Supramolecular Hemoprotein Assembly with a Periodic Structure Showing Heme-Heme Exciton Coupling.

A supramolecular assembly of units of cytochrome b562 with externally attached heme having intermolecular linkages formed via the heme-heme pocket interaction was investigated in an effort to construct a well-defined structure. The engineered site for surface attachment of heme at Cys80 in an N80C mutant of cytochrome b562 provides the primary basis for the formation of the periodic assembly structure, which is characterized herein by circular dichroism (CD) spectroscopy and high-speed atomic force microscopy (AFM). This assembly represents the first example of the observation of a split-type Cotton effect by heme-heme exciton coupling in an artificial hemoprotein assembly system. Molecular dynamics simulations validated by simulated CD spectra, AFM images, and mutation experiments reveal that the assembly has a periodic helical structure with 3 nm pitches, suggesting the formation of the assembled structure is driven not only by the heme-heme pocket interaction but also by additional secondary hydrogen bonding and/or electrostatic interactions at the protein interfaces of the assembly.

Catalytic asymmetric synthesis of α-methyl-p-boronophenylalanine.

p-Boronophenylalanine (l-BPA) is applied in clinical settings as a boron carrier for boron neutron capture therapy (BNCT) to cure malignant melanomas. Structural modification or derivatization of l-BPA, however, to improve its uptake efficiency into tumor cells has scarcely been investigated. We successfully synthesized (S)-2-amino-3-(4-boronophenyl)-2-methylpropanoic acid in enantioenriched form as a novel candidate molecule for BNCT. Key steps to enhance the efficiency of this synthesis were enantioselective alkylation of N-protected alanine tert-butyl ester with a Maruoka catalyst and Miyaura borylation reaction to install the boron functionality.

Evaluation of expression systems for recombinant protein production in chicken egg bioreactors.

Chicken eggs have gained attention as excellent bioreactors because of their genetic modifications. However, the development of chicken egg bioreactors requires a long time from the construction of the production system to the evaluation of the products. Therefore, in this study, a chicken cell line producing ovalbumin (OVA) was established and constructed a system for the rapid evaluation of the production system. First, the EF1α promoter was knocked in upstream of the OVA locus in chicken DF-1 cells for continuous OVA expression. Furthermore, an ideal position at the OVA locus for the insertion of useful protein genes to maximize recombinant protein yield was analyzed and identified. The knocking in the EF1α promoter upstream of exon1 yielded the maximum production of OVA protein was achieved. In addition, Linking a recombinant hFGF2 cDNA to the 5' side of the OVA was found to increase production efficiency. Therefore, an OVA-expressing cell line and an evaluation system for proteins in chicken egg bioreactors was established. The findings may improve the efficiency of chicken expression systems and expand their applications in protein production.

Wnt signaling blockade is essential for maintaining the pluripotency of chicken embryonic stem cells.

Activation of Wnt/ß-catenin signaling supports the self-renewal of mouse embryonic stem cells. We aimed to understand the effects of Wnt signaling activation or inhibition on chicken embryonic stem cells (chESCs), as these effects are largely unknown. When the glycogen synthase kinase-3 ß inhibitor CHIR99021-which activates Wnt signaling-was added to chESC cultures, the colony shape flattened, and the expression levels of pluripotency-related (NANOG, SOX2, SOX3, OCT4, LIN28A, DNMT3B, and PRDM14) and germ cell (CVH and DAZL) markers showed a decreasing trend, and the growth of chESCs was inhibited after approximately 7 d. By contrast, when the Wnt signaling inhibitor XAV939 was added to the culture, dense and compact multipotent colonies (morphologically similar to mouse embryonic stem cell colonies) showing stable expression of pluripotency-related and germline markers were formed. The addition of XAV939 stabilized the proliferation of chESCs in the early stages of culture and promoted their establishment. Furthermore, these chESCs formed chimeras. In conclusion, functional chESCs can be stably cultured using Wnt signaling inhibitors. These findings suggest the importance of Wnt/ß-catenin signaling in avian stem cells, offering valuable insights for applied research using chESCs.

Lipofection with Lipofectamine™ 2000 in a heparin-free growth medium results in high transfection efficiency in chicken primordial germ cells.

