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Proper regulation of N-methyl-D-aspartate-type glutamate receptor (NMDA receptor) expression is responsible for excitatory synaptic functions in the mammalian brain. NMDA receptor dysfunction can cause various neuropsychiatric disorders and neurodegenerative diseases. Posttranslational protein S-palmitoylation, the covalent attachment of palmitic acid to intracellular cysteine residues via thioester bonds, occurs in the carboxyl terminus of GluN2B, which is the major regulatory NMDA receptor subunit. Mutations of three palmitoylatable cysteine residues in the membrane-proximal cluster of GluN2B to non-palmitoylatable serine (3CS) lead to the dephosphorylation of GluN2B Tyr1472 in the hippocampus and cerebral cortex, inducing a reduction in the surface expression of GluN2B-containig NMDA receptors. Furthermore, adult GluN2B 3CS homozygous mice demonstrated a definite clasping response without abnormalities in the gross brain structure, other neurological reflexes, or expression levels of synaptic proteins in the cerebrum. This behavioral disorder, observed in the GluN2B 3CS knock-in mice, indicated that complex higher brain functions are coordinated through the palmitoylation-dependent regulation of NMDA receptors in excitatory synapses., (© 2024. The Author(s).)

Imine reduction is a useful reaction in the preparation of amine derivatives. Various catalysts have been reported to promote this reaction and photoredox catalysts are promising candidates for sustainable amine synthesis. Improvement of this reaction using biomolecule-based reaction scaffolds is expected to increase the utility of the reaction. In this context, we have recently investigated photoredox Ru complexes with pentapeptide scaffolds via coordination bonds as catalysts for photoreduction of dihydroisoquinoline derivatives. First, Ru bipyridine terpyridine complexes coordinated with five different pentapeptides (XVHVV: X = V, F, W, Y, C) were prepared and characterized by mass spectrometry. Catalytic activities of the Ru complexes with XVHVV were evaluated for photoreduction of dihydroisoquinoline derivatives in the presence of ascorbate and thiol compounds as sacrificial reagents and hydrogen sources. Interestingly, the turnover number of the Ru complex with VVHVV is 531, which is two-fold higher than that of a simple Ru complex with an imidazole ligand. The detailed emission lifetime measurements indicate that the enhanced catalytic activity provided by the peptide scaffold is caused by an efficient reaction with the thiol derivative to accelerate reductive quenching of Ru complex. The quenching behavior suggests formation of an active species such as a Ru(I) complex. These findings reveal that the simple pentapeptide serves as an effective scaffold to enhance the photocatalytic activity of a photoactive Ru complex., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Design of metal cofactor ligands is essential for controlling the reactivity of metalloenzymes. We investigated a carbene transfer reaction catalyzed by myoglobins containing iron porphyrin cofactors with one and two trifluoromethyl groups at peripheral sites (FePorCF 3 and FePor(CF 3 ) 2 , respectively), native heme and iron porphycene (FePc). These four myoglobins show a wide range of Fe(II)/Fe(III) redox potentials in the protein of +147 mV, +87 mV, +42 mV and -198 mV vs. NHE, respectively. Myoglobin reconstituted with FePor(CF 3 ) 2 has a more positive potential, which enhances the reactivity of a carbene intermediate with alkenes, and demonstrates superior cyclopropanation of inert alkenes, such as aliphatic and internal alkenes. In contrast, engineered myoglobin reconstituted with FePc has a more negative redox potential, which accelerates the formation of the intermediate, but has low reactivity for inert alkenes. Mechanistic studies indicate that myoglobin with FePor(CF 3 ) 2 generates an undetectable active intermediate with a radical character. In contrast, this reaction catalyzed by myoglobin with FePc includes a detectable iron-carbene species with electrophilic character. This finding highlights the importance of redox-focused design of the iron porphyrinoid cofactor in hemoproteins to tune the reactivity of the carbene transfer reaction., (© 2024 The Author(s). Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

We here report the purification of a novel member of the galectin family, the β-galactoside-binding lectin hRTL, from the marine sponge Chondrilla australiensis . The hRTL lectin is a tetrameric proto-type galectin with a subunit molecular weight of 15.5 kDa, consisting of 141 amino acids and sharing 92% primary sequence identity with the galectin CCL from the congeneric species C. caribensis . Transcriptome analysis allowed for the identification of additional sequences belonging to the same family, bringing the total number of hRTLs to six. Unlike most other galectins, hRTLs display a 23 amino acid-long signal peptide that, according to Erdman degradation, is post-translationally cleaved, leaving an N-terminal end devoid of acetylated modifications, unlike most other galectins. Moreover, two hRTLs display an internal insertion, which determines the presence of an unusual loop region that may have important functional implications. The characterization of the glycan-binding properties of hRTL revealed that it had high affinity towards TF-antigen, sialyl TF, and type-1 N-acetyl lactosamine with a Galβ1-3 structure. When administered to DLD-1 cells, a colorectal carcinoma cell line expressing mucin-associated TF-antigen, hRTL could induce glycan-dependent cytotoxicity.

A series of isostructural reticular frameworks with systematic differences on chemical structures allows us to disclose correlations between specific structural factors and properties, providing insights for designing novel porous materials. However, even slight differences in the molecular structure often lead to non-isostructural polymorphic frameworks particularly in the case of hydrogen-bonded organic frameworks (HOFs) because the structures of HOFs are based on a subtle balance of reversible interactions. In this study, we found that three simple analogues of tetracarboxylic acids with naphthalene, quinoxaline, and pyrazinopyrazine cores (NT, QX, and PP, respectively) yielded isostructural solvated HOFs (NT-1, QX-1, and PP-1, respectively), where hydrogen-bonded sql-networked sheets were slip-stacked with closely similar manners. More importantly, these isostructural HOFs underwent structural transformations in different manners upon removal of the guest solvents. Comparison of the crystal structures of the HOFs before and after the transformation revealed that intermolecular interactions of the core significantly affected on rearrangements of hydrogen bonds in the transformation. The results suggest the potential to control the properties and functions of isostructural HOFs by elements in the core., (© 2024 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Increased levels of lactate, an end-product of glycolysis, have been proposed as a potential surrogate marker for metabolic changes during neuronal excitation. These changes in lactate levels can result in decreased brain pH, which has been implicated in patients with various neuropsychiatric disorders. We previously demonstrated that such alterations are commonly observed in five mouse models of schizophrenia, bipolar disorder, and autism, suggesting a shared endophenotype among these disorders rather than mere artifacts due to medications or agonal state. However, there is still limited research on this phenomenon in animal models, leaving its generality across other disease animal models uncertain. Moreover, the association between changes in brain lactate levels and specific behavioral abnormalities remains unclear. To address these gaps, the International Brain pH Project Consortium investigated brain pH and lactate levels in 109 strains/conditions of 2294 animals with genetic and other experimental manipulations relevant to neuropsychiatric disorders. Systematic analysis revealed that decreased brain pH and increased lactate levels were common features observed in multiple models of depression, epilepsy, Alzheimer's disease, and some additional schizophrenia models. While certain autism models also exhibited decreased pH and increased lactate levels, others showed the opposite pattern, potentially reflecting subpopulations within the autism spectrum. Furthermore, utilizing large-scale behavioral test battery, a multivariate cross-validated prediction analysis demonstrated that poor working memory performance was predominantly associated with increased brain lactate levels. Importantly, this association was confirmed in an independent cohort of animal models. Collectively, these findings suggest that altered brain pH and lactate levels, which could be attributed to dysregulated excitation/inhibition balance, may serve as transdiagnostic endophenotypes of debilitating neuropsychiatric disorders characterized by cognitive impairment, irrespective of their beneficial or detrimental nature., Competing Interests: HH, HS, SH, GS, YT, MT, MI, MS, IH, KH, MH, AN, YM, SO, MM, AU, YK, AM, KN, SK, TS, TI, HN, YG, MR, TT, KY, NT, HK, JI, IN, TK, TT, MD, HN, KT, KS, KN, TT, HF, YS, KY, LR, NH, HM, TY, SW, TA, KT, TN, HK, FH, AH, HH, KT, TT, TK, TK, IG, GC, NA, HH, SK, TK, MH, YH, RM, KO, AH, LB, IY, YH, HK, YM, KI, HK, BK, HO, YY, AT, NK, SG, MI, MN, KM, EO, IO, TS, KY, HA, KT, KI, TS, AK, MF, NU, TK, TT, YY, MI, TH, KI, KN, SH, KE, KH, YT, SN, NS, TY, NU, TY, YK, RO, NS, KS, SH, KO, HM, TF, MT, HS, DS, YI, TI, YK, TY, KS, TM No competing interests declared, SY, NN Employee of Takeda Pharmaceutical Company, Ltd

