Akio Matsuda

Genome-Wide and Functional Annotation of Human E3 Ubiquitin Ligases Identifies MULAN, a Mitochondrial E3 that Regulates the Organelle's Dynamics and Signaling

Specificity of protein ubiquitylation is conferred by E3 ubiquitin (Ub) ligases. We have annotated ∼617 putative E3s and substrate-recognition subunits of E3 complexes encoded in the human genome. The limited knowledge of the function of members of the large E3 superfamily prompted us to generate genome-wide E3 cDNA and RNAi expression libraries designed for functional screening. An imaging-based screen using these libraries to identify E3s that regulate mitochondrial dynamics uncovered MULAN/FLJ12875, a RING finger protein whose ectopic expression and knockdown both interfered with mitochondrial trafficking and morphology. We found that MULAN is a mitochondrial protein – two transmembrane domains mediate its localization to the organelles outer membrane. MULAN is oriented such that its E3-active, C-terminal RING finger is exposed to the cytosol, where it has access to other components of the Ub system. Both an intact RING finger and the correct subcellular localization were required for regulation of mitochondrial dynamics, suggesting that MULANs downstream effectors are proteins that are either integral to, or associated with, mitochondria and that become modified with Ub. Interestingly, MULAN had previously been identified as an activator of NF-κB, thus providing a link between mitochondrial dynamics and mitochondria-to-nucleus signaling. These findings suggest the existence of a new, Ub-mediated mechanism responsible for integration of mitochondria into the cellular environment.

Factors Associated with Steroid Phobia in Caregivers of Children with Atopic Dermatitis

Abstract:  Topical corticosteroids (TCS) are first‐line therapeutic agents for atopic dermatitis (AD). Some patients express irrational fear and anxiety about using TCS, which leads to poor outcomes for AD. Although it is important to understand the factors underlying steroid phobia so that its effects can be minimized, few studies have addressed this subject. Here, we used a questionnaire to investigate predictive factors for steroid phobia in the caregivers (usually mothers) of children with AD. We studied 436 children with AD (mean age 47.6 mos, range 2–236 mos) who newly visited our AD outpatient unit. The caregivers were asked to complete a medical history questionnaire regarding AD. Steroid phobia was analyzed for correlations with other patient and caregiver variables. Overall, 38.3% of the caregivers were reluctant to use TCS on their children’s skin. Patient characteristics female sex (odds ratio [OR] = 1.85 vs male; p = 0.005), child’s paternal history of AD (OR = 1.94; p = 0.03), and frequent changing of clinics (OR = 1.25; p = 0.03) were predictive factors for steroid phobia. AD severity did not correlate with steroid phobia. Our findings suggest that greater attention to the patient’s sex and clinical background of patients with AD is important to the success of AD therapy, regardless of AD severity.

Characterization and Cloning of an Enantioselective Amidase from Comamonas acidovorans KPO-2771-4

Abstract The characteristics and amino acid sequence of an enantiomer-selective amidase active on R-(−)-2-(3′-benzoyl phenyl)propionamide [R-(−)-ketoprofen amide] purified previously from Comamonas acidovorans KPO-2771-4 were studied. On gel filtration, this amidase appeared to be a monomer with a molecular mass of 55 kDa. It had maximal activity at 35°C and at pH values from 8.5 to 10.0. Except for Cu2+, Zn2+, Pb2+, and p-chloromercuribenzoate, it was not affected by chelating reagents, carbonyl reagents, reductants, most metal ions, or thiol reagents. The amidase had hydrolyzing activity against a broad range of aliphatic, aromatic, and amino acid amides, evidence that it is a wide-spectrum amidase. Oligonucleotide probes designed from limited peptide sequence information were used to clone the corresponding gene. The nucleotide-determined sequence indicated that the amidase consists of 473 amino acids (Mw 50,464 Da). Significant homologies were found at the amino acid level between the R-enantiomer-selective amidase of C. acidovorans KPO-2771-4 and the S-enantiomer-selective enzymes from Rhodococcus sp., Brevibacterium sp., and Pseudomonas sp. Only 39% homology was conserved in the consensus region of these amidases.

