Publications by authors named "Hironobu Hojo"

75 Publications

Total synthesis and structural characterization of caveolin-1.

Angew Chem Int Ed Engl 2021 Apr 6. Epub 2021 Apr 6.

RIKEN, Cluster for Pioneering Research, JAPAN.

Caveolin-1, which is an essential protein for caveola formation, was chemically synthesized. It is composed of 177 amino acid residues, triply palmitoylated at the C-terminus region and supposed to be inserted into the lipid bilayer forming a V-shaped structure in the middle of the polypeptide chain. The entire sequence was divided into 5 peptide segments and each of them was synthesized by the solid-phase method. To improve the solubility of the C-terminal region, the O-acyl isopeptide structures were incorporated. After the ligation by the thioester method and the introduction of the palmitoyl groups, all the protecting groups were removed and the isopeptide structures were converted to the native peptide bond. Finally, the obtained polypeptide was successfully inserted into the bicelle, thus showing the success of the synthesis.
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http://dx.doi.org/10.1002/anie.202100826DOI Listing
April 2021

Chemical synthesis of ferredoxin with 4 selenocysteine residues using a segment condensation method.

Chem Commun (Camb) 2020 Nov 29;56(91):14239-14242. Epub 2020 Oct 29.

Institute for Protein Research, Osaka University, Osaka 565-0871, Japan.

Ferredoxin (Fd) is an electron carrier protein containing a [2Fe-2S] cluster. In this paper, we synthesized Se-Fd, in which four Cys residues coordinated to the cluster are substituted to selenocysteine. After the one-pot segment coupling by the thioester method, followed by deprotection and cluster loading, the desired Se-Fd was successfully obtained.
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http://dx.doi.org/10.1039/d0cc06252aDOI Listing
November 2020

Copper stabilizes antiparallel β-sheet fibrils of the amyloid β40 (Aβ40)-Iowa variant.

J Biol Chem 2020 07 6;295(27):8914-8927. Epub 2020 May 6.

National Synchrotron Light Source II, Brookhaven National Laboratory, Upton, New York, USA; Department of Chemistry, Laufer Center for Physical and Quantitative Biology, Stony Brook University, Stony Brook, New York, USA.

Cerebral amyloid angiopathy (CAA) is a vascular disorder that primarily involves deposition of the 40-residue-long β-amyloid peptide (Aβ40) in and along small blood vessels of the brain. CAA is often associated with Alzheimer's disease (AD), which is characterized by amyloid plaques in the brain parenchyma enriched in the Aβ42 peptide. Several recent studies have suggested a structural origin that underlies the differences between the vascular amyloid deposits in CAA and the parenchymal plaques in AD. We previously have found that amyloid fibrils in vascular amyloid contain antiparallel β-sheet, whereas previous studies by other researchers have reported parallel β-sheet in fibrils from parenchymal amyloid. Using X-ray fluorescence microscopy, here we found that copper strongly co-localizes with vascular amyloid in human sporadic CAA and familial Iowa-type CAA brains compared with control brain blood vessels lacking amyloid deposits. We show that binding of Cu(II) ions to antiparallel fibrils can block the conversion of these fibrils to the more stable parallel, in-register conformation and enhances their ability to serve as templates for seeded growth. These results provide an explanation for how thermodynamically less stable antiparallel fibrils may form amyloid in or on cerebral vessels by using Cu(II) as a structural cofactor.
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http://dx.doi.org/10.1074/jbc.RA119.011955DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7335782PMC
July 2020

Two distinct modes of DNMT1 recruitment ensure stable maintenance DNA methylation.

Nat Commun 2020 03 6;11(1):1222. Epub 2020 Mar 6.

Division of Cancer Cell Biology, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, Japan.

Stable inheritance of DNA methylation is critical for maintaining differentiated phenotypes in multicellular organisms. We have recently identified dual mono-ubiquitylation of histone H3 (H3Ub2) by UHRF1 as an essential mechanism to recruit DNMT1 to chromatin. Here, we show that PCNA-associated factor 15 (PAF15) undergoes UHRF1-dependent dual mono-ubiquitylation (PAF15Ub2) on chromatin in a DNA replication-coupled manner. This event will, in turn, recruit DNMT1. During early S-phase, UHRF1 preferentially ubiquitylates PAF15, whereas H3Ub2 predominates during late S-phase. H3Ub2 is enhanced under PAF15 compromised conditions, suggesting that H3Ub2 serves as a backup for PAF15Ub2. In mouse ES cells, loss of PAF15Ub2 results in DNA hypomethylation at early replicating domains. Together, our results suggest that there are two distinct mechanisms underlying replication timing-dependent recruitment of DNMT1 through PAF15Ub2 and H3Ub2, both of which are prerequisite for high fidelity DNA methylation inheritance.
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http://dx.doi.org/10.1038/s41467-020-15006-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7060239PMC
March 2020

Modern Peptide and Protein Chemistry: Reaching New Heights.