C-H bond amination is an effective way to obtain nitrogen-containing products. In this work, we demonstrate that myoglobin reconstituted with iron porphycene (rMb(FePc)) catalyzes intramolecular C(sp 3 )-H bond amination of arylsulfonyl azides to yield corresponding sultam analogs. The total turnover number of rMb(FePc) is up to 5.7 × 10 4 for the C-H bond amination of 2,4,6-triisopropylbenzenesulfonyl azide. Moreover, rMb(FePc) exhibits higher selectivity for the desired C-H bond amination than the competing azide reduction compared to native myoglobin. Kinetic studies reveal that the k cat value of rMb(FePc) is 4-fold higher than that of native myoglobin. Furthermore, H64A, H64V and H64I mutants of rMb(FePc) enhance the turnover number (TON) and enantioselectivity for the C-H bond amination of 2,4,6-triethylbenzenesulfonyl azide. The present findings indicate that iron porphycene is an attractive artificial cofactor for myoglobin toward the C-H bond amination reaction., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Hydrogels containing synthetic polymers and supramolecular cross-linking units are expected to exhibit unique functions and properties. The heme-heme pocket interaction in hemeproteins may be useful for development of a cross-linking unit because heme binding depends on the redox states of the iron center. In this work, hexameric tyrosine-coordinated hemoprotein (HTHP) is employed as a cross-linking unit in a polyacrylamide gel to create redox-responsive hydrogels. First, redox-dependent stability of the heme-heme pocket interaction in HTHP was evaluated, and it was found that the heme affinity dramatically decreases in the Fe(ii) state. Second, the polymerization of acrylamide and engineered HTHP possessing acryloyl group-tethering heme moieties provided a polyacrylamide gel containing HTHP as a cross-linking unit. A reduction-triggered gel-sol transition in the presence of apomyoglobin was observed. Furthermore, the mechanical properties of the gels containing the engineered HTHP and methylene bisacrylamide were evaluated by a tensile test, and the Young's modulus value was determined to be 14 kPa, which is higher than that of the control gel containing only methylene bisacrylamide (8.5 kPa). Compression tests of the gels revealed redox-responsive mechanical behavior, resulting in a decrease in the compressive modulus upon the addition of a reductant. This behavior is qualitatively consistent with the redox-responsive heme binding of HTHP in a solution state. This finding is expected to contribute to the development of redox-responsive materials for biomedical and biological applications., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Affinity purification of recombinant proteins is an essential technique in biotechnology. However, current affinity purification methods are very cost-intensive, and this imposes limits on versatile use of affinity purification for obtaining purified proteins for a variety of applications. To overcome this problem, we developed a new affinity purification system which we call CSAP (chitin- and streptavidin-mediated affinity purification) for low-cost purification of Strep-tag II fusion proteins. The CSAP system is designed to utilize commercially available chitin powder as a chromatography matrix, thereby significantly improving the cost-efficiency of protein affinity purification. We investigated the CSAP system for protein screening in 96-well format as a demonstration. Through the screening of 96 types of purified hemoproteins, several proteins capable of the catalytic diastereodivergent synthesis of cyclopropanes were identified as candidates for an abiotic carbene transfer reaction., (© 2023 Wiley-VCH GmbH.)

A bipyridine-strapped porphyrin was prepared using a remote template effect of alkali or transition metal cations in the bipyridine subunit to enhance the yield 10-fold. The flexibility of the bipyridine-strap also allowed the synthesis of a doubly strapped porphyrin.

Evolutionary engineering of our previously reported Cp*Rh(III)-linked artificial metalloenzyme was performed based on a DNA recombination strategy to improve its catalytic activity toward C(sp 2 )-H bond functionalization. Improved scaffold design was achieved with α-helical cap domains of fatty acid binding protein (FABP) embedded within the β-barrel structure of nitrobindin (NB) as a chimeric protein scaffold for the artificial metalloenzyme. After optimization of the amino acid sequence by directed evolution methodology, an engineered variant, designated NB HLH1 (Y119A/G149P) with enhanced performance and enhanced stability was obtained. Additional rounds of metalloenzyme evolution provided a Cp*Rh(III)-linked NB HLH1 (Y119A/G149P) variant with a >35-fold increase in catalytic efficiency ( k cat / K M ) for cycloaddition of oxime and alkyne. Kinetic studies and MD simulations revealed that aromatic amino acid residues in the confined active-site form a hydrophobic core which binds to aromatic substrates adjacent to the Cp*Rh(III) complex. The metalloenzyme engineering process based on this DNA recombination strategy will serve as a powerful method for extensive optimization of the active-sites of artificial metalloenzymes.

Natural protein assemblies have encouraged scientists to create large supramolecular systems consisting of various protein motifs. In the case of hemoproteins containing heme as a cofactor, several approaches have been reported to form artificial assemblies with various structures such as fibers, sheets, networks, and cages. This chapter describes the design, preparation, and characterization of cage-like micellar assemblies for chemically modified hemoproteins including hydrophilic protein units attached to hydrophobic molecules. Detailed procedures are described for constructing specific systems using cytochrome b 562 and hexameric tyrosine-coordinated heme protein as hemoprotein units with heme-azobenzene conjugate and poly-N-isopropylacrylamide as attached molecules., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Long-lasting fear-related disorders depend on the excessive retention of traumatic fear memory. We previously showed that the palmitoylation-dependent removal of synaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors prevents hyperexcitation-based epileptic seizures and that AMPA receptor palmitoylation maintains neural network stability. In this study, AMPA receptor subunit GluA1 C-terminal palmitoylation-deficient (GluA1C811S) mice were subjected to comprehensive behavioral battery tests to further examine whether the mutation causes other neuropsychiatric disease-like symptoms. The behavioral analyses revealed that palmitoylation-deficiency in GluA1 is responsible for characteristic prolonged contextual fear memory formation, whereas GluA1C811S mice showed no impairment of anxiety-like behaviors at the basal state. In addition, fear generalization gradually increased in these mutant mice without affecting their cued fear. Furthermore, fear extinction training by repeated exposure of mice to conditioned stimuli had little effect on GluA1C811S mice, which is in line with augmentation of synaptic transmission in pyramidal neurons in the basolateral amygdala. In contrast, locomotion, sociability, depression-related behaviors, and spatial learning and memory were unaffected by the GluA1 non-palmitoylation mutation. These results indicate that impairment of AMPA receptor palmitoylation specifically causes posttraumatic stress disorder (PTSD)-like symptoms., (© 2022. The Author(s).)

