Tertiary and quaternary structures of photoreactive Fe-type nitrile hydratase from Rhodococcus sp. N-771: roles of hydration water molecules in stabilizing the structures and the structural origin of the substrate specificity of the enzyme.

The crystal structure analysis of the Fe-type nitrile hydratase from Rhodococcus sp. N-771 revealed the unique structure of the enzyme composed of the alpha- and beta-subunits and the unprecedented structure of the non-heme iron active center [Nagashima, S., et al. (1998) Nat. Struct. Biol. 5, 347-351]. A number of hydration water molecules were identified both in the interior and at the exterior of the enzyme. The study presented here investigated the roles of the hydration water molecules in stabilizing the tertiary and the quaternary structures of the enzyme, based on the crystal structure and the results from a laser light scattering experiment for the enzyme in solution. Seventy-six hydration water molecules between the two subunits significantly contribute to the alphabeta heterodimer formation by making up the surface shape, forming extensive networks of hydrogen bonds, and moderating the surface charge of the beta-subunit. In particular, 20 hydration water molecules form the extensive networks of hydrogen bonds stabilizing the unique structure of the active center. The amino acid residues hydrogen-bonded to those hydration water molecules are highly conserved among all known nitrile hydratases and even in the homologous enzyme, thiocyanate hydrolase, suggesting the structural conservation of the water molecules in the NHase family. The crystallographic asymmetric unit contained two heterodimers connected by 50 hydration water molecules. The heterotetramer formation in crystallization was clearly explained by the concentration-dependent aggregation state of NHase found in the light scattering measurement. The measurement proved that the dimer-tetramer equilibrium shifted toward the heterotetramer dominant state in the concentration range of 10(-2)-1.0 mg/mL. In the tetramer dominant state, 50 water molecules likely glue the two heterodimers together as observed in the crystal structure. Because NHase exhibits a high abundance in bacterial cells, the result suggests that the heterotetramer is physiologically relevant. In addition, it was revealed that the substrate specificity of this enzyme, recognizing small aliphatic substrates rather than aromatic ones, came from the narrowness of the entrance channel from the bulk solvent to the active center. This finding may give a clue for changing the substrate specificity of the enzyme. Under the crystallization condition described here, one 1,4-dioxane molecule plugged the channel. Through spectroscopic and crystallographic experiments, we found that the molecule prevented the dissociation of the endogenous NO molecule from the active center even when the crystal was exposed to light.     

Identification of significant regions of transcription factor DP-1 (TFDP-1) involved in stability/instability of the protein

We previously reported the identification of DP-1 isoforms (α and β), which are structurally C-terminus-deleted ones, and revealed the low-level expression of these isoforms. It is known that wild-type DP-1 is degraded by the ubiquitin-proteasome system, but few details are known about the domains concerned with the protein stability/instability for the proteolysis of these DP-1 isoforms. Here we identified the domains responsible for the stability/instability of DP-1. Especially, the DP-1 “Stabilon” domain was a C-terminal acidic motif and was quite important for DP-1 stability. Moreover, we propose that this DP-1 Stabilon may be useful for the stability of other nuclear proteins when fused to them.     

Signal transduction in blue light‐induced oxygen uptake in Chlorella cells

In DCMU‐poisoned wild‐type and in non‐photosynthetic pigment mutant cells of Chlorella kessleri , grown heterotrophically with glucose as a carbon source and with nitrate as sole nitrogen source, the known blue light‐enhanced uptake of oxygen and breakdown of starch were reduced by staurosporine and K252a, both potent inhibitors of protein kinase C. This corresponded to sensitivity to these inhibitors of blue light‐induced uptake of nitrate of such organisms. Cells grown with ammonia as sole nitrogen source responded to short wavelength visible irradiation with an increase in oxygen uptake, and this, too, was inhibited by staurosporine and K252a. However, these cells did not show any blue light‐enhanced uptake of nitrate. From these results, enhanced consumption of oxygen under blue light cannot be a consequence of blue light‐induced protein phosphorylation involved in the light‐dependent uptake of nitrate. However, existence of a specific protein phosphorylation within the process of enhancement of oxygen uptake under blue light is not yet proven by the data. There might be a master reaction that induces both processes independently, or there may be influences of other light‐induced processes which lead to enhanced starch breakdown, thereby supplying the glucose for oxidative degradation.     

Mutations of the CD40 ligand gene in 13 Japanese patients with X-linked hyper-IgM syndrome

X-linked hyper-IgM syndrome (XHIM) is a rare primary immunodeficiency caused by a defective CD40 ligand. We identified mutations of the CD40 ligand gene in 13 unrelated Japanese XHIM patients. Of the four patients with missense mutations, one had a mutation within the transmembrane domain, and the three others had mutations affecting the TNF homology region of the extracellular domain. Two of the missense mutations resulted in the substitution of amino acids that are highly conserved in TNF family proteins. Three patients had nonsense mutations, all of which resulted in the truncation of the TNF homology domain of the CD40 ligand. Three patients had genomic DNA deletions of 2, 3 or 4 nucleotides, respectively. All of the deletions were flanked by direct repeat sequences, suggesting that these deletions were caused by slipped mispairing. Three patients had mutations within introns resulting in altered splicing, and multiple splicing products were found in one patient. Thus, each of the 13 Japanese patients had different mutations, 9 of them being novel mutations. These results indicate that mutations in XHIM are highly heterogeneous, although codon 140 seems to be a hot spot of the CD40 ligand gene since two additional point mutations were located at Trp 140, bringing the total numbers of mutations affecting codon 140 to six. In one XHIM family with a missense mutation, prenatal diagnosis was performed by single-strand conformation polymorphism analysis of genomic DNA of a male fetus. Received: 20 August 1996     

