Redox Engineering of Myoglobin by Cofactor Substitution to Enhance Cyclopropanation Reactivity.

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 (FePorCF3 and FePor(CF3)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(CF3)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(CF3)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.

Limb-Clasping Response in NMDA Receptor Palmitoylation-Deficient Mice.

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.

Intramolecular C-H bond amination catalyzed by myoglobin reconstituted with iron porphycene.

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(sp3)-H bond amination of arylsulfonyl azides to yield corresponding sultam analogs. The total turnover number of rMb(FePc) is up to 5.7 × 104 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 kcat 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.

A polyacrylamide gel containing an engineered hexameric hemoprotein as a cross-linking unit toward redox-responsive materials.

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.

Chitin- and Streptavidin-Mediated Affinity Purification Systems: A Screening Platform for Enzyme Discovery.

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.

Preparation of Cage-Like Micellar Assemblies of Engineered Hemoproteins.

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 b562 and hexameric tyrosine-coordinated heme protein as hemoprotein units with heme-azobenzene conjugate and poly-N-isopropylacrylamide as attached molecules.

Remote template effect in the synthesis of bipyridine-strapped porphyrins.

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 a Cp*Rh(III) Complex-Linked Artificial Metalloenzyme with a Chimeric ß-Barrel Protein Scaffold.

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(sp2)-H bond functionalization. Improved scaffold design was achieved with α-helical cap domains of fatty acid binding protein (FABP) embedded within the ß-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 NBHLH1(Y119A/G149P) with enhanced performance and enhanced stability was obtained. Additional rounds of metalloenzyme evolution provided a Cp*Rh(III)-linked NBHLH1(Y119A/G149P) variant with a >35-fold increase in catalytic efficiency (kcat/KM) 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.

A disulphide bond-mediated hetero-dimer of a hemoprotein and a fluorescent protein exhibiting efficient energy transfer.

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.

Membrane lipid rafts are required for AMPA receptor tyrosine phosphorylation.

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.

Calcium sparks enhance the tissue fluidity within epithelial layers and promote apical extrusion of transformed cells.

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.

Reactivity of Myoglobin Reconstituted with Cobalt Corrole toward Hydrogen Peroxide.

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.

Prolonged contextual fear memory in AMPA receptor palmitoylation-deficient mice.

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.

Tiling mechanisms of the Drosophila compound eye through geometrical tessellation.

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.

Palmitoylation couples the kinases DLK and JNK3 to facilitate prodegenerative axon-to-soma signaling.

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.

Focusing on a nickel hydrocorphinoid in a protein matrix: methane generation by methyl-coenzyme M reductase with F430 cofactor and its models.

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 (CH3S-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 CH3-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.

DNA-Mediated Protein Shuttling between Coacervate-Based Artificial Cells.

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.

One-Step Preparation of Fe/N/C Single-Atom Catalysts Containing Fe-N4 Sites from an Iron Complex Precursor with 5,6,7,8-Tetraphenyl-1,12-Diazatriphenylene Ligands.

Fe/N/C single-atom catalysts containing Fe-Nx 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-N4 sites in an Fe/N/C catalyst. One-step pyrolysis of the iron complex with carbon black forms atomically dispersed Fe-N4 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-N4 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-N4 sites is controlled by precise tuning of the chemical structure of the iron complex precursor.

Ubiquitin-dependent rapid degradation conceals a cell-protective function of cytoplasmic SIRT3 against oxidative stress.

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-ß (Aß), and overexpression of SIRT3ct suppressed the toxic function of Aß in PC12 cells. These results indicate that SIRT3ct promotes cell survival under physiological and pathological conditions.

Evolutionarily Established Palmitoylation-Dependent Regulatory Mechanisms of the Vertebrate Glutamatergic Synapse and Diseases Caused by Their Disruption.

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.