Membrane lipid rafts are sphingolipids and cholesterol-enriched membrane microdomains, which form a center for the interaction or assembly of palmitoylated signaling molecules, including Src family non-receptor type protein tyrosine kinases. Lipid rafts abundantly exist in neurons and function in the maintenance of synapses. Excitatory synaptic strength is largely controlled by the surface expression of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors in the mammalian brain. AMPA receptor endocytosis from the synaptic surface is regulated by phosphorylation of the GluA2 subunit at tyrosine 876 by Src family kinases. Here, I revealed that tyrosine phosphorylated GluA2 is concentrated in the lipid rafts fraction. Furthermore, stimulation-induced upregulation of GluA2 tyrosine phosphorylation is disrupted by the treatment of neurons with a cholesterol-depleting compound, filipin III. These results indicate the importance of lipid rafts as enzymatic reactive sites for AMPA receptor tyrosine phosphorylation and subsequent AMPA receptor internalization from the synaptic surface., Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hayashi.)

Artificial protein hetero-dimerization is one of the promising strategies to construct protein-based chemical tools. In this work, cytochrome b 562 , an electron transfer hemoprotein, and green fluorescent protein (GFP) mutants with cysteine residues added to their surfaces were conjugated via a pyridyl disulphide-based thiol-disulfide exchange reaction. The eight hetero-dimers, which have cysteine residues at different positions to form the disulphide bonds, were obtained and characterized by gel-electrophoresis, mass spectrometry and size exclusion chromatography. The fluorescence properties of the hetero-dimers were evaluated by fluorescence spectroscopy and fluorescence lifetime measurements. Efficient photoinduced energy transfer from the GFP chromophore to the heme cofactor was observed in each of the hetero-dimers. The energy transfer efficiency is strongly dependent on the cross-linking residues, reaching 96%. Furthermore, the estimated Förster distance and the structure-based maximum possible distances of the donor and acceptor suggest that one of the hetero-dimers has a rigid protein-protein structure with favourable properties for energy transfer. The disulphide bond-mediated protein hetero-dimerization is useful for screening functional protein systems towards further developments., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

In vertebrates, newly emerging transformed cells are often apically extruded from epithelial layers through cell competition with surrounding normal epithelial cells. However, the underlying molecular mechanism remains elusive. Here, using phospho-SILAC screening, we show that phosphorylation of AHNAK2 is elevated in normal cells neighboring RasV12 cells soon after the induction of RasV12 expression, which is mediated by calcium-dependent protein kinase C. In addition, transient upsurges of intracellular calcium, which we call calcium sparks, frequently occur in normal cells neighboring RasV12 cells, which are mediated by mechanosensitive calcium channel TRPC1 upon membrane stretching. Calcium sparks then enhance cell movements of both normal and RasV12 cells through phosphorylation of AHNAK2 and promote apical extrusion. Moreover, comparable calcium sparks positively regulate apical extrusion of RasV12-transformed cells in zebrafish larvae as well. Hence, calcium sparks play a crucial role in the elimination of transformed cells at the early phase of cell competition., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Tiling patterns are observed in many biological structures. The compound eye is an interesting example of tiling and is often constructed by hexagonal arrays of ommatidia, the optical unit of the compound eye. Hexagonal tiling may be common due to mechanical restrictions such as structural robustness, minimal boundary length, and space-filling efficiency. However, some insects exhibit tetragonal facets. 1-4 Some aquatic crustaceans, such as shrimp and lobsters, have evolved with tetragonal facets. 5-8 Mantis shrimp is an insightful example as its compound eye has a tetragonal midband region sandwiched between hexagonal hemispheres. 9 , 10 This casts doubt on the naive explanation that hexagonal tiles recur in nature because of their mechanical stability. Similarly, tetragonal tiling patterns are also observed in some Drosophila small-eye mutants, whereas the wild-type eyes are hexagonal, suggesting that the ommatidial tiling is not simply explained by such mechanical restrictions. If so, how are the hexagonal and tetragonal patterns controlled during development? Here, we demonstrate that geometrical tessellation determines the ommatidial tiling patterns. In small-eye mutants, the hexagonal pattern is transformed into a tetragonal pattern as the relative positions of neighboring ommatidia are stretched along the dorsal-ventral axis. We propose that the regular distribution of ommatidia and their uniform growth collectively play an essential role in the establishment of tetragonal and hexagonal tiling patterns in compound eyes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The protein matrix of natural metalloenzymes regulates the reactivity of metal complexes to establish unique catalysts. We describe the incorporation of a cobalt complex of corrole (CoCor), a trianionic porphyrinoid metal ligand, into an apo-form of myoglobin to provide a reconstituted protein (rMb(CoCor)). This protein was characterized by UV-vis, EPR, and mass spectroscopic measurements. The reaction of rMb(CoCor) with hydrogen peroxide promotes an irreversible oxidation of the CoCor cofactor, whereas the same reaction in the presence of a phenol derivative yields the cation radical form of CoCor. Detailed kinetic investigations indicate the formation of a transient hydroperoxo complex of rMb(CoCor) which promotes the oxidation of the phenol derivatives. This mechanism is significantly different for native heme-dependent peroxidases, which generate a metal-oxo species as an active intermediate in a reaction with hydrogen peroxide. The present findings of unique reactivity will contribute to further design of artificial metalloenzymes.

The regulation of protein uptake and secretion is crucial for (inter)cellular signaling. Mimicking these molecular events is essential when engineering synthetic cellular systems. A first step towards achieving this goal is obtaining control over the uptake and release of proteins from synthetic cells in response to an external trigger. Herein, we have developed an artificial cell that sequesters and releases proteinaceous cargo upon addition of a coded chemical signal: single-stranded DNA oligos (ssDNA) were employed to independently control the localization of a set of three different ssDNA-modified proteins. The molecular coded signal allows for multiple iterations of triggered uptake and release, regulation of the amount and rate of protein release and the sequential release of the three different proteins. This signaling concept was furthermore used to directionally transfer a protein between two artificial cell populations, providing novel directions for engineering lifelike communication pathways inside higher order (proto)cellular structures., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Dual leucine-zipper kinase (DLK; a MAP3K) mediates neuronal responses to diverse injuries and insults through the c-Jun N-terminal kinase (JNK) family of mitogen-activated protein kinases (MAPKs). Here, we identified two ways through which DLK is coupled to the neural-specific isoform JNK3 to control prodegenerative signaling. JNK3 catalyzed positive feedback phosphorylation of DLK that further activated DLK and locked the DLK-JNK3 module in a highly active state. Neither homologous MAP3Ks nor a homologous MAPK could support this positive feedback loop. Unlike the related JNK1 isoform JNK2 and JNK3 promote prodegenerative axon-to-soma signaling and were endogenously palmitoylated. Moreover, palmitoylation targeted both DLK and JNK3 to the same axonal vesicles, and JNK3 palmitoylation was essential for axonal retrograde signaling in response to optic nerve crush injury in vivo. These findings provide previously unappreciated insights into DLK-JNK signaling relevant to neuropathological conditions and answer long-standing questions regarding the selective prodegenerative roles of JNK2 and JNK3.

