Feedback Response to Selective Depletion of Endogenous Carbon Monoxide in the Blood.

The physiological roles of endogenous carbon monoxide (CO) have not been fully understood because of the difficulty in preparing a loss-of-function phenotype of this molecule. Here, we have utilized in vivo CO receptors, hemoCDs, which are the supramolecular 1:1 inclusion complexes of meso-tetrakis(4-sulfonatophenyl)porphinatoiron(II) with per-O-methylated ß-cyclodextrin dimers. Three types of hemoCDs (hemoCD1, hemoCD2, and hemoCD3) that exhibit different CO-affinities have been tested as CO-depleting agents in vivo. Intraperitoneally administered hemoCD bound endogenous CO within the murine circulation, and was excreted in the urine along with CO in an affinity-dependent manner. The sufficient administration of hemoCD that has higher CO-affinity than hemoglobin (Hb) produced a pseudoknockdown state of CO in the mouse in which heme oxygenase-1 (HO-1) was markedly induced in the liver, causing the acceleration of endogenous CO production to maintain constant CO-Hb levels in the blood. The contents of free hemin and bilirubin in the blood plasma of the treated mice significantly increased upon removal of endogenous CO by hemoCD. Thus, a homeostatic feedback model for the CO/HO-1 system was proposed as follows: HemoCD primarily removes CO from cell-free CO-Hb. The resulting oxy-Hb is quickly oxidized to met-Hb by oxidant(s) such as hydrogen peroxide in the blood plasma. The met-Hb readily releases free hemin that directly induces HO-1 in the liver, which metabolizes the hemin into iron, biliverdin, and CO. The newly produced CO binds to ferrous Hb to form CO-Hb as an oxidation-resistant state. Overall, the present system revealed the regulatory role of CO for maintaining the ferrous/ferric balance of Hb in the blood.

Zn(II) binding and DNA binding properties of ligand-substituted CXHH-type zinc finger proteins.

CCHH-type zinc fingers are among the most common DNA binding motifs found in eukaryotes. In a previous report, we substituted the second ligand cysteine residue with aspartic acid, producing a Zn(II)-responsive transcription factor; this indicates that a ligand substitution is a possible design target of an engineered zinc finger peptide. Despite the importance of Zn(II) binding with respect to the folding and DNA binding properties of a zinc finger peptide, no study about the effects of ligand substitution on both Zn(II) binding and DNA binding properties has been reported. Here, we substituted a conserved cysteine (C) with other zinc-coordinated amino acid residues, histidine (H), aspartic acid (D), and glutamic acid (E), to create CXHH-type zinc finger peptides (X = C, H, D, and E). The Zn(II)-dependent conformational change was observed in all peptides; however, the Zn(II) binding affinity and metal coordination geometry of the peptides were different. Gel mobility shift assays showed that the Zn(II)-bound forms of the ligand-substituted derivatives retain DNA binding ability, while the DNA binding affinity decreased in the following manner: CCHH > CDHH > CEHH ≫ CHHH. The DNA binding sequence preferences of the ligand-substituted derivatives were similar to that of the wild type in the context of the full three-finger DNA-binding domain of transcription factor Zif268. These results indicate that artificial zinc finger proteins with various DNA binding affinities that respond to a diverse range of Zn(II) concentrations can be designed by substituting the Zn(II) ligand.

Non-FokI-based zinc finger nucleases.

The design of functional proteins is one of the most challenging areas of protein research. We have constructed zinc finger peptides with metal-dependent hydrolytic abilities by mutating the zinc ligands in classical zinc fingers, without the need to add a FokI or other DNA cleavage domain. The designed peptides acquired DNA cleavage ability successfully, retaining the proper zinc finger folding and DNA targeting ability. We have also succeeded in site-specific DNA cleavage in the presence of cerium ions by introducing a lanthanide ion-binding loop as a linker of zinc finger motifs.

Alpha-helical linker of an artificial 6-zinc finger peptide contributes to selective DNA binding to a discontinuous recognition sequence.