J Org Chem 2020 02;85(3):1328-1330

Leibniz-Institut fur Molekulare Pharmakologie , Robert-Roessle-Strasse 10 , Berlin 13125 , Germany.

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http://dx.doi.org/10.1021/acs.joc.0c00104DOI Listing
February 2020

On-resin synthesis of cyclic peptides via tandem N-to-S acyl migration and intramolecular thiol additive-free native chemical ligation.

Chem Commun (Camb) 2020 Jan;56(6):956-959

Química Farmacéutica, Departamento de Química Orgánica, Facultad de Química, Universidad de la República, General Flores 2124, CC 1157, Montevideo, Uruguay.

On-resin intramolecular native chemical ligation (NCL) assisted by N-ethylcysteine using Fmoc/SPPS to obtain cyclic peptides is described. N-terminal cysteine-containing peptides were subjected to NCL conditions leading to cyclization-cleavage reactions and consecutive S → N shift, rendering cyclic peptides in good yields and purities. The compounds were evaluated against P. falciparum 3D7.
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http://dx.doi.org/10.1039/c9cc07783aDOI Listing
January 2020

One Step Synthesis of Fmoc-Aminoacyl--alkylcysteine via the Ugi Four-Component Condensation Reaction.

J Org Chem 2020 02 16;85(3):1458-1465. Epub 2019 Dec 16.

Institute for Protein Research , Osaka University , Yamadaoka 3-2 , Suita 565-0871 , Japan.

A prompt preparation method of the Fmoc-aminoacyl--alkylcysteine dipeptide by an Ugi four-component condensation reaction is described. Through a reaction with a commercially available Fmoc-amino acid, an amine, an isocyanide, and a mercaptoacetaldehyde derivative, one step synthesis of dipeptides containing 20 kinds of natural amino acid residues was achieved, which avoided the problematic N-alkylation of -tritylcysteine and its coupling reaction. The dipeptide was applied to the Fmoc-solid-phase peptide synthesis, and peptide thioesters were successfully obtained in high efficiency via -alkylcysteine (NAC)-assisted thioesterification.
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http://dx.doi.org/10.1021/acs.joc.9b02433DOI Listing
February 2020

Enhanced processivity of Dnmt1 by monoubiquitinated histone H3.

Genes Cells 2020 Jan 3;25(1):22-32. Epub 2019 Dec 3.

Laboratory of Epigenetics, Institute for Protein Research, Osaka University, Suita, Japan.

DNA methylation controls gene expression, and once established, DNA methylation patterns are faithfully copied during DNA replication by the maintenance DNA methyltransferase Dnmt1. In vivo, Dnmt1 interacts with Uhrf1, which recognizes hemimethylated CpGs. Recently, we reported that Uhrf1-catalyzed K18- and K23-ubiquitinated histone H3 binds to the N-terminal region (the replication focus targeting sequence, RFTS) of Dnmt1 to stimulate its methyltransferase activity. However, it is not yet fully understood how ubiquitinated histone H3 stimulates Dnmt1 activity. Here, we show that monoubiquitinated histone H3 stimulates Dnmt1 activity toward DNA with multiple hemimethylated CpGs but not toward DNA with only a single hemimethylated CpG, suggesting an influence of ubiquitination on the processivity of Dnmt1. The Dnmt1 activity stimulated by monoubiquitinated histone H3 was additively enhanced by the Uhrf1 SRA domain, which also binds to RFTS. Thus, Dnmt1 activity is regulated by catalysis (ubiquitination)-dependent and -independent functions of Uhrf1.
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http://dx.doi.org/10.1111/gtc.12732DOI Listing
January 2020

Chemical synthesis of the ubiquitinated form of histone H3 and its effect on DNA methyltransferase 1.

J Pept Sci 2019 Sep 15;25(9):e3200. Epub 2019 Jul 15.

Institute for Protein Research, Osaka University, Suita, Japan.