Methyl-coenzyme M reductase (MCR) containing a nickel hydrocorphinoid cofactor, F430, is an essential enzyme that catalyzes anaerobic methane generation and oxidation. The active Ni(I) species in MCR converts methyl-coenzyme M (CH 3 S-CoM) and coenzyme B (HS-CoB) to methane and heterodisulfide (CoM-S-S-CoB). Extensive experimental and theoretical studies focusing on the substrate-binding cavity including the F430 cofactor in MCR have suggested two principally different reaction mechanisms involving an organonickel CH 3 -Ni(III) species or a transient methyl radical species. In parallel with research on native MCR itself, the functionality of MCR has been investigated in the context of model complexes of F430 and recent protein-based functional models, which include a nickel complex. In the latter case, hemoproteins reconstituted with tetradehydro- and didehydrocorrinoid nickel complexes have been found to represent useful model systems that are responsible for methane generation. These efforts support the proposed mechanism of the enzymatic reaction and provide important insight into replicating the MCR-like methane-generation process. Furthermore, the modeling of MCR described here is expected to lead to understanding of protein-supported nickel porphyrinoid chemistry as well as the creation of MCR-inspired catalysis.

SIRT3 is an NAD+-dependent protein deacetylase localized in mitochondria. Several studies reported localization of SIRT3 in the cytoplasm or nucleus, but data of these studies were not consistent. We detected expression of mitochondrial (SIRT3mt) and cytoplasmic (SIRT3ct) Sirt3 mRNAs in the mouse brain, and we also found SIRT3 immunostaining of mitochondria and cytoplasm in the brain and cultured neural cells. However, expression levels of SIRT3ct in COS cells transfected with SIRT3ct cDNA were much lower than those of SIRT3mt. We found that SIRT3ct but not SIRT3mt was promptly degraded by ubiquitin-dependent degradation, in which SIRT3ct degradation was mediated mainly by ubiquitination of NH2-terminal methionine and partly by that of lysine residues of SIRT3ct. SIRT3ct expression level was significantly enhanced by the treatment of cells with staurosporine or H2O2. H2O2 treatment promoted nuclear translocation of SIRT3ct and induced histone H3 deacetylation and superoxide dismutase 2 expression. Overexpression of SIRT3ct decreased cell death caused by H2O2 at levels similar to those achieved by overexpression of SIRT3mt. Knockdown of Sirt3 mRNA increased cell death caused by amyloid-β (Aβ), and overexpression of SIRT3ct suppressed the toxic function of Aβ in PC12 cells. These results indicate that SIRT3ct promotes cell survival under physiological and pathological conditions., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Fe/N/C single-atom catalysts containing Fe-N x sites prepared by pyrolysis are promising cathode materials for fuel cells and metal-air batteries due to their high oxygen reduction reaction (ORR) activities. We have developed iron complexes containing N2- or N3-chelating coordination structures with preorganized aromatic rings in a 1,12-diazatriphenylene framework tethering bromo substituents as precursors to precisely construct Fe-N 4 sites in an Fe/N/C catalyst. One-step pyrolysis of the iron complex with carbon black forms atomically dispersed Fe-N 4 sites without iron aggregates. X-ray absorption spectroscopy (XAS) and electrochemical measurements revealed that the iron complex with N3-coordination is more effectively converted to Fe-N 4 sites catalyzing ORR with a TOF value of 0.21 e site -1  s -1 at 0.8 V vs. RHE. This indicates that the formation of Fe-N 4 sites is controlled by precise tuning of the chemical structure of the iron complex precursor., (© 2021 Wiley-VCH GmbH.)

A 70-year-old woman presented with a 6-year history of cognitive dysfunction, neurogenic bladder, constipation and recurrent vomiting, and gradual worsening of symptoms. At the first admission to our department, she was also found to have hepatic encephalopathy due to intrahepatic portosystemic shunt. Head MRI revealed abnormal signal intensity at the corticomedullary junction, the splenium of the corpus callosum, and bilateral middle cerebellar peduncles on DWI. She was diagnosed with intranuclear inclusion disease (NIID) based on skin biopsy and genetic testing of NOTCH2NLC. In a retrospective review of serial head MRI findings for ten years, abnormal signal intensity at the corticomedullary junction and the splenium of the corpus callosum on MRI existed prior to the onset of cognitive dysfunction, and expanded gradually. For early diagnosis of NIID, it is important to focus not only on the characteristic high signal intensity at the corticomedullary junction, but also on the signal at the splenium of the corpus callosum from the early stage.

Glutamate is the major excitatory neurotransmitter in the vertebrate brain and various modifications have been established in the glutamatergic synapses. Generally, many neuronal receptors and ion channels are regulated by S -palmitoylation, a reversible post-translational protein modification. Genome sequence databases show the evolutionary acquisition and conservation concerning vertebrate-specific palmitoylation of synaptic proteins including glutamate receptors. Moreover, palmitoylation of some glutamate receptor-binding proteins is subsequently acquired only in some mammalian lineages. Recent progress in genome studies has revealed that some palmitoylation-catalyzing enzymes are the causative genes of neuropsychiatric disorders. In this review, I will summarize the evolutionary development of palmitoylation-dependent regulation of glutamatergic synapses and their dysfunctions which are caused by the disruption of palmitoylation mechanism., Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hayashi.)

AMPA receptors are responsible for fast excitatory synaptic transmission in the mammalian brain. Post-translational protein S -palmitoylation of AMPA receptor subunits GluA1-4 reversibly regulates synaptic AMPA receptor expression, resulting in long-lasting changes in excitatory synaptic strengths. Our previous studies have shown that GluA1 C-terminal palmitoylation-deficient (GluA1C811S) mice exhibited hyperexcitability in the cerebrum and elevated seizure susceptibility without affecting brain structure or basal synaptic transmission. Moreover, some inhibitory GABAergic synapses-targeting anticonvulsants, such as valproic acid, phenobarbital, and diazepam, had less effect on these AMPA receptor palmitoylation-deficient mutant mice. This work explores pharmacological effect of voltage-gated ion channel-targeted anticonvulsants, phenytoin and trimethadione, on GluA1C811S mice. Similar to GABAergic synapses-targeting anticonvulsants, anticonvulsive effects were also reduced for both sodium channel- and calcium channel-blocking anticonvulsants, which suppress excess excitation. These data strongly suggest that the GluA1C811S mice generally underlie the excessive excitability in response to seizure-inducing stimulation. AMPA receptor palmitoylation site could be a novel target to develop unprecedented type of anticonvulsants and GluA1C811S mice are suitable as a model animal for broadly evaluating pharmacological effectiveness of antiepileptic drugs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Iizumi, Oota-Ishigaki, Yamashita and Hayashi.)