Although many zinc finger motifs have been developed to recognize specific DNA triplets, a rational way to selectively skip a particular non-recognized gap in the DNA sequence has never been established. We have now created a 6-zinc finger peptide with an alpha-helix linker, Sp1ZF6(EAAAR)4, which selectively binds to the discontinuous recognition sites in the same phase (10 bp gap) against the opposite phase (5 bp gap) of the DNA helix. The linker peptide (EAAAR)4 forms an alpha-helix structure stabilized by salt bridges, and the helical length is estimated to be about 30 A, corresponding to that of the 10 bp DNA. The gel shift assays demonstrate that Sp1ZF6(EAAAR)4 preferably binds to the 10 bp-gapped target rather than the 5 bp-gapped target. The CD spectra show that the alpha-helical content of the (EAAAR)4 linker is higher in the complex with the 10 bp-gapped target than in the complex with the 5 bp-gapped target. The present results indicate that the alpha-helical linker is suitable for binding to the recognition sites in the same phase and that the linker induces the loss of binding affinity to the recognition sites with the opposite phase. The engineering of a helix-structured linker in the 6-zinc finger peptides should be one of the most promising approaches for selectively targeting discontinuous recognition sites depending on their phase situations.

Interaction of arginine-rich peptides with membrane-associated proteoglycans is crucial for induction of actin organization and macropinocytosis.

Arginine-rich peptides, including octaarginine (R8), HIV-1 Tat, and branched-chain arginine-rich peptides, belong to one of the major classes of cell-permeable peptides which deliver various proteins and macromolecules to cells. The importance of the endocytic pathways has recently been demonstrated in the cellular uptake of these peptides. We have previously shown that macropinocytosis is one of the major pathways for cellular uptake and that organization of the F-actin accompanies this process. In this study, using proteoglycan-deficient CHO cells, we have demonstrated that the membrane-associated proteoglycans are indispensable for the induction of the actin organization and the macropinocytic uptake of the arginine-rich peptides. We have also demonstrated that the cellular uptake of the Tat peptide is highly dependent on heparan sulfate proteoglycan (HSPG), whereas the R8 peptide uptake is less dependent on HSPG. This suggests that the structure of the peptides may determine the specificity for HSPG, and that HSPG is not the sole receptor for macropinocytosis. Comparison of the HSPG specificity of the branched-chain arginine-rich peptides in cellular uptake has suggested that the charge density of the peptides may determine the specificity. The activation of the Rac protein and organization of the actin were observed within a few minutes after the peptide treatment. These data strongly suggest the possibility that the interaction of the arginine-rich peptides with the membrane-associated proteoglycans quickly activates the intracellular signals and induces actin organization and macropinocytotis.

Acid wash in determining cellular uptake of Fab/cell-permeating peptide conjugates.

Successful intracellular delivery of various bioactive molecules has been reported using cell-permeating peptides (CPPs) as delivery vectors. To determine the effects of CPPs on the cellular uptake of immunoglobulin Fab fragment, conjugates of a radio-iodinated Fab fragment with CPPs (CPP-(125)I-Fab) derived from HIV-1 TAT, HIV-1 REV, and Antennapedia (ANP) were prepared. These vectors are rich in basic amino acids, and their strong adsorption on cell surfaces often results in overestimation of internalized peptides. Cell wash with an acidic buffer (0.2M glycine-0.15M NaCl, pH 3.0) was thus employed in this study to remove cell-surface adsorbed CPP-(125)I-Fab conjugates. This procedure enabled clearer understanding of the methods of internalization of CPP-(125)I-Fab conjugates. The kinetics of internalization of REV-(125)I-Fab conjugate was rapid, and a considerable fraction of REV-(125)I-Fab was taken up by HeLa cells as early as 5 min after administration. It was also shown that cellular uptake of these conjugates was significantly inhibited in the presence of endocytosis/ macropinocytosis inhibitors, in the order REV-(125)I-Fab > or = TAT-(125)I-Fab > or = ANP-(125)I-Fab; this order was the same as for effectiveness of intracellular delivery. Simultaneous cell washing with phosphate-buffered saline (PBS) and this acidic buffer effectively separated the internalized conjugates from the cell-surface-adsorbed ones, and considerable differences were observed in these amounts dependent on the employed CPPs.

