is one of the most common DNA‐binding motifs in eukaryotes. A simple mode of DNA recognition by the Cys2His2‐type zinc‐finger domain provides an ideal framework for designing proteins with new functions. Our laboratory has utilized several design strategies to create new zinc‐finger peptides/proteins by redesigning the Cys2His2‐type zinc‐finger motif. This review focuses on the aspects of design strategies, mainly from our recent results, for the creation of artificial zinc‐finger proteins, and discusses the possible application of zinc‐finger technology for gene regulation and gene therapy.
Designer zinc fingers: Zinc fingers are the DNA‐binding domains that occur in nature as part of transcription factors conferring DNA sequence specificity. The zinc‐finger motif has a stable and compact structure, and gives a versatile framework for the design of artificial zinc‐finger proteins. In this concept article, redesigned strategy and medicinal application of artificial zinc‐finger proteins are discussed. The picture shows the binding of the zinc‐finger proteins to the DNA consensus sequence.
Basic peptides such as human immunodeficiency virus type 1 (HIV-1) Tat-(48–60) and DrosophilaAntennapedia-(43–58) have been reported to have a membrane permeability and a carrier function for ...intracellular protein delivery. We have shown that not only Tat-(48–60) but many arginine-rich peptides, including HIV-1 Rev-(34–50) and octaarginine (Arg8), efficiently translocated through the cell membranes and worked as protein carriers (Futaki, S., Suzuki, T., Ohashi, W., Yagami, T., Tanaka, S., Ueda, K., and Sugiura, Y. (2001) J. Biol. Chem. 276, 5836–5840). Quantification and time course analyses of the cellular uptake of the above peptides by mouse macrophage RAW264.7, human cervical carcinoma HeLa, and simian kidney COS-7 cells revealed that Rev-(34–50) and Arg8 had a comparable translocation efficiency to Tat-(48–60). Internalization of Tat-(48–60) and Rev-(34–50) was saturable and inhibited by the excess addition of the other peptide. Typical endocytosis and metabolic inhibitors had little effect on the internalization. The uptake of these peptides was significantly inhibited in the presence of heparan sulfate or chondroitin sulfates A, B, and C. Treatment of the cells with the anti-heparan sulfate antibody or heparinase III also lowered the translocation of these peptides. These results strongly suggest that the arginine-rich basic peptides share a certain part of the internalization pathway.
Intracellular delivery of bioactive molecules using arginine-rich peptides, including oligoarginine and HIV-1 Tat peptides, is a recently developed technology. Here, we report a dramatic change in ...the methods of internalization for these peptides brought about by the presence of pyrenebutyrate, a counteranion bearing an aromatic hydrophobic moiety. In the absence of pyrenebutyrate, endocytosis plays a major role in cellular uptake. However, the addition of pyrenebutyrate results in direct membrane translocation of the peptides yielding diffuse cytosolic peptide distribution within a few minutes. Using this method, rapid and efficient cytosolic delivery of the enhanced green fluorescent protein (EGFP) was achieved in cells including rat hippocampal primary cultured neurons. Enhancement of bioactivity on the administration of anapoptosis-inducing peptide is also demonstrated. Thus, coupling arginine-rich peptides with this hydrophobic anion dramatically improved their ability to translocate cellular membranes, suggesting the great impact of this approach on exploring and controlling cell function.
We investigated the cell penetration of Sp1 zinc finger proteins (Sp1 ZF) and the mechanism
via
which the total cationic charge and distribution of cationic residues on the protein surface affect ...intracellular trafficking. Sp1 ZFs showed intrinsic cell membrane permeability. The intracellular transfer of Sp1 ZFs other than 1F3 was dependent on the total cationic charge. Investigation of the effect of cationic residue distribution on intracellular membrane permeability revealed that the cellular uptake of unfolded Zn
2+
-non-coordinating Ala mutants was lower than that of the wild type. Therefore, the total cationic charge and distribution of cationic residues on the protein played crucial roles in intracellular translocation. Mutational studies revealed that the two-dimensional cation cluster on the protein surface significantly improved their cellular uptake. This study will contribute to the design of artificial cargoes that can efficiently transport target substances into cells.