Posttranslational modifications of histone proteins, which form nucleosome cores, play an important role in gene regulation. Ubiquitination is one such modification. We previously reported on the synthesis of ubiquitinated histone H3 with an isopeptide mimetic structure. In this report, we describe the preparation of ubiquitinated histone H3 peptides with a native isopeptide structure, which showed a slightly weaker effect on the enzymatic activity of DNA methyltransferase 1 than the previous ubiquitinated H3 peptide analogs. These findings show that a native structure is important for determining the mechanism of the function, although ubiquitinated H3 peptide analogs can mimic the role of the original ubiquitinated H3. We also report on the successful preparation of the ubiquitinated full length histone H3.
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http://dx.doi.org/10.1002/psc.3200DOI Listing
September 2019

Revalidation of recombinant aequorin as a light emission standard: Estimation of specific activity of Gaussia luciferase.

Biochem Biophys Res Commun 2018 12 10;507(1-4):242-245. Epub 2018 Nov 10.

Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka, 565-0871, Japan.

To validate the use of recombinant aequorin (reAequorin) as a light emission standard, the protein concentrations of highly purified reAequorin were determined by amino acid composition analysis, and the presence of active reAequorin was confirmed by the ratio of absorbance peak at 460 nm to that at 280 nm. The high correlation of the luminescence intensity with the protein concentration showed that reAequorin could be used for a light emission standard to study the luminescence properties of luciferases and to evaluate the detection sensitivity of luminometers. The specific activity of Gaussia luciferase with I was 7.5-fold higher than that of reAequorin and was calculated to be 3.8 × 10 quanta/mg protein.
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http://dx.doi.org/10.1016/j.bbrc.2018.11.015DOI Listing
December 2018

Synthesis of selenocysteine-containing cyclic peptides via tandem N-to-S acyl migration and intramolecular selenocysteine-mediated native chemical ligation.

Chem Commun (Camb) 2018 Oct 2;54(83):11737-11740. Epub 2018 Oct 2.

Department of Chemistry, School of Science, Tokai University, Kitakaname, Hiratsuka-shi, Kanagawa 259-1292, Japan.

Selenocysteine-containing cyclic 8-mer peptides, which were designed to mimic the plausible catalytic tetrad of glutathione peroxidase, were successfully synthesized in one pot via tandem N-to-S acyl migration of N-alkylcysteine (NAC)-containing selenopeptides and intramolecular selenocysteine-mediated native chemical ligation (Sec-NCL) of the generated thioesters.
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http://dx.doi.org/10.1039/c8cc06805dDOI Listing
October 2018

Heat-Induced Aggregation of Hen Ovalbumin Suggests a Key Factor Responsible for Serpin Polymerization.

Biochemistry 2018 09 6;57(37):5415-5426. Epub 2018 Sep 6.

Institute for Protein Research , Osaka University , Yamadaoka 3-2 , Suita , Osaka 565-0871 , Japan.

Although ovalbumin (OVA), a main component of hen egg white and a non-inhibitory serpin superfamily protein, has been reported to form fibrillar aggregates, its relationship with amyloid fibrils associated with various degenerative diseases is unclear. We studied the heat-induced aggregation of intact OVA using an amyloid-specific thioflavin T assay with a fluorometer or direct imaging with a light-emitting diode lamp and several physicochemical approaches, and the results confirmed that intact OVA forms aggregates with a small part of amyloid cores and dominantly amorphous aggregates. We isolated the amyloidogenic core peptide by proteolysis with trypsin. The isolated 23-residue peptide, pOVA, with marked amyloidogenicity, corresponded to one (β-strand 3A) of the key regions involved in serpin latency transition and domain-swap polymerization leading to serpinopathies. Although the strong amyloidogenicity of pOVA was suppressed in a mixture of tryptic digests, it was observed under acidic conditions in the presence of various salts, with which pOVA has a positive charge. Cytotoxicity measurements suggested that, although heat-treated OVA aggregates exhibited the strongest toxicity, it was attributed to a general property of amorphous aggregates rather than amyloid toxicity. Predictions indicated that the high amyloidogenicity of the β-strand 3A region is common to various serpins. This suggests that the high amyloidogenicity of β-strand 3A that is important for serpin latency transition and domain-swap polymerization is retained in OVA and constitutes β-spine amyloid cores upon heat aggregation.
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http://dx.doi.org/10.1021/acs.biochem.8b00619DOI Listing
September 2018

Sialyl Tn Unit with TFA-Labile Protection Realizes Efficient Synthesis of Sialyl Glycoprotein.

Chemistry 2018 Feb 25;24(11):2593-2597. Epub 2018 Jan 25.

Institute for Protein Research, Osaka University, Osaka, 565-0871, Japan.

