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Mast cells (MCs) act as primary effectors in inflammatory and allergic reactions by releasing intracellularly-stored inflammatory mediators in diseases. The two major pathways for MC activation are known to be immunoglobulin E (IgE)-dependent and -independent. Although IgE-dependent signaling is the main pathway to MC activation, IgE-independent pathways have also been found to serve pivotal roles in the pathophysiology of various inflammatory conditions. Recent studies have shown that human and mouse MCs express several regulatory receptors such as toll-like receptors (TLRs), CD48, C300a, and GPCRs, including mas-related GPCR-X2 (MRGPRX2). MRGPRX2 has been reported as a novel GPCR that is expressed in MCs activated by basic secretagogues, neurokinin peptides, host defense antimicrobial peptides, and small molecule compounds (e.g., neuromuscular blocking agents) and leads to MC degranulation and eicosanoids release under in vitro experimental condition. Functional analyses of MRGPRX2 and Mrgprb2 (mouse ortholog) indicate that MRGPRX2 is involved in MC hypersensitivity reactions causing neuroinflammation such as postoperative pain, type 2 inflammation, non-histaminergic itch, and drug-induced anaphylactic-like reactions. In this review, we discuss the roles in innate immunity through functional studies on MRGPRX2-mediated IgE-independent MC activation and also the therapeutic potential of MRGPRX2 inhibitors on allergic and inflammatory diseases.

NADPH-cytochrome P450 oxidoreductase (CPR) supplies electrons to various heme proteins including heme oxygenase (HO), which is a key enzyme for heme degradation. Electrons from NADPH flow first to flavin adenine dinucleotide, then to flavin mononucleotide (FMN), and finally to heme in the redox partner. For electron transfer from CPR to its redox partner, the ‘‘closed-open transition’’ of CPR is indispensable. Here, we demonstrate that a hinge-shortened CPR variant, which favors an open conformation, makes a stable complex with heme–HO-1 and can support the HO reaction, although its efficiency is extremely limited. Furthermore, we determined the crystal structure of the CPR variant in complex with heme–HO-1 at 4.3-Å resolution. The crystal structure of a complex of CPR and its redox partner was previously unidentified. The distance between heme and FMN in this complex (6 Å) implies direct electron transfer from FMN to heme.

The discovery of new chlorophyllous pigments would provide greater understanding of the mechanisms and evolution of photosynthesis. Bacteriochlorophyll f has never been observed in nature, although this name was proposed ~40 years ago based on structurally related compounds. We constructed a bacteriochlorophyll f –accumulating mutant of the green sulfur bacterium Chlorobaculum limnaeum , which originally produced bacteriochlorophyll e , by knocking out the bchU gene encoding C-20 methyltransferase based on natural transformation. This novel pigment self-aggregates in an in vivo light-harvesting antenna, the chlorosome and exhibits a Q y peak of 705 nm, more blue-shifted than any other chlorosome reported so far; the peak overlaps the maximum (~700 nm) of the solar photon flux spectrum. Bacteriochlorophyll f chlorosomes can transfer light energy from core aggregated pigments to another bacteriochlorophyll in the chlorosomal envelope across an energy gap of ~100 nm and is thus a promising material for development of new bioenergy applications.

The gene named bciD, which encodes the enzyme involved in C7-formylation in bacteriochlorophyll e biosynthesis, was found and investigated by insertional inactivation in the brown-colored green sulfur bacterium Chlorobaculum limnaeum (previously called Chlorobium phaeobacteroides). The bciD mutant cells were green in color, and accumulated bacteriochlorophyll c homologs bearing the 7-methyl group, compared to C7-formylated BChl e homologs in the wild type. BChl-c homolog compositions in the mutant were further different from those in Chlorobaculum tepidum which originally produced BChl c: (3(1) S)-8-isobutyl-12-ethyl-BChl c was unusually predominant.