A water-soluble strapped iron(III)tetraarylporphyrin ( Fe III Por-1 ) bearing two propylpyridinium groups at the side chains and a carboxylic acid group at the overhanging position of the strap was synthesized to mimic the function of myoglobin with the distal polar functionality in aqueous solution. Fe III Por-1 forms a stable 1:1 inclusion complex with a per- O -methylated β-cyclodextrin dimer having a pyridine linker ( Py3OCD ), providing a hydrophobic environment and a proximal fifth ligand to stabilize the O 2 -complex. The ferrous complex ( Fe II PorCD-1 ) binds both O 2 and CO in aqueous solution. The O 2 and CO binding affinities ( P 1/2 O2 and P 1/2 CO ) and half-life time ( t 1/2 ) of the O 2 complex of Fe II PorCD-1 are 6.3 and 0.021 Torr, and 7 h, respectively, at pH 7 and 25 °C. The control compound without the strap structure ( Fe II PorCD-2 ) has similar oxygen binding characteristics ( P 1/2 O2 = 8.0 Torr), but much higher CO binding affinity ( P 1/2 CO = 3.8 × 10 -4 Torr), and longer t 1/2 (30 h). The O 2 and CO kinetics indicate that the strapped structure in Fe II PorCD-1 inhibits the entrance of these gaseous ligands into the iron(II) center, as evidenced by lower k on O2 and k on CO values. Interestingly, the CO complex of Fe II PorCD-1 is significantly destabilized (relatively larger k off CO ), while the k off O2 value is much smaller than that of Fe II PorCD-2 , resulting in significantly increased O 2 /CO selectivity (reduced M value, where M = P 1/2 O2 / P 1/2 CO = 320) in Fe II PorCD-1 compared to Fe II PorCD-2 ( M = 21000).

Hexameric hemoprotein (HTHP) is employed as a scaffold protein for the supramolecular assembly and activation of the apoptotic signalling enzyme caspase-9, using short DNA elements as modular recruitment domains. Caspase-9 assembly and activation on the HTHP platform due to enhanced proximity is followed by combinatorial inhibition at high scaffold concentrations. The DNA recruitment domains allow for reversible switching of the caspase-9 assembly and activity state using short modulatory DNA strands. Tuning of the recruitment domain affinity allows for generating kinetically trapped active enzyme complexes, as well as for dynamic repositioning of caspases over scaffold populations and inhibition using monovalent sink platforms. The conceptual combination of a highly structured multivalent protein platform with modular DNA recruitment domains provides emergent biomimicry properties with advanced levels of control over protein assembly., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Pyrolytically prepared iron and nitrogen codoped carbon (Fe/N/C) catalysts are promising nonprecious metal electrocatalysts for the oxygen reduction reaction (ORR) in fuel cell applications. Fabrication of the Fe/N/C catalysts with Fe-N x active sites having precise structures is now required. We developed a strategy for thermally controlled construction of the Fe-N x structure in Fe/N/C catalysts by applying a bottom-up synthetic methodology based on a N-doped graphene nanoribbon (N-GNR). The preorganized aromatic rings within the precursors assist graphitization during generation of the N-GNR structure with iron-coordinating sites. The Fe/N/C catalyst prepared from the N-GNR precursor, iron ion, and the carbon support Vulcan XC-72R provides a high onset potential of 0.88 V (vs reversible hydrogen electrode (RHE)) and promotes efficient four-electron ORR. X-ray absorption fine structure (XAFS) and X-ray photoelectron spectroscopy (XPS) studies reveal that the N-GNR precursor induces the formation of iron-coordinating nitrogen species during pyrolysis. The details of the graphitization process of the precursor were further investigated by analyzing the precursors pyrolyzed at various temperatures using MgO particles as a sacrificial template, with the results indicating that the graphitized structure was obtained at 700 °C. The preorganized N-GNR precursors and its pyrolysis conditions for graphitization are found to be important factors for generation of the Fe-N x active sites along with the N-GNR structure in high-performance Fe/N/C catalysts for the ORR.

A whole-cell biohybrid catalyst where a (pentamethylcyclopentadienyl)rhodium(III) (Cp*Rh(III)) complex was covalently incorporated into the cavity of nitrobindin (NB), a β-barrel protein, was prepared on an E. coli cell surface to produce isoquinolines via C(sp 2 )-H bond activation. In this whole-cell biohybrid system, the Cp*Rh(III)-dithiophosphate complex with latent catalytic activity was utilized as a precursor of the metal cofactor. Strong chelation of the dithiophosphate ligands protects the rhodium complex from being deactivated by abundant nucleophiles in cellular environments during conjugation of the cofactor with the protein scaffold. The whole-cell biohybrid catalyst was then activated upon addition of Ag + ion to dissociate the dithiophosphate ligands and promoted cycloaddition of acetophenone oxime with diphenylacetylene. Furthermore, the activity of the Cp*Rh(III)-linked whole-cell biohybrid catalyst was enhanced 2.1-fold by introducing glutamate residues at positions adjacent to the Cp*Rh(III) cofactor. These results indicate that the use of the Cp*Rh(III)-dithiophosphate complex with switchable activity from a "latent" form to an "active" form provides a new strategy for generating whole-cell biohybrid catalysts., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Directed evolution of Cp*Rh III -linked nitrobindin (NB), a biohybrid catalyst, was performed based on an in vitro screening approach. A key aspect of this effort was the establishment of a high-throughput screening (HTS) platform that involves an affinity purification step employing a starch-agarose resin for a maltose binding protein (MBP) tag. The HTS platform enables efficient preparation of the purified MBP-tagged biohybrid catalysts in a 96-well format and eliminates background influence of the host E. coli cells. Three rounds of directed evolution and screening of more than 4000 clones yielded a Cp*Rh III -linked NB(T98H/L100K/K127E) variant with a 4.9-fold enhanced activity for the cycloaddition of acetophenone oximes with alkynes. It is confirmed that this HTS platform for directed evolution provides an efficient strategy for generating highly active biohybrid catalysts incorporating a synthetic metal cofactor., (© 2020 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Metalloenzymes naturally achieve various reactivities by assembling limited types of cofactors with endogenous amino acid residues. Enzymes containing metal porphyrinoid cofactors such as heme, cobalamin and F430 exert precise control over the reactivities of the cofactors with protein matrices. This perspective article focuses on our recent efforts to assemble metal complexes of non-natural porphyrinoids within the protein matrix of myoglobin, an oxygen storage hemoprotein. Engineered myoglobins with suitable metal complexes as artificial cofactors demonstrate unique reactivities toward C-H bond hydroxylation, olefin cyclopropanation, methyl group transfer and methane generation. In these cases, the protein matrix enhances the catalytic activities of the cofactors and allows us to monitor the active intermediates. The present findings indicate that placing artificial cofactors in protein matrices provides a useful strategy for creating artificial metalloenzymes that catalyse otherwise unfavourable reactions and providing enzyme models for elucidating the complicated reaction mechanisms of natural enzymes.