Amino acids bearing 4-methylbenzyl (MBn) and 4-methoxybenzyl (MPM)-protected sialic acid were synthesized and used for the 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase synthesis of a glycopeptide. The α-sialyl linkage of the MBn-protected unit was partially cleaved under the final deprotection by trifluoroacetic acid (TFA). In addition, the removal of several MBn groups were incomplete. On the other hand, the MPM-protected unit gave the desired glycopeptide without decomposition of the α-sialyl linkage. Using this unit, peptide thioesters of the tandem repeat unit of MUC1 mucin were synthesized by the N-alkylcysteine (NAC) method and used for the one-pot ligation by the thioester method. As a result, the three tandem repeats of MUC1 carrying sialyl Tn antigens were successfully synthesized.
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http://dx.doi.org/10.1002/chem.201706127DOI Listing
February 2018

Development of SAAP3D force field and the application to replica-exchange Monte Carlo simulation for chignolin and C-peptide.

J Comput Aided Mol Des 2017 12 17;31(12):1039-1052. Epub 2017 Nov 17.

Genomic Sciences Center, RIKEN, Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan.

Single amino acid potential (SAAP) would be a prominent factor to determine peptide conformations. To prove this hypothesis, we previously developed SAAP force field for molecular simulation of polypeptides. In this study, the force field was renovated to SAAP3D force field by applying more accurate three-dimensional main-chain parameters, instead of the original two-dimensional ones, for the amino acids having a long side-chain. To demonstrate effectiveness of the SAAP3D force field, replica-exchange Monte Carlo (REMC) simulation was performed for two benchmark short peptides, chignolin (H-GYDPETGTWG-OH) and C-peptide (CHO-AETAAAKFLRAHA-NH). For chignolin, REMC/SAAP3D simulation correctly produced native β-turn structures, whose minimal all-atom root-mean-square deviation value measured from the native NMR structure (except for H) was 1.2 Å, at 300 K in implicit water, along with misfolded β-hairpin structures with unpacked aromatic side chains of Tyr2 and Trp9. Similar results were obtained for chignolin analog [G1Y,G10Y], which folded more tightly to the native β-turn structure than chignolin did. For C-peptide, on the other hand, the α-helix content was larger than the β content on average, suggesting a significant helix-forming propensity. When the imidazole side chain of His12 was protonated (i.e., [His12Hip]), the α content became larger. These observations as well as the representative structures obtained by clustering analysis were in reasonable agreement not only with the structures of C-peptide that were determined in this study by NMR in 30% CDCD in HO at 298 K but also with the experimental and theoretical behaviors having been reported for protonated C-peptide. Thus, accuracy of the SAAP force field was improved by applying three-dimensional main-chain parameters, supporting prominent importance of SAAP for peptide conformations.
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http://dx.doi.org/10.1007/s10822-017-0084-8DOI Listing
December 2017

Structure of the Dnmt1 Reader Module Complexed with a Unique Two-Mono-Ubiquitin Mark on Histone H3 Reveals the Basis for DNA Methylation Maintenance.

Mol Cell 2017 Oct;68(2):350-360.e7

Division of Cancer Cell Biology, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan; Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan. Electronic address:

The proper location and timing of Dnmt1 activation are essential for DNA methylation maintenance. We demonstrate here that Dnmt1 utilizes two-mono-ubiquitylated histone H3 as a unique ubiquitin mark for its recruitment to and activation at DNA methylation sites. The crystal structure of the replication foci targeting sequence (RFTS) of Dnmt1 in complex with H3-K18Ub/23Ub reveals striking differences to the known ubiquitin-recognition structures. The two ubiquitins are simultaneously bound to the RFTS with a combination of canonical hydrophobic and atypical hydrophilic interactions. The C-lobe of RFTS, together with the K23Ub surface, also recognizes the N-terminal tail of H3. The binding of H3-K18Ub/23Ub results in spatial rearrangement of two lobes in the RFTS, suggesting the opening of its active site. Actually, incubation of Dnmt1 with H3-K18Ub/23Ub increases its catalytic activity in vitro. Our results therefore shed light on the essential role of a unique ubiquitin-binding module in DNA methylation maintenance.
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http://dx.doi.org/10.1016/j.molcel.2017.09.037DOI Listing
October 2017

One-Pot Four-Segment Ligation Using Seleno- and Thioesters: Synthesis of Superoxide Dismutase.

Angew Chem Int Ed Engl 2017 12 3;56(49):15708-15711. Epub 2017 Nov 3.

Institute for Protein Research, Osaka University, Osaka, 565-0871, Japan.