Design and synthesis of artificial zinc finger proteins.

Of the DNA-binding motifs, the (Cys)2(His)2-type zinc finger motif has great potential for manipulation. The zinc finger motif offers an attractive framework for the design of novel DNA-binding proteins. Specially, a structure-based design strategy is valuable for the creation of new artificial zinc finger proteins that have novel DNA-binding properties, namely, long-DNA recognition, DNA bending, and AT-rich sequence recognition. Herein, new strategies for the design of multi-zinc finger proteins for the recognition of a target DNA sequence, a DNA-bending zinc finger protein, a (His)4-type zinc finger protein, and an AT-recognizing zinc finger protein are described based on recent experimental results.

Direct and rapid cytosolic delivery using cell-penetrating peptides mediated by pyrenebutyrate.

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.

Transmission of extramembrane conformational change into current: construction of metal-gated ion channel.

Interaction with Fe(III) induces the reversible conformational switch of the extramembrane segment in the artificial receptor channel, which is transmitted into membranes as an increase in channel current (ion flux).

Effects of cell-permeating peptide binding on the distribution of 125I-labeled Fab fragment in rats.

The peptides comprising the sequence of HIV-1 Tat protein (positions 48-60), Antennapedia (positions 43-58), and HIV-1 Rev protein (positions 34-50) are known to be cell-permeating. In this study, we examined how the distribution of Fab fragments in rats is affected by conjugation with these peptides. Fab fragment was iodinated by a chloramine-T method and then chemically conjugated with cell-permeating peptide. The complex of 125I-Fab and cell-permeating peptide was administered to male rats intravenously at a dose of 1 mg/kg, and whole-body autoradiography was performed at 4 and 24 h after administration. The patterns of distribution of 125I-Fab exhibited remarkable variation depending on the cell-permeating peptide used. In particular, at 4 h, high concentrations of radioactivity were observed in the spleen, adrenal gland, renal medulla, and liver with Rev peptide-Fab complex, in the liver and spleen with Tat peptide-Fab complex, and in the spleen, adrenal gland, and liver with Antennapedia peptide-Fab complex. Even at 24 h, high concentrations of radioactivity were still observed in the spleen and renal medulla of rat with Rev peptide-Fab complex, and in the spleen and renal cortex of rat with Antennapedia peptide-Fab complex. These findings demonstrate that the patterns of distribution of peptide-125I-Fab complexes can be modulated by selection of cell-penetrating peptides. Moreover, the patterns of retention of peptide-125I-Fab complexes in internal organs also differed at 24 h after administration. These findings provide valuable information for the development of novel antibody pharmaceuticals and therapeutic systems.

Unique features of a pH-sensitive fusogenic peptide that improves the transfection efficiency of cationic liposomes.

BACKGROUND: One of the critical steps in intracellular gene delivery using cationic liposomes is the endosomal escape of the plasmid/liposome complexes to the cytosol. The addition of GALA, a pH-sensitive fusogenic peptide, is a promising method to accelerate this step in order to enhance the expression of the desired proteins. Detailed studies on the methods of enhancement would broaden the horizon of its application. METHODS: Using representative commercially available cationic liposomes (Lipofectin, Lipofectamine, and Lipofectamine 2000), the effects of GALA on transfection efficiency were studied by luciferase assay and confocal microscopic observations. RESULTS: A concentration-dependent increase in the transfection efficiency was observed for GALA. Addition of 0.1 microM GALA to the plasmid/liposome complex significantly increased the transfection efficiency, especially in the case of Lipofectin, but higher concentration of GALA decreased transfection efficiency. Successful reduction in the liposomal dosage was attained by employing GALA while maintaining a high transfection efficiency. Interestingly, although the transfection efficiency was higher in the presence of GALA, a lower amount of the plasmid DNA was taken up by the cells. Confocal microscopic observations of the rhodamine-labeled plasmid did not show a significant difference in the cellular localization among cells incubated in the presence or absence of GALA, suggesting that a slight increase in GALA-induced release of the plasmid to the cytosol may cause a significant change in the transfection efficiency. CONCLUSION: The unique features of GALA to mediate improved transfection efficiencies were identified.