Until recently the total synthesis of insulin, with its characteristic heterodimeric structure crosslinked by two interchain and one intrachain disulfide (SS) bridge, remained largely an unsolved challenge. By optimizing the synthesis and directed disulfide crosslinking of the two chains, and by applying biomimetic monocomponent proinsulin approaches, efficient insulin syntheses have been realized. Here we report the optimization and characterisation of an alternative strategy, oxidative native chain assembly. In this method unprotected A- and B-chains assemble oxidatively under thermodynamic control to afford bovine pancreatic insulin in 39% yield. Folding is found to proceed predominantly via structured 1SS* and 2SS* intermediates with a common interchain Cys A20 ‒Cys B19 disulfide. These results suggest that native chain assembly, long considered inefficient, may represent a reasonable strategy to access insulin variants. This is supported by the synthesis of human insulin and human type-II relaxin in yields of up to 49 and 47%, respectively, although the application to human insulin Val A16 variant is unsuccessful. The synthesis and folding pathways of insulin and related proteins are of wide interest. Here the authors characterise the major two-chain oxidative folding pathways of bovine pancreatic insulin, and develop synthetic conditions applicable to related foldable insulin variants

Heme oxygenase (HO) catalyzes heme degradation using electrons supplied by NADPH–cytochrome P450 oxidoreductase (CPR). Electrons from NADPH flow first to FAD, then to FMN, and finally to the heme in the redox partner. Previous biophysical analyses suggest the presence of a dynamic equilibrium between the open and the closed forms of CPR. We previously demonstrated that the open-form stabilized CPR (ΔTGEE) is tightly bound to heme–HO-1, whereas the reduction in heme–HO-1 coupled with ΔTGEE is considerably slow because the distance between FAD and FMN in ΔTGEE is inappropriate for electron transfer from FAD to FMN. Here, we characterized the enzymatic activity and the reduction kinetics of HO-1 using the closed-form stabilized CPR (147CC514). Additionally, we analyzed the interaction between 147CC514 and heme–HO-1 by analytical ultracentrifugation. The results indicate that the interaction between 147CC514 and heme–HO-1 is considerably weak, and the enzymatic activity of 147CC514 is markedly weaker than that of CPR. Further, using cryo-electron microscopy, we confirmed that the crystal structure of ΔTGEE in complex with heme–HO-1 is similar to the relatively low-resolution structure of CPR complexed with heme–HO-1 in solution. We conclude that the “open–close” transition of CPR is indispensable for electron transfer from CPR to heme–HO-1.

Monogalactosyldiacylglycerol (MGDG), which is conserved in almost all photosynthetic organisms, is the most abundant natural polar lipid on Earth. In plants, MGDG is highly accumulated in the chloroplast membranes and is an important bulk constituent of thylakoid membranes. However, precise functions of MGDG in photosynthesis have not been well understood. Here, we report a novel MGDG synthase from the green sulfur bacterium Chlorobaculum tepidum. This enzyme, MgdA, catalyzes MGDG synthesis using UDP-Gal as a substrate. The gene encoding MgdA was essential for this bacterium; only heterozygous mgdA mutants could be isolated. An mgdA knockdown mutation affected in vivo assembly of bacteriochlorophyll aggregates, suggesting the involvement of MGDG in the construction of the light-harvesting complex called chlorosome. These results indicate that MGDG biosynthesis has been independently established in each photosynthetic organism to perform photosynthesis under different environmental conditions. We complemented an Arabidopsis thaliana MGDG synthase mutant by heterologous expression of MgdA. The complemented plants showed almost normal levels of MGDG, although they also had abnormal morphological phenotypes, including reduced chlorophyll content, no apical dominance in shoot growth, atypical flower development, and infertility. These observations provide new insights regarding the importance of regulated MGDG synthesis in the physiology of higher plants.