Primordial germ cells (PGCs) that can differentiate into gametes are used to produce genome-edited chickens. However, the transfection efficiency into PGCs is low in chickens; therefore, the yield efficiency of PGCs modified via genome editing is problematic. In this study, we improved transfection efficiency and achieved highly efficient genome editing in chicken PGCs. For transfection, we used lipofection, which is convenient for gene transfer. Chicken PGC cultures require adding heparin to support growth; however, heparin significantly reduces lipofection efficiency (p < 0.01). Heparin-induced lipofection efficiency was restored by adding protamine. Based on these results, we optimized gene transfer into chicken PGCs. Lipofectamine 2000 and our PGC medium were the most efficient transfection reagent and medium, respectively. Finally, based on established conditions, we compared the gene knock-out efficiencies of ovomucoid, a major egg allergen, and gene knock-in efficiencies at the ACTB locus. These results indicate that optimized lipofection is useful for CRISPR/Cas9-mediated knock-out and knock-in. Our findings may contribute to the generation of genome-edited chickens and stimulate research in various applications involving them.

Photoinduced electron transfer within supramolecular hemoprotein co-assemblies and heterodimers containing Fe and Zn porphyrins.

Electron transfer (ET) events occurring within metalloprotein complexes are among the most important classes of reactions in biological systems. This report describes a photoinduced electron transfer between Zn porphyrin and Fe porphyrin within a supramolecular cytochrome b562 (Cyt b562) co-assembly or heterodimer with a well-defined rigid structure formed by a metalloporphyrin-heme pocket interaction and a hydrogen-bond network at the protein interface. The photoinduced charge separation (CS: kCS = 320-600 s-1) and subsequent charge recombination (CR: kCR = 580-930 s-1) were observed in both the Cyt b562 co-assembly and the heterodimer. In contrast, interestingly, no ET events were observed in a system comprised of a flexible and structurally-undefined co-assembly and heterodimers which lack the key hydrogen-bond interaction at the protein interface. Moreover, analysis of the kinetic constants of CS and CR of the heterodimer using the Marcus equation suggests that a single-step ET reaction occurs in the system. These findings provide strong support that the rigid hemoprotein-assembling system containing an appropriate hydrogen-bond network at the protein interface is essential for monitoring the ET reaction.

A ring-shaped hemoprotein trimer thermodynamically controlled by the supramolecular heme-heme pocket interaction.

Engineered cytochrome b562, a small hemoprotein, with an externally-attached heme moiety via a moderately long linker at a suitable position predominantly forms a thermodynamically stable ring-shaped trimer in dilute solution. In an equilibrium between supramolecular polymerization and depolymerization, the ring-shaped trimer is kinetically trapped even in a concentrated solution.

Molecular-Iodine-Catalyzed Cyclization of 2-Alkynylanilines via Iodocyclization-Protodeiodination Sequence.

Molecular iodine catalyzes a cyclization of N-aryl-2-alkynylanilines, which proceeds through the iodocyclization of 2-alkynylanilines followed by the protodeiodination of the iodocyclized intermediates at room temperature. Furthermore, the molecular iodine catalysis can be applied to the cascade cyclization of 2-(enynyl)aniline and to the tandem cyclization-addition reaction of 2-alkynylanilines with α,ß-enones.

Gold-Catalyzed Domino Synthesis of Functionalized Benzofurans and Tetracyclic Isochromans via Formal Carboalkoxylation.

A domino synthesis of benzofurans with the modification of side chains from α-alkoxyalkyl o-alkynylaryl ethers (n = 0) and electron-rich arenes has been developed. In the present domino reaction, which would proceed via the α-alkoxyalkylation of arenes with an intermediate in the migratory cycloisomerization of o-alkynylaryl ethers followed by the nucleophilic addition of benzofurans to benzyl ethers, a cationic Au(III) catalyst activates the C-C π bond and the C-O σ bond. The present method could be extended to Au(I)-catalyzed domino synthesis of tetracyclic isochromans from α-alkoxyalkyl (o-alkynylaryl)methyl ethers (n = 1) and aryl methoxymethyl ethers.