Two-dimensional cation clusters formed on the surface of proteins play an important role in their intracellular translocation.
A basic peptide derived from HIV-1 Tat has been reported to have the ability to translocate through cell membranes and to bring exogenous proteins into cells. We have demonstrated that these features ...could be observed among many arginine-rich peptides, and the presence of a ubiquitous internalization mechanism for arginine-rich oligopeptides has been suggested. In this report, we report that these features are also applicable to the peptides having branched-chain structures. Peptides that have arginine residues on four branched chains (R n )4 n (number of arginine residues) = 0−6 were prepared. Fluorescence microscopic observation revealed that the (R2)4 peptide exhibited the most efficient translocation. The dependence on the number of arginine residues of the translocation efficiency and cellular localization was also observed for the branched-chain peptides as was seen in the linear peptides. Quite interestingly, efficient translocation was also recognized in the (RG3R)4 peptide, where three glycine residues intervened between two arginine residues on each chain of (R2)4. The results strongly suggested that a linear structure was not indispensable for the translocation of arginine-rich peptides and that there could be considerable flexibility in the location of the arginine residue in the molecules.
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In this study, we sought to improve the hydrolytic activity of a His4-type single finger domain (f2), which was previously derived from the second finger domain (f2′) of the Sp1 zinc ...finger protein (Sp1wt), which has 3 tandem finger domains (f1′, f2′, and f3′). To this end, 2 His4-type single finger domains were generated by mutating 2 Cys residues participating in Zn(II) coordination with the His residues in the first (f1′) and third finger (f3′) domains of Sp1wt. Circular dichroism spectroscopy results showed that the first and second His4-type zinc finger domains (f1 and f2) adopted folded ββα structures in the presence of Zn(II), but that the third His4-type zinc finger domain (f3) did not. Non-FokI-type zinc finger nucleases containing 3 or 4 finger domains were also prepared by combining a His4-type zinc finger domain with the Sp1wt scaffold. We studied their DNA-binding abilities and hydrolytic activities against DNA oligonucleotides by performing gel-mobility-shift assays. The results showed that f1 had higher hydrolytic activity for a DNA oligonucleotide with a GC box (5′-GGG GCG GGG-3′), compared with that of f2, although both His4-type single finger domains had similar DNA-binding affinities. The difference in the hydrolytic activity between f1 and f2 was ascribed not only to the zinc coordinate structure, but also to its folding structure and the stability of finger domain.
The Cys2His2-type zinc finger is a common DNA binding motif that is widely used in the design of artificial zinc finger proteins. In almost all Cys2His2-type zinc fingers, position 4 of the α-helical ...DNA-recognition site is occupied by a Leu residue involved in formation of the minimal hydrophobic core. However, the third zinc finger domain of native Zif268 contains an Arg residue instead of the conserved Leu. Our aim in the present study was to clarify the role of this Arg in the formation of a stable domain structure and in DNA binding by substituting it with a Lys, Leu, or Hgn, which have different terminal side-chain structures. Assessed were the metal binding properties, peptide conformations, and DNA-binding abilities of the mutants. All three mutant finger 3 peptides exhibited conformations and thermal stabilities similar to the wild-type peptide. In DNA-binding assays, the Lys mutant bound to target DNA, though its affinity was lower than that of the wild-type peptide. On the other hand, the Leu and Hgn mutants had no ability to bind DNA, despite the similarity in their secondary structures to the wild-type. Our results demonstrate that, as with the Leu residue, the aliphatic carbon side chain of this Arg residue plays a key role in the formation of a stable zinc finger domain, and its terminal guanidinium group appears to be essential for DNA binding mediated through both electrostatic interaction and hydrogen bonding with DNA phosphate backbone.
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