In the mammalian CNS, glutamate is the major excitatory neurotransmitter. Ionotropic glutamate receptors (iGluRs) are responsible for the glutamate-mediated postsynaptic excitation of neurons. Regulation of glutamatergic synapses is critical for higher brain functions including neural communication, memory formation, learning, emotion, and behaviour. Many previous studies have shown that post-translational protein S-palmitoylation, the only reversible covalent attachment of lipid to protein, regulates synaptic expression, intracellular localization, and membrane trafficking of iGluRs and their scaffolding proteins in neurons. This modification mechanism is extremely conserved in the vertebrate lineages. The failure of appropriate palmitoylation-dependent regulation of iGluRs leads to hyperexcitability that reduces the maintenance of network stability, resulting in brain disorders, such as epileptic seizures. This review summarizes advances in the study of palmitoylation of iGluRs, especially AMPA receptors and NMDA receptors, and describes the current understanding of palmitoylation-dependent regulation of excitatory glutamatergic synapses. LINKED ARTICLES: This article is part of a themed issue on Neurochemistry in Japan. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.4/issuetoc., (© 2020 The British Pharmacological Society.)

Proteins have been used as building blocks to provide various supramolecular structures in efforts to develop nano-biomaterials possessing broad biological functionalities. A series of unique structures have been obtained from the engineering of hemoproteins which contain the iron porphyrin known as heme, as a prosthetic group. This work in developing assembling systems is extended using cytochrome b 562 , a small electron transfer hemoprotein engineered to include an externally-attached heme moiety. The engineered units, which form a one-dimensional assembly via interprotein heme-heme pocket interactions, are conjugated to an apo-form of hexameric tyrosine-coordinated hemoprotein (apoHTHP) to provide a branching unit promoting the assembly of a star-shaped structure. The incorporation of the heme moiety attached to the protein surface of cytochrome b 562 into apoHTHP can be accelerated by elevating the reaction temperature to generate a new assembly. The formation of a new larger assembly structure was confirmed by size exclusion chromatography. The ratio of the heme-containing units in the assemblies was analyzed by UV-Vis spectroscopy and the population of protein units estimated from SDS PAGE suggests the presence of plausible star-shaped structures, which are supported by hydrodynamic diameter data obtained by dynamic light scattering.

This paper studies aggregation of preferences under ignorance, in which everybody knows that the true probability distribution over outcomes lies in some objective set but knows nothing about which one in it is true or which one in it is more likely to be true. We consider two decision models which express the precautionary principle under ignorance, the maximin criterion and the α -maximin criterion. We show that the Pareto axiom implies dictatorship, in each decision model. The impossibility results force us to choose between two options, one is to give up the precautionary principle as modelled at the social level, the other is to weaken the Pareto axiom. We provide possibility results for each of the options., (© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2021.)

A Cp*Rh(III)-dithiophosphate cofactor with "latent" catalytic activity was developed to construct an artificial metalloenzyme representing a new type of biohybrid catalyst which is capable of promoting C(sp 2 )-H bond functionalization within the β-barrel structure of nitrobindin (NB). To covalently conjugate the Cp*Rh(III) cofactor into a specific position of the hydrophobic cavity of NB via a maleimide-Cys linkage, strong chelation of the dithiophosphate ligand is employed to protect the rhodium metal center against attack by nucleophilic amino acid residues in the protein. It is found that subsequent addition of the Ag + ion induces dissociation of the dithiophosphate ligands, thereby activating the catalytic activity of the Cp*Rh(III) cofactor. The resulting "active" biohybrid catalyst promotes cycloaddition of acetophenone oxime with diphenylacetylene via C(sp 2 )-H bond activation. This catalytic activity is enhanced 2.3-fold with the introduction of two glutamate residues (A100E/L125E) adjacent to the Cp*Rh(III) cofactor. The Cp*Rh(III) cofactor with switchable activity from a "latent" form to an "active" form provides a new strategy for generating biohybrid catalysts incorporating a variety of highly reactive transition metal complexes specifically within its protein scaffolds.

Significant advancement of chemoproteomics has contributed to uncovering the mechanism of action (MoA) of small-molecule drugs by characterizing drug-protein interactions in living systems. However, cell-membrane proteins such as G protein-coupled receptors (GPCRs) and ion channels, due to their low abundance and unique biophysical properties associated with multiple transmembrane domains, can present challenges for proteome-wide mapping of drug-receptor interactions. Herein, we describe the development of novel tetrafunctional probes, consisting of (1) a ligand of interest, (2) 2-aryl-5-carboxytetrazole (ACT) as a photoreactive group, (3) a hydrazine-labile cleavable linker, and (4) biotin for enrichment. In live cell labeling studies, we demonstrated that the ACT-based probe showed superior reactivity and selectivity for labeling on-target GPCR by mass spectrometry analysis compared with control probes including diazirine-based probes. By leveraging ACT-based cleavable probes, we further identified a set of representative ionotropic receptors, targeted by CNS drugs, with remarkable selectivity and precise binding site information from mouse brain slices. We anticipate that the robust chemoproteomic platform using the ACT-based cleavable probe coupled with phenotypic screening should promote identification of pharmacologically relevant target receptors of drug candidates and ultimately development of first-in-class drugs with novel MoA.

Methyl-coenzyme M reductase (MCR), which contains the nickel hydrocorphinoid cofactor F430, is responsible for biological methane generation under anaerobic conditions via a reaction mechanism which has not been completely elucidated. In this work, myoglobin reconstituted with an artificial cofactor, nickel(I) tetradehydrocorrin (Ni I (TDHC)), is used as a protein-based functional model for MCR. The reconstituted protein, rMb(Ni I (TDHC)), is found to react with methyl donors such as methyl p -toluenesulfonate and trimethylsulfonium iodide with methane evolution observed in aqueous media containing dithionite. Moreover, rMb(Ni I (TDHC)) is found to convert benzyl bromide derivatives to reductively debrominated products without homocoupling products. The reactivity increases in the order of primary > secondary > tertiary benzylic carbons, indicating steric effects on the reaction of the nickel center with the benzylic carbon in the initial step. In addition, Hammett plots using a series of para -substituted benzyl bromides exhibit enhancement of the reactivity with introduction of electron-withdrawing substituents, as shown by the positive slope against polar substituent constants. These results suggest a nucleophilic S N 2-type reaction of the Ni(I) species with the benzylic carbon to provide an organonickel species as an intermediate. The reaction in D 2 O buffer at pD 7.0 causes a complete isotope shift of the product by +1 mass unit, supporting our proposal that protonation of the organonickel intermediate occurs during product formation. Although the turnover numbers are limited due to inactivation of the cofactor by side reactions, the present findings will contribute to elucidating the reaction mechanism of MCR-catalyzed methane generation from activated methyl sources and dehalogenation.

The brain is organized morphologically and functionally into a columnar structure. According to the radial unit hypothesis, neurons from the same lineage form a radial unit that contributes to column formation. However, the molecular mechanisms that link neuronal lineage and column formation remain elusive. Here, we show that neurons from the same lineage project to different columns under control of Down syndrome cell adhesion molecule (Dscam) in the fly brain. Dscam1 is temporally expressed in newly born neuroblasts and is inherited by their daughter neurons. The transient transcription of Dscam1 in neuroblasts enables the expression of the same Dscam1 splice isoform within cells of the same lineage, causing lineage-dependent repulsion. In the absence of Dscam1 function, neurons from the same lineage project to the same column. When the splice diversity of Dscam1 is reduced, column formation is significantly compromised. Thus, Dscam1 controls column formation through lineage-dependent repulsion.