The synthesis of a peptide selenoester was efficiently carried out by the 9-fluorenylmethoxycarbonyl (Fmoc) method using N-alkylcysteine, at the C-terminus of the peptide, as the N-to-S acyl shift device. The selenoester selectively reacted with the terminal amino group of the peptide aryl thioester in the presence of N,N-diisopropylethylamine and dipyridyldisulfide, thus leaving the aryl thioester intact. Combined with silver-ion-promoted and silver-ion-free thioester activation methods, a one-pot four-segment ligation was realized. The method was successfully used to assemble the entire sequence of superoxide dismutase (SOD), which is composed of 153 amino-acid residues, in one pot. After the folding reaction, the fully active SOD was obtained.
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http://dx.doi.org/10.1002/anie.201709418DOI Listing
December 2017

RFTS-dependent negative regulation of Dnmt1 by nucleosome structure and histone tails.

FEBS J 2017 10 11;284(20):3455-3469. Epub 2017 Sep 11.

Laboratory of Epigenetics, Institute for Protein Research, Osaka University, Suita, Japan.

DNA methylation in promoter regions represses gene expression and is copied over mitotic divisions by Dnmt1. Dnmt1 activity is regulated by its replication foci targeting sequence (RFTS) domain which masks the catalytic pocket. It has been shown that Dnmt1 activity on unmethylated DNA is inhibited in nucleosome cores. In the present study, we aimed to assess the effect of nuclesome formation on maintenance methylation at single CpG resolution. We show that Dnmt1 fully methylates naked linker DNA in dinucleosomes, whereas maintenance methylation was repressed at all CpG sites in nucleosome core particles. Deletion of RFTS partly released obstruction of Dnmt1 activity in core particles. Histone H3 tail peptides inhibited Dnmt1 in an RFTS-dependent manner and repression was modulated by acetylation or methylation. We propose a novel function of RFTS to regulate Dnmt1 activity in nucleosomes.
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http://dx.doi.org/10.1111/febs.14205DOI Listing
October 2017

Regulation of CCR7-dependent cell migration through CCR7 homodimer formation.

Sci Rep 2017 08 17;7(1):8536. Epub 2017 Aug 17.

Laboratory of Immune Molecular Function, Faculty of Science & Engineering, Kindai University, 3-4-1 Kowakae, Higashiosaka, Osaka, 577-8502, Japan.

The chemokine receptor CCR7 contributes to various physiological and pathological processes including T cell maturation, T cell migration from the blood into secondary lymphoid tissues, and tumor cell metastasis to lymph nodes. Although a previous study suggested that the efficacy of CCR7 ligand-dependent T cell migration correlates with CCR7 homo- and heterodimer formation, the exact extent of contribution of the CCR7 dimerization remains unclear. Here, by inducing or disrupting CCR7 dimers, we demonstrated a direct contribution of CCR7 homodimerization to CCR7-dependent cell migration and signaling. Induction of stable CCR7 homodimerization resulted in enhanced CCR7-dependent cell migration and CCL19 binding, whereas induction of CXCR4/CCR7 heterodimerization did not. In contrast, dissociation of CCR7 homodimerization by a novel CCR7-derived synthetic peptide attenuated CCR7-dependent cell migration, ligand-dependent CCR7 internalization, ligand-induced actin rearrangement, and Akt and Erk signaling in CCR7-expressing cells. Our study indicates that CCR7 homodimerization critically regulates CCR7 ligand-dependent cell migration and intracellular signaling in multiple cell types.
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http://dx.doi.org/10.1038/s41598-017-09113-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5561199PMC
August 2017

Total Synthesis and Antibacterial Investigation of Plusbacin A.

Org Lett 2017 07 11;19(14):3771-3774. Epub 2017 Jul 11.

Faculty of Pharmaceutical Sciences, Hokkaido University , Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan.

The total synthesis of plusbacin A (1) has been accomplished using a solvent-dependent diastereodivergent Joullié-Ugi three-component reaction (JU-3CR) as a key step. Two trans-3-hydroxy-l-proline residues were constructed by combining the JU-3CR with a convertible isocyanide strategy. Subsequent peptide coupling and macrolactamization afforded plusbacin A. Investigating the antibacterial activity of 1 compared with that of its dideoxy analogue revealed that the threo-β-hydroxyaspartic acid residues are essential for antibacterial activity. Notably, there is a low potential for the development of resistance in S. aureus against plusbacin A.
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http://dx.doi.org/10.1021/acs.orglett.7b01629DOI Listing
July 2017

Preparation of Selenoinsulin as a Long-Lasting Insulin Analogue.

Angew Chem Int Ed Engl 2017 05 10;56(20):5522-5526. Epub 2017 Apr 10.

Department of Chemistry, School of Science, Tokai University, Kitakaname, Hiratsuka-shi, Kanagawa, 259-1292, Japan.