A Portable Lipid Bilayer System for Environmental Sensing with a Transmembrane Protein

This paper describes a portable measurement system for current signals of an ion channel that is composed of a planar lipid bilayer. A stable and reproducible lipid bilayer is formed in outdoor environments by using a droplet contact method with a micropipette. Using this system, we demonstrated that the single-channel recording of a transmembrane protein (alpha-hemolysin) was achieved in the field at a high-altitude (∼3623 m). This system would be broadly applicable for obtaining environmental measurements using membrane proteins as a highly sensitive sensor.     

Helical transformations of Salmonella flagella in vitro.

The direct regulation of fatty acid synthesis by the relA gene has been demonstrated in vivo by use of a system in which fatty acid synthesis is uncoupled from membrane phospholipid synthesis.     

Formation of a flagella-like but straight polymer of Salmonella flagellin

Salmonella flagellin (monomer) polymerizes into flagellar filaments with the addition of (NH₄)₂SO₄ (Ada et al., 1963; Wakabayashi et al., 1969). When, however, this process was allowed to take place in the presence of a high concentration of NaCl (about 1.5 m), the product consisted of flagella-like but straight filaments. This phenomenon was common to four kinds of flagellins derived from strains SJ670, SJ25, SJ30 and SJ814. When the straight filament, suspended in 0.15 m-NaCl, was heated, it depolymerized to the monomer, which could in turn be polymerized into flagellar filaments by the addition of short fragments of flagella at room temperature. Nevertheless, attempts at direct transformation between the two types of filaments were unsuccessful. In 0.15 m-NaCl, straight filaments prepared from the four kinds of flagellins had markedly different heat stabilities, which were much lower than that of any kind of flagella. When monomeric flagellin dissolved in 3.5 m-NaCl was seeded with short fragments of straight filaments, the monomer polymerized onto the ends of the short fragments, which consequently grew into long straight filaments. In this type of experiment, monomers and seeds derived from the four strains were able to interact in any combination, suggesting that straight filaments consisting of the four kinds of flagellins have the same substructures. Whether the concentration of added NaCl was 0.15 m or 3.5 m, fragments of flagella (or straight filaments) were unable to act as seeds for the formation of straight filaments (or flagellar filaments). From this and other experimental results, it was concluded that in the two filamentous structures, flagellin molecules may be packed in different ways.     

Type 2 diabetes mellitus in obese mouse model induced by monosodium glutamate.

The number of diabetic patients is increasing every year, and new model animals are required to study the diverse aspects of this disease. An experimental obese animal model has reportedly been obtained by injecting monosodium glutamate (MSG) to a mouse. We found that ICR-MSG mice on which the same method was used developed glycosuria. Both female and male mice were observed to be obese but had no polyphagia, and were glycosuric by 29 weeks of age, with males having an especially high rate of incidence (70.0%). Their blood concentrations of glucose, insulin, total cholesterol, and triglycerides were higher than in the control mice at 29 weeks. These high concentrations appeared in younger males more often than in females, and were severe in adult males. Also, the mice at 54 weeks of age showed obvious obesity and increased concentrations of glucose, insulin, and total cholesterol in the blood. The pathological study of ICR-MSG female and male mice at 29 weeks of age showed hypertrophy of the pancreatic islet. This was also observed in most of these mice at 54 weeks. It was recognized as a continuation of the condition of diabetes mellitus. From the above results, these mice are considered to be useful as new experimental model animals developing a high rate of obese type 2 (non-insulin dependent) diabetes mellitus without polyphagia.     

Ethyl gallate attenuates acute lung injury through Nrf2 signaling

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) is the clinical syndrome of persistent lung inflammation caused by various direct and indirect stimuli. Despite advances in the understanding of disease pathogenesis, few therapeutic have emerged for ALI/ARDS. Thus, in the present study we evaluated the therapeutic potential of ethyl gallate (EG), a plant flavanoid in the context of ALI using in vivo (BALB/c) and in vitro models (human monocytes). Our in vivo data supports the view that EG alleviates inflammatory condition in ALI as significant reduction in BALF neutrophils, ROS, proinflammatory cytokines and albumin levels were observed with the single i.p of EG post LPS exposure. Also, histochemical analysis of mice lung tissue demonstrated that EG restored LPS stimulated cellular influx inside the lung airspaces. Unraveling the mechanism of action, our RT-PCR and western blot analysis suggest that enhanced expression of HO-1 underlies the protective effect of EG on ROS level in mice lung tissue. Induction of HO-1 in turn appears to be mediated by Nrf2 nuclear translocation and consequent activation and ablation of Nrf2 activity through siRNA notably abrogated the EG induced protective effect in LPS induced human monocytes. Furthermore, our results indicate that EG generated moderate amounts of H₂O₂ could induce Nrf2 translocation in the in vitro systems. However, given the insignificant amount of H₂O₂ recorded in the injected material in the in vivo system, additional mechanism for EG action could not be excluded. Nevertheless our results highlight the protective role of EG in ALI and provide the novel insight into its usefulness as a therapeutic tool for the treatment of ALI.