Previous studies have revealed that, at the initial step of carcinogenesis, transformed cells are often eliminated from epithelia via cell competition with the surrounding normal cells. In this study, we performed cell competition-based high-throughput screening for chemical compounds using cultured epithelial cells and confocal microscopy. PLX4720 was identified as a hit compound that promoted apical extrusion of RasV12-transformed cells surrounded by normal epithelial cells. Knockdown/knockout of ZAK, a target of PLX4720, substantially enhanced the apical elimination of RasV12 cells in vitro and in vivo. ZAK negatively modulated the accumulation or activation of multiple cell competition regulators. Moreover, PLX4720 treatment promoted apical elimination of RasV12-transformed cells in vivo and suppressed the formation of potentially precancerous tumors. This is the first report demonstrating that a cell competition-promoting chemical drug facilitates apical elimination of transformed cells in vivo, providing a new dimension in cancer preventive medicine., Competing Interests: Declaration of Interests The authors declare no competing of interest., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Site-specific modification of peptides and proteins is a key aspect of protein engineering. We developed a method for modification of the N terminus of proteins using 1H-1,2,3-triazole-4-carbaldehyde (TA4C) derivatives, which can be prepared in one step. The N-terminal specific labeling of bioactive peptides and proteins with the TA4C derivatives proceeds under mild reaction conditions in excellent conversion (angiotensin I: 92 %, ribonuclease A: 90 %). This method enables site-specific conjugation of various functional molecules such as fluorophores, biotin, and polyethylene glycol attached to the triazole ring to the N terminus. Furthermore, a functional molecule modified with a primary amine moiety can be directly converted into a TA4C derivative through a Dimroth rearrangement reaction with 1-(4-nitrophenyl)-1H-1,2,3-triazole-4-carbaldehyde. This method can be used to obtain N-terminal-modified proteins via only two steps: 1) convenient preparation of a TA4C derivative with a functional group and 2) modification of the N terminus of the protein with the TA4C derivative., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The beneficial effects of omega (ω)-3 polyunsaturated fatty acid (PUFA) supplementation on major depressive disorder have been actively studied, but the underlying mechanism remains unknown. The present study examined the involvement of the nucleus accumbens (NAc) dopaminergic systems in behavioral changes in mice fed a diet high in ω-3 PUFAs. Mice fed a diet containing about double the amount of ω-3 PUFAs (krill oil (KO) diet) exerted shorter immobility times in the forced swim test (FST) than mice fed a control diet, containing only α-linolenic acid (ALA) as ω-3 PUFAs. The shorter immobility times were observed in both male and female mice. A dopamine metabolite, 3,4-dihydroxyphenylacetic acid, increased in the NAc in male mice fed the KO diet when compared with those fed the control diet. In addition, dopamine, 3-methoxytyramine, and homovanillic acid increased in the NAc in female mice fed the KO diet. Notably, the effects of the KO diet on the immobility time in the FST were abolished by microinjection of sulpiride, an antagonist of D2-like receptors, into the NAc. A similar microinjection of an antagonist selective for D1-like receptors, SKF83566, also abolished the reduction in immobility in the FST. Moreover, we found that tyrosine hydroxylase-positive cells increased in the ventral tegmental area (VTA) in mice fed the KO diet. These results suggest that modulation of the VTA-NAc dopaminergic pathway is one of the mechanisms by which a KO diet rich in ω-3 PUFAs reduces the immobility behavior in the mouse FST., Competing Interests: The authors have declared that no competing interests exist.

Artificial protein assemblies inspired by nature have significant potential in development of emergent functional materials. In order to construct an artificial protein assembly, we employed a mutant of a thermostable hemoprotein, hexameric tyrosine-coordinated heme protein (HTHP), as a building block. The HTHP mutant which has cysteine residues introduced on the bottom surface of its columnar structure was reacted with maleimide-tethering thermoresponsive poly( N -isopropylacrylamide), PNIPAAm, to generate the protein assembly upon heating. The site-specific modification of the cysteine residues with PNIPAAm on the protein surface was confirmed by SDS-PAGE and analytical size exclusion chromatography (SEC). The PNIPAAm-modified HTHP (PNIPAAm-HTHP) is found to provide a 43 nm spherical structure at 60 °C, and the structural changes observed between the assembled and the disassembled forms were duplicated at least five times. High-speed atomic force microscopic measurements of the micellar assembly supported by cross-linkage with glutaraldehyde indicate that the protein matrices are located on the surface of the sphere and cover the inner PNIPAAm core. Furthermore, substitution of heme with a photosensitizer, Zn protoporphyrin IX (ZnPP), in the micellar assembly provides an artificial light-harvesting system. Photochemical measurements of the ZnPP-substituted micellar assembly demonstrate that energy migration among the arrayed ZnPP molecules occurs within the range of several tens of picoseconds. Our present work represents the first example of an artificial light-harvesting system based on an assembled hemoprotein oligomer structure to replicate natural light-harvesting systems.

Introduction: In patients receiving chronic anticoagulation agents, antiplatelet agents, or both, perioperative antithrombotic therapy for inguinal hernia repair requires an understanding of potential side-effects-specifically, the postoperative bleeding risks. In the present study, we evaluated postoperative bleeding complications after transabdominal preperitoneal patch plasty (TAPP) in patients undergoing antithrombotic therapy., Methods: We retrospectively reviewed 413 patients who had undergone TAPP between February 2013 and June 2017. Individuals in the antithrombotic group received one of three regimens of perioperative antithrombotic therapy. The clinical indications for chronic anticoagulation agents (ie bridging therapy with unfractionated heparin), antiplatelet agents (ie continuation of aspirin), or both were followed. The antithrombotic group was compared to the control group in terms of surgical outcomes. We primarily focused on the incidence of postoperative bleeding complications., Results: A total of 83 patients received antithrombotic therapy. We observed significant differences between the groups in terms of mean age, ASA physical status, and length of postoperative stay. In contrast, postoperative complications were not significantly different between the antithrombotic and control groups (4.8% vs 5.5%, P = 0.818). In addition, a significantly greater postoperative bleeding rate was not observed in the antithrombotic group than in the control group (1.2% vs 0.6%, P = 0.566). Likewise, other complications were similar in both groups., Conclusions: Antithrombotic therapy is not a risk factor for postoperative bleeding complications in patients who have undergone TAPP, suggesting its safety and efficacy in this patient population. Indeed, this group has the same incidence rates of morbidity and postoperative bleeding complications as patients who have not undergone antithrombotic therapy., (© 2019 Japan Society for Endoscopic Surgery, Asia Endosurgery Task Force and John Wiley & Sons Australia, Ltd.)