Synthetic insulin analogues with a long lifetime are current drug targets for the therapy of diabetic patients. The replacement of the interchain disulfide with a diselenide bridge, which is more resistant to reduction and internal bond rotation, can enhance the lifetime of insulin in the presence of the insulin-degrading enzyme (IDE) without impairing the hormonal function. The [C7U ,C7U ] variant of bovine pancreatic insulin (BPIns) was successfully prepared by using two selenocysteine peptides (i.e., the C7U analogues of A- and B-chains, respectively). In a buffer solution at pH 10 they spontaneously assembled under thermodynamic control to the correct insulin fold. The selenoinsulin (Se-Ins) exhibited a bioactivity comparable to that of BPIns. Interestingly, degradation of Se-Ins with IDE was significantly decelerated (τ ≈8 h vs. ≈1 h for BPIns). The lifetime enhancement could be due to both the intrinsic stability of the diselenide bond and local conformational changes induced by the substitution.
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http://dx.doi.org/10.1002/anie.201701654DOI Listing
May 2017

Pyrrole-Based Macrocyclic Small-Molecule Inhibitors That Target Oocyte Maturation.

ChemMedChem 2017 04 28;12(8):580-589. Epub 2017 Mar 28.

Division of Magnetic Resonance, Korea Basic Science Institute, Ochang, Chung-Buk, 363-883, Republic of Korea.

Polo-like kinase 1 (PLK1) plays crucial roles in various stages of oocyte maturation. Recently, we reported that the peptidomimetic compound AB103-8, which targets the polo box domain (PBD) of PLK1, affects oocyte meiotic maturation and the resumption of meiosis. However, to overcome the drawbacks of peptidic compounds, we designed and synthesized a series of pyrrole-based small-molecule inhibitors and tested them for their effects on the rates of porcine oocyte maturation. Among them, the macrocyclic compound (E/Z)-3-(2,16-dioxo-19-(4-phenylbutyl)-3,19-diazabicyclo[15.2.1]icosa-1(20),6,17-trien-3-yl)propyl dihydrogen phosphate (4) showed the highest inhibitory activity with enhanced inhibition against embryonic blastocyst formation. Furthermore, the addition of this compound to culture media efficiently blocked the maturation of porcine and mouse oocytes, indicating its ability to penetrate the zona pellucida and cell membrane. We investigated mouse oocytes treated with compound 4, and the resulting impairment of spindle formation confirmed PLK1 inhibition. Finally, molecular modeling studies with PLK1 PBD also confirmed the presence of significant interactions between compound 4 and PLK1 PBD binding pocket residues, including those in the phosphate, tyrosine-rich, and pyrrolidine binding pockets. Collectively, these results suggest that the macrocyclic compound 4 may serve as a promising template for the development of novel contraceptive agents.
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http://dx.doi.org/10.1002/cmdc.201700048DOI Listing
April 2017

Synthesis of ubiquitylated histone H3 using a thiirane linker for chemical ligation.

J Pept Sci 2017 Jul 31;23(7-8):532-538. Epub 2017 Jan 31.

Institute for Protein Research, Osaka University, Suita, Osaka, 565-0871, Japan.

Post-translational modifications of histone proteins, which form nucleosome cores, play an important role in gene regulation. Ubiquitin modification is one such modification. We previously reported on the use of a thiirane linker to introduce a 1,2-aminothiol moiety at a cysteine residue for native chemical ligation with peptide thioesters, which permitted isopeptide mimetics to be produced. In this report, we describe the preparation of the ubiquitylated full length histone H3 at the 18 position and the construction of tetranucleosomes with recombinant histones H2A, H2B, H4, and DNA, which are slightly more stable than those that are prepared without ubiquitin modification. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/psc.2976DOI Listing
July 2017

One-pot native chemical ligation by combination of two orthogonal thioester precursors.

Chem Commun (Camb) 2017 Feb;53(13):2114-2117

Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan.

We developed a one-pot peptide ligation method using two orthogonal thioester precursors and a protecting group for the ligation reaction between Asp and Cys. Combination of the two precursors facilitated the one-pot operation and yielded the entire polypeptide. The usefulness of this method was successfully demonstrated by the total synthesis of histone H4.
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http://dx.doi.org/10.1039/c6cc10243cDOI Listing
February 2017

Variation of free-energy landscape of the p53 C-terminal domain induced by acetylation: Enhanced conformational sampling.

J Comput Chem 2016 12 13;37(31):2687-2700. Epub 2016 Oct 13.

Institute for Protein Research Osaka University, 3-2 Yamadaoka, Suita, Osaka, 565-0871, Japan.