An artificial six-zinc finger peptide with polyarginine linker: selective binding to the discontinuous DNA sequences.

Artificial DNA binding peptides recognizing separated sequences would expand varieties of the target genes for desirable transcriptional control. Here we demonstrated that polyarginine linker between two 3-zinc finger domains gives DNA binding selectivity to the separated target sequences. We created a six-zinc finger peptide, Sp1ZF6(Arg)8, by connecting two DNA binding domains of transcription factor Sp1 with a bulky and cationic polyarginine linker. The DNA binding properties to continuous and discontinuous target sequences were examined and compared to those of Sp1ZF6(Gly)10 containing a flexible and neutral polyglycine linker. The dissociation constants indicate that Sp1ZF6(Arg)8 has an obvious DNA binding preference to discontinuous target sequences but not Sp1ZF6(Gly)10. Footprinting analyses also showed that Sp1ZF6(Arg)8 binds properly only to the discontinuous target sites, while Sp1ZF6(Gly)10 does not distinguish them. The results provide helpful information for linker design of future zinc finger peptides to various states of DNA as gene expression regulators.

Site-specific DNA cleavage by artificial zinc finger-type nuclease with cerium-binding peptide.

The addition of a new function to native proteins is one of the most attractive protein-based designs. In this study, we have converted a C(2)H(2)-type zinc finger as a DNA-binding motif into a novel zinc finger-type nuclease by connecting two distinct zinc finger proteins (Sp1 and GLI) with a functional linker possessing DNA cleavage activity. As a DNA cleavage domain, we chose an analogue of the metal-binding loop (12 amino acid residues), peptide P1, which has been reported to exhibit a strong binding affinity for a lanthanide ion and DNA cleavage ability in the presence of Ce(IV). Our newly designed nucleases, Sp1(P1)GLI and Sp1(P1G)GLI, can strongly bind to a lanthanide ion and show a unique DNA cleavage pattern, in which certain positions between the two DNA-binding sites are specifically cleaved. The present result provides useful information for expanding the design strategy for artificial nucleases.

Swapping of the beta-hairpin region between Sp1 and GLI zinc fingers: significant role of the beta-hairpin region in DNA binding properties of C2H2-type zinc finger peptides.

The recent design strategy of zinc finger peptides has mainly focused on the alpha-helix region, which plays a direct role in DNA recognition. On the other hand, the study of non-DNA-contacting regions is extremely scarce. By swapping the beta-hairpin regions between the Sp1 and GLI zinc fingers, in this study, we investigated how the beta-hairpin region of the C(2)H(2)-type zinc finger peptides contributes to the DNA binding properties. Surprisingly, the Sp1 mutant with the GLI-type beta-hairpin had a higher DNA binding affinity than that of the wild-type Sp1. The result of the DNase I footprinting analyses also showed the change in the DNA binding pattern. In contrast, the GLI zinc finger completely lost DNA binding ability as a result of exchanging the beta-hairpin region. These results strongly indicate that the beta-hairpin region appears to function as a scaffold and has an important effect on the DNA binding properties of the C(2)H(2)-type zinc finger peptides.

Control of peptide structure and recognition by Fe(III)-induced helix destabilization.

