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ABSTRACT: All change at the C terminus: We have established a novel methodology for the ribosomal synthesis of peptides featuring C-terminal cyclization and various modifications, including macrocyclization, by making use of genetic code reprogramming. The C-terminal amide modification of linear and cyclic peptides should enhance their physiological stabilities, and open up the possibility of developing new drug-like peptides. The C terminus of a peptide expressed by the translation apparatus generally ends in a carboxylate group. On the other hand, the C termini of some naturally occurring peptides have amide moieties instead of carboxylates, which are believed to give better biostability. Here, we describe a new strategy for the ribosomal synthesis of peptides featuring C-terminal lactam, thiolactone, and alkylamide units. The method was based on the concept of genetic code reprogramming involving the flexizymes (flexible tRNA acylation ribozymes) and the PURE (peptide synthesis using recombinant elements) system, in which vacant codons are reassigned to nonproteinogenic amino acids; this enabled us to convert the C termini of peptides into the above functionalities. We have also applied this method to the synthesis of a macrocyclic peptide closed by an amide bond formed between a lysine side chain and the peptide C terminus. This method thus offers us new opportunities to express various peptides with C-terminal modifications as well as macrocyclic peptides using the translation apparatus, and potentially to accelerate the discovery of peptidic drugs designed for various therapeutic targets.
ChemBioChem 05/2009; 10(7):1186-92. · 3.94 Impact Factor
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ABSTRACT: Here we report a new methodology for the synthesis of bicyclic peptides by using a reconstituted cell-free translation system under the reprogrammed genetic code. Cysteine (Cys) and three different nonproteinogenic amino acids, Cab, Aha, and Pgl, were simultaneously incorporated into a peptide chain. The first cyclization occurred between the chloroacetyl group of Cab and the sulfhydryl group in Cys in situ of translation, and the second cyclization on the side chains of Aha-Pgl via Cu(I)-catalyzed azide-alkyne cycloaddition was performed. This offers us a powerful means of mRNA-programmed synthesis of various peptides with uniform bicyclic scaffolds.
Journal of the American Chemical Society 07/2008; 130(23):7232-4. · 9.91 Impact Factor
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ABSTRACT: Here we report a new enabling technology for the synthesis of peptidase-resistant cyclic peptides by means of genetic code reprogramming involving the flexizyme (a tRNA acylation ribozyme) and PURE (a reconstituted cell-free translation) systems. In this work, we have developed a new nonproteinogenic amino acid bearing a chloroacetyl group in the side chain, which forms a physiologically stable thioether bond by intramolecular reaction with the sulfhydryl group of a Cys residue in the peptide chain upon translation. Significantly, this chemistry takes place spontaneously in situ of the translation solution, giving the corresponding cyclic peptides independent of ring sizes. We have used this method to convert human urotensin II, known as a potent vasoconstrictor, to its analogue containing a thioether bond, showing that this new analogue retains biological activity. Moreover, this peptide exhibits remarkable resistance against peptidases under reducing conditions. Thus, this technology offers a new means to accelerate the discovery of therapeutic peptidic drugs.
ACS Chemical Biology 05/2008; 3(4):241-9. · 6.45 Impact Factor
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ABSTRACT: The initiation codon dictates that the translation initiation event exclusively begins with methionine. We report here a new technology to reprogram the initiation event, where various amino acids and those bearing N (alpha)-acyl groups can be used as an initiator for peptide synthesis. The technology is built upon the concept of genetic code reprogramming, where methionine is depleted from the translation system and the initiation codon is reassigned to the desired amino acid. We have applied this technology to the synthesis of an antitumor cyclic peptide, G7-18NATE, closed by a physiologically stable bond, and it is also extended to the custom synthesis of its analogues with various ring sizes. Significantly, cyclization occurs spontaneously upon translation of the precursor linear peptides. To demonstrate the practicality of this methodology, we also prepared a small cyclic peptide library designated by 160 distinct mRNAs. Thus, this technology offers a new means to prepare a wide array of in vivo compatible cyclic peptide libraries for the discovery of peptidic drug candidates against various therapeutic targets.
ACS Chemical Biology 03/2008; 3(2):120-9. · 6.45 Impact Factor
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Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 11/2007; 52(13 Suppl):1643-8.
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ABSTRACT: Protein-based fluorescent and functional probes are widely used for real-time visualization, purification, and regulation of a variety of biological molecules. The protein-based probes can generally be targeted into subcellular compartments of eukaryotic cells by a particular short peptide sequence. Little is known, however, about the sequence that targets probes into the mitochondrial intermembrane space (IMS). To identify the IMS-targeting sequence, we developed a simple genetic screening method to discriminate the proteins localized in the IMS from those in the mitochondrial matrix, thereby revealing the minimum requisite sequence for the IMS targeting. An IMS-localized protein, Smac/DIABLO, was randomly mutated, and the mitochondrial localization of each mutant was analyzed. We found that the four residues of Ala-Val-Pro-Ile are required for IMS localization, and a sequence of these four residues fused with matrix-targeting signals is sufficient for targeting the Smac/DIABLO into the IMS. The sequence was shown to readily direct three dissimilar proteins of interest to the IMS, which will open avenues to elucidating the functions of the IMS in live cells.