The service life of many buildings and houses built using asbestos-containing materials is coming to an end and their demolition will lead to a great deal of asbestos-containing waste (ACW). Conventionally, the disposal of such waste is conducted by isolation under controlled landfill procedures; however problems with this method exist, such as the risk at the time of re-utilization of landfill sites and the depletion of lands to be reclaimed. Melting treatment is a promising technology that could be used to solve these problems; a thermal process involving temperatures exceeding the melting points of asbestos, it transforms them into non-hazardous minerals. This technology may be applicable not only for friable ACW but also for nonfriable ACW. We performed a demonstration test of melting treatment of nonfriable ACW using a gasification and melting furnace of the shaft furnace type, which is a typical method for high temperature melting. Detailed observation using transmission electron microscopy as well as general analyses verified that the same level of asbestos remained in the slag obtained by high temperature melting as that of the background soil. In addition, the asbestos concentration in the exhaust gas and the dust from the facility were at sufficiently low levels, and it was thus verified that the asbestos concentration in the atmosphere in the vicinity of the facility during the melting treatment was comparable to that of Japan’s background level reported by the Ministry of the Environment in 2007.

In nature, heme is a prosthetic group that is universally used as a cofactor for heme proteins. It is necessary for the execution of fundamental biological processes including electron transfer, oxidation and metabolism. However, free heme is toxic to cells, because of its capability to enhance oxidative stress, hence its cellular concentration is strictly regulated through multiple mechanisms. Heme oxygenase (HO) serves as an irreplaceable member in the heme degradation system. It is a ubiquitous protein, existing in many species including mammals, higher plants, and interestingly, certain pathogenic bacteria. In the HO reaction, HO catalyzes oxidative cleavage of heme to generate biliverdin and release carbon monoxide and ferrous iron. Because of the beneficial effects of these heme catabolism products, HO plays a key role in iron homeostasis and in defense mechanism against oxidative stress. HO is composed of an N-terminal structured region and a C-terminal membrane-bound region. Furthermore, the soluble form of HO, which is obtainable by excision of the membrane-bound region, retains its catalytic activity. Here, we present the backbone resonance assignments of the soluble form (residues 1–232) of HO-1 in the free and Zn(II) protoporphyrin IX (ZnPP)-bound states, and analyzed the structural differences between the states. ZnPP is a potent enzyme inhibitor, and the ZnPP-bound structure of HO-1 mimics the heme-bound structure. These assignments provide the structural basis for a detailed investigation of the HO-1 function.

A water-soluble selenoxide (DHSox) having a five-membered ring structure enables rapid and selective conversion of cysteinyl SH groups in a polypeptide chain into SS bonds in a wide pH and temperature range. It was previously demonstrated that the second-order rate constants for the SS formation with DHSox would be proportional to the number of the free SH groups present in the substrate if there is no steric congestion around the SH groups. In the present study, kinetics of the SS formation with DHSox was extensively studied at pH 4–10 and 25 °C by using reduced ribonuclease A, recombinant hirudin variant (CX-397), insulin A- and B-chains, and relaxin A-chain, which have two to eight cysteine residues, as polythiol substrates. The obtained rate constants showed stochastic SS formation behaviors under most conditions. However, the rate constants for CX-397 at pH 8.0 and 10.0 were not proportional to the number of the free SH groups, suggesting that the SS intermediate ensembles possess densely packed structures under weakly basic conditions. The high two-electron redox potential of DHSox (375 mV at 25 °C) compared to l-cystine supported the high ability of DHSox for SS formation in a polypeptide chain. Interestingly, the rate constants of the SS formation jumped up at a pH around the pKa value of the cysteinyl SH groups. The SS formation velocity was slightly decreased by addition of a denaturant due probably to the interaction between the denaturant and the peptide. The stochastic behaviors as well as the absolute values of the second-order rate constants in comparison to dithiothreitol (DTTred) are useful to probe the chemical reactivity and conformation, hence the folding, of polypeptide chains. [Display omitted] ▸ DHSox was applied as a SS-forming reagent for five polythiol peptides and DTTred. ▸ The SS formation velocities depend on the kind of substrate and solvent conditions. ▸ The SS-formation rate constants are proportional to the number of free SH groups. ▸ Folded structures and SH pKa modify the stochastic nature and absolute values of the rate constants. ▸ DHSox is a useful probe of chemical reactivity and intermediate structures in oxidative protein folding.