The C-terminal domain (CTD) of tumor suppressor protein p53 is an intrinsically disordered region that binds to various partner proteins, where lysine of CTD is acetylated/nonacetylated and histidine neutralized/non-neutralized. Because of the flexibility of the unbound CTD, a free-energy landscape (FEL) is a useful quantity for determining its statistical properties. We conducted enhanced conformational sampling of CTD in the unbound state via virtual system coupled multicanonical molecular dynamics, in which the lysine was acetylated or nonacetylated and histidine was charged or neutralized. The fragments were expressed by an all-atom model and were immersed in an explicit solvent. The acetylation and charge-neutralization varied FEL greatly, which might be convenient to exert a hub property. The acetylation slightly enhanced alpha-helix structures that are more compact than sheet/loop conformations. The charge-neutralization produced hairpins. Additionally, circular dichroism experiments confirmed the computational results. We propose possible binding mechanisms of CTD to partners by investigating FEL. © 2016 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/jcc.24494DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5242334PMC
December 2016

A strategy for the synthesis of hydrophobic proteins and glycoproteins.

Authors:
Hironobu Hojo

Org Biomol Chem 2016 Jul;14(27):6368-74

Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.

Our synthetic strategy for the hydrophobic glycoprotein is summarized. The reverse-polarity protection strategy, in which the side chain carboxy group is protected as a basic picolyl ester, combined with the O-acylisopeptide method proved to be efficient for the preparation of the hydrophobic glycoprotein by ligation methods, and would be applicable for the synthesis of membrane proteins in the future.
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http://dx.doi.org/10.1039/c6ob00827eDOI Listing
July 2016

Enthalpy-driven interactions with sulfated glycosaminoglycans promote cell membrane penetration of arginine peptides.

Biochim Biophys Acta 2016 Jun 19;1858(6):1339-49. Epub 2016 Mar 19.

Institute of Biomedical Sciences, Graduate School of Pharmaceutical Sciences, Tokushima University, 1-78-1 Shoumachi, Tokushima 770-8505, Japan; Department of Biophysical Chemistry, Kyoto Pharmaceutical University, 5 Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan. Electronic address:

The first step of cell membrane penetration of arginine peptides is thought to occur via electrostatic interactions between positive charges of arginine residues and negative charges of sulfated glycosaminoglycans (GAGs) on the cell surface. However, the molecular interaction of arginine peptides with GAG still remains unclear. Here, we compared the interactions of several arginine peptides of Tat, R8, and Rev and their analogues with heparin in relation to the cell membrane penetration efficiency. The high-affinity binding of arginine peptides to heparin was shown to be driven by large favorable enthalpy contributions, possibly reflecting multidentate hydrogen bondings of arginine residues with sulfate groups of heparin. Interestingly, the lysine peptides in which all arginine residues are substituted with lysine residues exhibited negligible binding enthalpy despite of their considerable binding to heparin. In CHO-K1 cells, arginine peptides exhibited a great cell-penetrating ability whereas their corresponding lysine peptides did not penetrate into cells. The degree of cell penetration of arginine peptides markedly decreased by the chlorate treatment of cells which prevents the sulfation of GAG chains. Significantly, the cell penetration efficiency of arginine peptides was found to be correlated with the favorable enthalpy of binding to heparin. These results suggest that the enthalpy-driven strong interaction with sulfated GAGs such as heparan sulfate plays a critical role in the efficient cell membrane penetration of arginine peptides.
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http://dx.doi.org/10.1016/j.bbamem.2016.03.021DOI Listing
June 2016

Site-specific labeling of synthetic peptide using the chemoselective reaction between N-methoxyamino acid and isothiocyanate.

J Pept Sci 2015 Oct;21(10):765-9

Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka, 565-0871, Japan.

Site-specific labeling of synthetic peptides carrying N-methoxyglycine (MeOGly) by isothiocyanate is demonstrated. A nonapeptide having MeOGly at its N-terminus was synthesized by the solid-phase method and reacted with phenylisothiocyanate under various conditions. In acidic solution, the reaction specifically gave a peptide having phenylthiourea structure at its N-terminus, leaving side chain amino group intact. The synthetic human β-defensin-2 carrying MeOGly at its N-terminus or the side chain amino group of Lys(10) reacted with phenylisothiocyanate or fluorescein isothiocyanate also at the N-methoxyamino group under the same conditions, demonstrating that this method is generally useful for the site-specific labeling of linear synthetic peptides as well as disulfide-containing peptides.
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http://dx.doi.org/10.1002/psc.2803DOI Listing
October 2015

Nucleosome compaction facilitates HP1γ binding to methylated H3K9.