ACS Chemical Biology 04/2007; 2(3):176-86. · 6.45 Impact Factor
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ABSTRACT: Here we describe a de novo tRNA acylation system consisting of artificial ribozymes. This system, the flexizyme (Fx) system, allows for the preparation of acyl-tRNAs with almost no limitation on the use of a variety of amino/hydroxy acids and tRNAs. To demonstrate its utility, we have carried out protein synthesis involving site-specific incorporation of nonnatural amino and hydroxy acids via amber or programmed frame-shift suppressions. We have also demonstrated peptide synthesis involving multiple nonnatural amino acids via sense codon suppression. The combination of the Fx system and appropriate cell-free translation is a powerful and flexible tool for mRNA-encoded synthesis of nonnatural polypeptides.
Nucleic Acids Symposium Series 02/2006;
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ABSTRACT: The appearance of the premature translation termination codons (PTCs) in the transcript is the major cause of human genetic diseases. PTC-containing transcripts are rapidly degraded through nonsense-mediated decay (NMD) pathway. If such mRNA transcripts were translated in frame like normal transcripts, it would afford not only restoration of the level of full-length protein but also prevention of mRNA degradation by the NMD pathway. Here we describe a novel approach to read through PTC-containing mRNAs using suppressor tRNA that is introduced to cells by transfection. Luciferase reporter gene assay showed that nonsense and four-base codons were suppressed by the corresponding suppressor tRNAs derived from human tRNA(Ser). We also demonstrated that transfection of the suppressor tRNA to Ullrich disease fibroblasts, possessing a frameshift mutation in the collagen VI alpha2 gene, induced the upregulation of the collagen VI alpha2 mRNA and accumulation of the collagen VI protein. PTC suppression potentially provides a novel therapeutic means to rescue various PTC-related diseases.
Nucleic Acids Symposium Series 02/2006;
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ABSTRACT: Bioactive peptides isolated from natural sources have diverse acyl groups on the N-terminus. It is difficult to synthesize these peptides in vitro translation system because ribosomal peptide synthesis generally limits the N-terminal group to be N-formylmethionine (fMet). To overcome this restriction, we developed a novel methodology for the ribosomal synthesis of peptides having various terminal N-acyl groups with desired amino acids. In this methodology, two technologies, Flexizyme system consisting of artificial ribozymes and a reconstitute E. coli cell-free translation system (PURE system), were used. First, an amino acid carrying a desired N-acyl group was charged onto an initiation tRNA by the Flexizyme system. The addition of this N-acyl-aminoacyl-tRNA (N-acyl-aa-tRNA) to the PURE system allowed us to initiate the peptide synthesis with the designated N-acyl-amino acid. By means of this methodology, the translation was exclusively initiated by various N-terminal acyl groups as well as amino acids without contamination of N-formylmethionine.
Nucleic Acids Symposium Series 02/2006;
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ABSTRACT: The compartments of eukaryotic cells maintain a distinct protein composition to perform a variety of specialized functions. We developed a new method for identifying the proteins that are transported to the endoplasmic reticulum (ER) in living mammalian cells. The principle is based on the reconstitution of two split fragments of enhanced green fluorescent protein (EGFP) by protein splicing with DnaE from Synechocystis PCC6803. Complementary DNA (cDNA) libraries fused to the N-terminal halves of DnaE and EGFP are introduced in mammalian cells with retroviruses. If an expressed protein is transported into the ER, the N-terminal half of EGFP meets its C-terminal half in the ER, and full-length EGFP is reconstituted by protein splicing. The fluorescent cells are isolated using fluorescence-activated cell sorting and the cDNAs are sequenced. The developed method was able to accurately identify cDNAs that encode proteins transported to the ER. We identified 27 novel proteins as the ER-targeting proteins. The present method overcomes the limitation of the previous GFP- or epitope-tagged methods, using which it was difficult to identify the ER-targeting proteins in a high-throughput manner.
Nucleic Acids Research 02/2005; 33(4):e34. · 8.03 Impact Factor
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ABSTRACT: The control of intricate networks within eukaryotic cells relies on differential compartmentalization of proteins. We have developed a method that allows rapid identification of novel proteins compartmentalized in mitochondria by screening large-scale cDNA libraries. The principle is based on reconstitution of split-enhanced green fluorescent protein (EGFP) by protein splicing of DnaE derived from Synechocystis sp. PCC6803. The cDNA libraries are expressed in mammalian cells following infection with retrovirus. If a test protein contains a functional mitochondrial targeting signal (MTS), it translocates into the mitochondrial matrix, where EGFP is then formed by protein splicing. The cells harboring this reconstituted EGFP are screened rapidly by fluorescence-activated cell sorting, and the cDNAs are isolated and identified from the cells. The analysis of 258 cDNAs revealed various MTSs, among which we identified new transcripts corresponding to mitochondrial proteins. This method should provide a means to map proteins distributed within intracellular organelles in a broad range of different tissues and disease states.
Nature Biotechnology 04/2003; 21(3):287-93. · 23.27 Impact Factor