Helical peptide segments that change their conformation due to external stimuli have often been employed in peptide-based molecular devices and materials. Using helices containing a pair of the iminodiacetic acid derivatives of lysine (Ida), we show that metal-induced helix destabilization is a promising approach to functional switching, especially for helices that are intrinsically stable. By i and i + 2 positioning of the Ida residues in a 17-residue model peptide, a significant decrease in the helical content was observed by the addition of Fe(III), whereas Fe(II) had no influence on the stability of the helix. The possibility of redox control of the helical structure was exemplified by the reduction of Fe(III) to Fe(II) using Na(2)S(2)O(4) followed by the subsequent reoxidation. Mutual recognition of the transcription factor Jun-derived leucine-zipper peptide segment with the Fos leucine-zipper segment containing Ida residues was also modulated in the presence of Fe(III).

Creation and characteristics of unnatural CysHis(3)-type zinc finger protein.

To investigate the properties of unnatural zinc finger peptides with CysHis(3)-type ligand combinations, the HCHH- and CHHH-type zinc finger proteins (zf(HCHH) and zf(CHHH), respectively) were created by mutating Cys to His in the Cys(2)His(2)-type zinc finger of the transcription factor Sp1 (zf(CCHH)). The CD measurements clearly show that the single-finger CysHis(3)-type zinc finger peptides (zf(HCHH)f2 and zf(CHHH)f2) are folded by complexation with Zn(II). From the gel mobility shift assays, the CysHis(3)-type zinc finger proteins (zf(HCHH) and zf(CHHH)) evidently bind to the GC-box DNA, though the DNA binding affinity is lower than that of the wild CCHH-type zinc finger protein. Furthermore, the zf(HCHH)f2 and zf(CHHH)f2 peptides catalyze the hydrolysis of the 4-nitrophenyl acetate in contrast to the catalytically inactive zf(CCHH) peptide. This is the first study of the CysHis(3)-type zinc finger proteins and also provides useful information for redesigning artificial metalloproteins.

Cellular uptake of arginine-rich peptides: roles for macropinocytosis and actin rearrangement.

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.

Arginine carrier peptide bearing Ni(II) chelator to promote cellular uptake of histidine-tagged proteins.

Arginine-rich peptide-mediated protein delivery into living cells is a novel technology for controlling cell functions with therapeutic potential. In this report, a novel approach for the intracellular delivery of histidine-tagged proteins was introduced where a Ni(II) chelate of octaarginine peptide bearing nitrilotriacetic acid [R8-NTA-Ni(II)] was used as a membrane-permeable carrier molecule. Significant internalization of histidine-tagged enhanced green fluorescent protein (EGFP) into HeLa cells was observed by confocal microscopic observation in the presence of R8-NTA-Ni(II). Nuclear condensation characteristic in apoptotic cell death was also induced in the cells treated with a histidine-tagged apoptosis-inducing peptide [pro-apoptotic domain peptide (PAD)], indicating that the cargo molecules really went through the membrane to reach the cytosol. The apoptosis-inducing activity of the peptide thus delivered was compared with that of the PAD peptide covalently connected with the octaarginine peptide.

Exchange of histidine spacing between Sp1 and GLI zinc fingers: distinct effect of histidine spacing-linker region on DNA binding.

In the DNA recognition mode of C(2)H(2)-type zinc fingers, the finger-finger connection region, consisting of the histidine spacing (HX(3-5)H) and linker, would be important for determining the orientation of the zinc finger domains. To clarify the influence of spacing between two ligand histidines in the DNA binding, we exchanged the histidine spacing between Sp1 and GLI zinc fingers, which have an HX(3)H-TGEKK linker (typical) and an HX(4)H-SNEKP linker (atypical), respectively. A significant decrease in the DNA binding affinity and specificity is found in Sp1-type peptides, whereas GLI-type peptides show a mild reduction. To evaluate the effect of the linker characteristics, we further designed Sp1-type mutants with an SNEKP linker. As a result, the significant effect of the histidine spacing in Sp1-type peptides was reduced. These results demonstrate that (1) the histidine spacing significantly affects the DNA binding of zinc finger proteins and (2) the histidine spacing and the following linker regions are one effective target for regulating the DNA recognition mode of zinc finger proteins.