Nucleic Acids Res 2015 Dec 28;43(21):10200-12. Epub 2015 Aug 28.

Laboratory of Epigenetics, Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan CREST, Japan Science and Technology Agency, Saitama 332-0012, Japan

The α, β and γ isoforms of mammalian heterochromatin protein 1 (HP1) selectively bind to methylated lysine 9 of histone H3 via their chromodomains. Although the phenotypes of HP1-knockout mice are distinct for each isoform, the molecular mechanisms underlying HP1 isoform-specific function remain elusive. In the present study, we found that in contrast to HP1α, HP1γ could not bind tri-methylated H3 lysine 9 in a reconstituted tetra-nucleosomes when the nucleosomes were in an uncompacted state. The hinge region connecting HP1's chromodomain and chromoshadow domain contributed to the distinct recognition of the nucleosomes by HP1α and HP1γ. HP1γ, but not HP1α, was strongly enhanced in selective binding to tri-methylated lysine 9 in histone H3 by the addition of Mg(2+) or linker histone H1, which are known to induce compaction of nucleosomes. We propose that this novel property of HP1γ recognition of lysine 9 in the histone H3 tail in different nucleosome structures plays a role in reading the histone code.
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http://dx.doi.org/10.1093/nar/gkv841DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4666388PMC
December 2015

The effects of chemically synthesized saposin C on glucosylceramide-β-glucosidase.

Clin Biochem 2015 Nov 9;48(16-17):1177-80. Epub 2015 Jun 9.

Institute of Glycoscience, Tokai University, Kanagawa 259-1292, Japan; Department of Pediatrics, Kawasaki Medical School, Okayama 701-0192, Japan. Electronic address:

Objective: Saposin C (SAP-C) is an essential activator of glucosylceramide (GlcCer)-β-glucosidase (GCase), the enzyme deficient in Gaucher's disease. In this study, we investigated the effects of chemically synthesized SAP-Cs (synthetic SAP-Cs) on GCase.

Methods: Enzymatic assays and western blot analyses were carried out to evaluate the effects of two kinds of synthetic SAP-Cs, a non-glycosylated form and a N-glycosylated form bearing a complex type nonasaccharide, on GCase with respect to its activation, stabilization, and protection. Imiglucerase (Cerezyme) was used as the GCase. To mimic physiological conditions, GCase activity was assayed in the presence of 4-nitrobenzo-2-oxa-1,3-diazole-labeled GlcCer-containing liposomes composed of bis(monoacylglycero)phosphate, l-α-phosphatidylcholine, and cholesterol.

Results: GCase activities increased depending on the concentration of synthetic SAP-Cs. SAP-Cs at a concentration of 1μM increased GCase activities significantly, by 14- to 22-fold (non-glycosylated SAP-C: 22.9±0.16; nona-glycosylated SAP-C: 14.9±0.19; without SAP-C: 1.05±0.035pmol/h/ng GCase). These values equaled or surpassed previously published values obtained using recombinant non-glycosylated SAP-C. Both synthetic SAP-Cs were as effective as bovine serum albumin in stabilizing GCase at 37°C. Western blot analysis revealed that synthetic SAP-Cs specifically protected GCase from cathepsin D digestion.

Conclusions: The results demonstrate that these novel, chemically synthesized SAP-Cs function as activators, stabilizers, and protectors of GCase, suggesting their utility in enzyme replacement therapy in patients with Gaucher's disease.
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http://dx.doi.org/10.1016/j.clinbiochem.2015.06.004DOI Listing
November 2015

Chemical Synthesis of O-Glycosylated Human Interleukin-2 by the Reverse Polarity Protection Strategy.

Angew Chem Int Ed Engl 2015 Jul 26;54(28):8226-30. Epub 2015 May 26.

Institute for Protein Research, Osaka University, Osaka 565-0871 (Japan).

The chemical synthesis of human interleukin-2 (IL-2) , having a core 1 sugar, by a ligation method is reported. Although IL-2 is a globular glycoprotein, its C-terminal region, in particular (99-133), is extremely insoluble when synthesized by solid-phase method. To overcome this problem, the side-chain carboxylic acid of the Glu residues was protected by a picolyl ester, thus reversing its polarity from negative to positive. This reverse polarity protection significantly increased the isoelectric point of the peptide segment and made it positive under acidic conditions and facilitated the purification. An efficient method to prepare the prolyl peptide thioester required for the synthesis of the (28-65) segment was also developed. These efforts resulted in the total synthesis of the glycosylated IL-2 having full biological activity.
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http://dx.doi.org/10.1002/anie.201501847DOI Listing
July 2015