ST2 Requires Th2-, but Not Th17-, Type Airway Inflammation in Epicutaneously Antigen-Sensitized Mice

BACKGROUND IL-33 is known to induce Th2-type cytokine production by various types of cells through its receptors, ST2 and IL-1RAcP. Polymorphism in the ST2 and/or IL-33 genes was found in patients with atopic dermatitis and asthma, implying that the IL-33/ST2 pathway is closely associated with susceptibility to these diseases. Exposure to allergens through damaged skin is suspected to be a trigger for allergen sensitization, resulting in development of such allergic disorders as asthma and atopic dermatitis. METHODS To elucidate the role(s) of the IL-33/ST2 pathway in asthma in individuals who had been epicutaneously sensitized to an antigen, wild-type and ST2-/- mice were epicutaneously sensitized with ovalbumin (OVA) and then were intranasally challenged with OVA. The degree of airway inflammation, the number of leukocytes and the activities of myeloperoxidase (MPO) and eosinophil peroxidase (EPO) in bronchoalveolar lavage fluids (BALFs), The levels of cytokines and chemokines in lungs and OVA-specific IgE levels in sera were determined by histological analysis, a hemocytometer, colorimetric assay, quantitative PCR or ELISA, respectively. RESULTS The number of eosinophils in BALFs, the levels of Th2 cytokines and chemoattractants in the lungs and OVA-specific IgE in sera from ST2-/- mice were significantly reduced compared with wild-type mice. Although the number of neutrophils in BALFs and the pulmonary levels of IL-17 were comparable in both mice, the levels of MPO activity in BALFs and neutrophil chemoattractants in the lung were reduced in ST2-/- mice. CONCLUSIONS The IL-33/ST2 pathway is crucial for Th2-cytokine-mediated eosinophilic, rather than Th17-cytokine-mediated neutrophilic, airway inflammation in mice that had been epicutaneously sensitized with antigens and then challenged with antigen.

Cell type‐dependent effects of corticosteroid on periostin production by primary human tissue cells

Overproduction of periostin, an IL‐13‐inducible matricellular protein, despite corticosteroid treatment is thought to be involved in the chronicity of allergic inflammation seen in corticosteroid‐refractory tissue fibrosis. Therefore, we hypothesized that some tissue cells must produce periostin in a corticosteroid‐insensitive manner. Here, we show that IL‐4 and IL‐13 each induced comparable levels of periostin production by primary normal human fibroblasts and microvascular endothelial cells derived from lung and skin. Dexamethasone, a corticosteroid, completely inhibited IL‐4/13‐induced, but did not affect TGF‐β‐induced, periostin production by fibroblasts. In contrast, dexamethasone synergistically enhanced IL‐4/13‐induced periostin production by microvascular endothelial cells. TGF‐β did not induce periostin production by microvascular endothelial cells. Our novel findings suggest that IL‐4/13‐induced microvascular endothelium‐derived and/or TGF‐β‐induced fibroblast‐derived periostin might play a pivotal role in corticosteroid‐refractory tissue fibrosis, leading to chronic allergic inflammation in the lung and/or skin.

Molecular cloning of acetyl coenzyme A: deacetylcephalosporin C o-acetyltransferase cDNA from Acremonium chrysogenum: sequence and expression of catalytic activity in yeast.