Objective: The purpose of this study was to clarify the influence of consuming yogurt fermented with Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 (1073R-1-yogurt) on influenza virus-bound salivary immunoglobulin A (IgA) levels, in the elderly residents of nursing homes. Methods: A double-blind, parallel-group, randomized controlled trial was conducted with 96 elderly volunteers residing in 2 nursing homes. During the trial, participants consumed 100 g of 1073R-1-yogurt every morning for 12 weeks, whereas the control participants consumed yogurt fermented with a different Lactobacillus strain (control yogurt). Saliva was collected before the trial and after 4, 8 and 12 weeks of yogurt ingestion. Results: Our data indicated that consumption of 1073R-1-yogurt affected influenza A virus subtype H3N2-bound IgA levels in saliva ( p  = .001). In addition, saliva flow rate and total IgA levels increased in response to the yogurt intake period in both the 1073R-1 and control yogurt groups ( p  = .04). Conclusions: Our study suggests that continuous daily ingestion of 1073R-1-yogurt may help prevent infection with influenza A virus subtype H3N2 in elderly subjects with weakened immunity, by increasing the production of influenza A virus subtype of H3N2-bound salivary IgA.

Myoglobin reconstituted with Ni tetradehydrocorrin was investigated as a model of F430-containing methyl-coenzyme M reductase, which catalyzes anaerobic methane generation. The Ni II tetradehydrocorrin complex has a Ni II /Ni I redox potential of -0.34 V vs. SHE and EPR spectroscopy indicates the formation of a Ni I species upon reduction by dithionite. This redox potential is approximately 0.31 V more positive than that of F430. The Ni I tetradehydrocorrin moiety is bound to the apo-form of myoglobin to yield the reconstituted protein. Methane gas is generated in the reaction of the model with methyl iodide in the presence of the reconstituted protein under reductive conditions, whereas the Ni I complex itself does not produce methane gas. This is the first example of a protein-based functional model of F430-containing methyl-coenzyme M reductase., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Site-specific modification of peptides and proteins is an important method for introducing an artificial function to the protein surface. Recently, we found that new bioconjugation reagents, 6-(azidomethyl)-2-pyridinecarbaldehyde (6AMPC) derivatives, allow specific N-terminal modification and enhance the reaction rate of the subsequent bioconjugation in a chelation-assisted CuAAC reaction. The N-terminal specific azide-labeling of bioactive peptides and proteins occurs under mild reaction conditions with 6AMPC derivatives (angiotensin I: 90%, ribonuclease A: 90%). Kinetic analysis of the CuAAC reaction with azide-labeled proteins reveals that the ligation is promoted in the presence of a copper-chelating pyridine moiety. Importantly, the introduction of an electron-donating methoxy group to the pyridine moiety further accelerates the CuAAC ligation. We demonstrate that this method enables site-specific conjugation of various functional molecules such as fluorophores, biotin, and polyethylene glycol.

In nature, heme cofactor-containing proteins participate not only in electron transfer and O 2 storage and transport but also in biosynthesis and degradation. The simplest and representative cofactor, heme b, is bound within the heme pocket via noncovalent interaction in many hemoproteins, suggesting that the cofactor is removable from the protein, leaving a unique cavity. Since the cavity functions as a coordination sphere for heme, it is of particular interest to investigate replacement of native heme with an artificial metal complex, because the substituted metal complex will be stabilized in the heme pocket while providing alternative chemical properties. Thus, cofactor substitution has great potential for engineering of hemoproteins with alternative functions. For these studies, myoglobin has been a focus of our investigations, because it is a well-known oxygen storage hemoprotein. However, the heme pocket of myoglobin has been only arranged for stabilizing the heme-bound dioxygen, so the structure is not suitable for activation of small molecules such as H 2 O 2 and O 2 as well as for binding an external substrate. Thus, the conversion of myoglobin to an enzyme-like biocatalyst has presented significant challenges. The results of our investigations have provided useful information for chemists and biologists. Our own efforts to develop functionalized myoglobin have focused on the incorporation of a chemically modified cofactor into apomyoglobin in order to (1) construct an artificial substrate-binding site near the heme pocket, (2) increase cofactor reactivity, or (3) promote a new reaction that has never before been catalyzed by a native heme enzyme. In pursuing these objectives, we first found that myoglobin reconstituted with heme having a chemically modified heme-propionate side chain at the exit of the heme pocket has peroxidase activity with respect to oxidation of phenol derivatives. Our recent investigations have succeeded in enhancing oxidation and oxygenation activities of myoglobin as well as promoting new reactions by reconstitution of myoglobin with new porphyrinoid metal complexes. Incorporation of suitable metal porphyrinoids into the heme pocket has produced artificial enzymes capable of efficiently generating reactive high valent metal-oxo and metallocarbene intermediates to achieve the catalytic hydroxylation of C(sp 3 )-H bonds and cyclopropanation of olefin molecules, respectively. In other efforts, we have focused on nitrobindin, an NO-binding hemoprotein, because aponitrobindin includes a β-barrel cavity, which provides a robust structure highly similar to that of the native holoprotein. It was expected that the aponitrobindin would be suitable for development as a protein scaffold for a metal complex. Recently, it was confirmed that several organometallic complexes can bind to this scaffold and function as catalysts promoting hydrogen evolution or C-C bond formation. The hydrophobic β-barrel structure plays a significant role in substrate binding as well as controlling the stereoselectivity of the reactions. Furthermore, these catalytic activities and stereoselectivities are remarkably improved by mutation-dependent modifications of the cavity structure for the artificial cofactor. This Account demonstrates how apoproteins of hemoproteins can provide useful protein scaffolds for metal complexes. Further development of these concepts will provide a useful strategy for generation of robust and useful artificial metalloenzymes.

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 b 562 (Cyt b 562 ) 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: k CS  = 320-600 s -1 ) and subsequent charge recombination (CR: k CR  = 580-930 s -1 ) were observed in both the Cyt b 562 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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: AMPA receptors predominantly mediate fast excitatory synaptic transmission in the mammalian brain. Post-translational protein S-palmitoylation of AMPA receptor GluA subunits at their C-termini reversibly controls the receptors trafficking to and from excitatory glutamatergic synapses. Excitatory inputs to neurons induce the expression of immediate early genes (IEGs), including Arc, with particular spatial patterns. In the hippocampal dentate gyrus, Arc is mainly expressed in the upper (dorsal) blade at the basal state. GluA1 C-terminal palmitoylation-deficient (GluA1C811S) mice showed enhanced seizure susceptibility and disturbed synaptic plasticity without impaired gross anatomy or basal synaptic transmission. These mutant mice also exhibited an increased expression of IEG products, c-Fos and Arc proteins, in the hippocampus and cerebral cortex. In this report, we further analyzed excitability and Arc expression pattern in the dentate gyrus of GluA1C811S mice., Methods and Results: Electrophysiological analysis of granule neurons to measure the evoked excitatory postsynaptic current/evoked inhibitory postsynaptic current ratio revealed that excitatory/inhibitory (E/I) balance was normal in GluA1C811S mice. In contrast, immunohistochemical staining showed an abnormal distribution of Arc-positive cells between upper and lower (ventral) blades of the dentate gyrus in these mutant mice. These data suggest that deficiency of GluA1 palmitoylation causes perturbed neuronal inputs from the entorhinal cortex to the dentate gyrus, which potentially underlies the excessive excitability in response to seizure-inducing stimulation., Conclusion: Our findings conclude that an appropriate regulation of Arc expression in the dentate gyrus, ensured by AMPA receptor palmitoylation, may be critical for stabilizing hippocampal neural circuits and may suppress excess excitation., (© 2018 The Authors. Neuropsychopharmacology Reports published by John Wiley & Sons Australia, Ltd on behalf of The Japanese Society of Neuropsychopharmacology.)