Acetyl CoA: deacetylcephalosporin C o-acetyltransferase(DCPC-ATF) catalyses the final step in the biosynthesis of cephalosporin C, the conversion of deacetylcephalosporin C to cephalosporin C. A cDNA encoding DCPC-ATF has been isolated from a cDNA library of a cephalosporin C producing fungus Acremonium chrysogenum using oligonucleotide probes based on N-terminal amino acid sequences of the enzyme. The cDNA contains a single large open reading frame for a putative precursor consisting of 12 amino acid(AA) leader peptide of unknown function, 274 AA large subunit and 126 AA small subunit at the carboxyl end. The cDNA was expressed in yeast exhibiting a functional DCPC-ATF activity. It was also indicated that the leader peptide was not essential for expression of the enzyme activity. The primary structure of DCPC-ATF shows significant homology with those of acetyl CoA: homoserine o-acetyltransferase in Saccharomyces cerevisiae and Ascobolas immersus.

Cloning and disruption of the cefG gene encoding acetyl coenzyme A: Deacetylcephalosporin C O-acetyltransferase from Acremonium chrysogenum

Acetyl CoA: deacetylcephalosporin C o-acetyltransferase(DCPC-ATF) catalyses the final step in the biosynthesis of cephalosporin C (CPC) in Acremonium chrysogenum. The gene encoding DCPC-ATF, cefG, has been isolated from an A. chrysogenum genomic library using a DCPC-ATF cDNA probe. Nucleotide sequence analysis revealed that cefG contains two short introns of 79bp and 65bp. The gene was found to be closely linked to the cefEF gene encoding deacetoxycephalosporin C synthetase/deacetylcephalosporin C synthetase, which catalyses the preceding two steps in the pathway. The two genes are separated by a 1114 bp segment from which they are divergently transcribed. Introduction of the cloned cefG gene to A.chrysogenum resulted in an increased level of DCPC-ATF activity. A plasmid carrying a cefG gene interrupted in the coding region by a selectable marker for resistance to hygromycin B was constructed and used to disrupt the cefG locus in A.chrysogenum. The cefG-disrupted strains were found to lack the ability to produce CPC, and accumulated its precursor, deacetylcephalosporin C in the culture broth. Southern hybridization analysis confirmed that the disruption resulted from a gene replacement event at the cefG locus.

Anti-inflammatory effects of high-dose IgG on TNF-α-activated human coronary artery endothelial cells

High‐dose infusion of IgG (IVIG) is used to treat autoimmune and inflammatory diseases, including Kawasaki disease (KD). Although the immunomodulatory effects of IVIG on blood cells such as macrophages have been well studied, its effects on tissue cells remain unclear. Here, we show that high‐dose IgG specifically and completely inhibited TNF‐α‐induced, but not IL‐1β‐induced, secretion of proinflammatory cytokines such as G‐CSF and IL‐6 by cultured human coronary artery endothelial cells (HCAECs). High‐dose IgG did not inhibit TNF‐α‐mediated early signaling events of the NF‐κB and MAPK pathways but it potently inhibited gene expression of G‐CSF and IL‐6 12 h after TNF‐α‐stimulation. Interestingly, suppression of the G‐CSF and IL‐6 gene expression correlated closely with functional inhibition of a transcription factor, C/EBPδ, whose binding sites in the promoters of G‐CSF and IL‐6 have been shown to be critical for their transcriptional activation. Furthermore, the inhibitory effect of intact IgG on HCAECs was exerted mainly via its F(ab’)2 fragment, and not its Fc fragment. These findings suggest that the clinical effects of IVIG on KD patients are at least in part due to its direct anti‐inflammatory effects on the coronary endothelium, which is a major lesion site in the pathogenesis of KD.

Galectin-9 Enhances Cytokine Secretion, but Suppresses Survival and Degranulation, in Human Mast Cell Line

Galectin-9 (Gal-9), a lectin having a β-galactoside-binding domain, can induce apoptosis of Th1 cells by binding to TIM-3. In addition, Gal-9 inhibits IgE/Ag-mediated degranulation of mast cell/basophilic cell lines by binding to IgE, thus blocking IgE/Ag complex formation. However, the role of Gal-9 in mast cell function in the absence of IgE is not fully understood. Here, we found that recombinant Gal-9 directly induced phosphorylation of Erk1/2 but not p38 MAPK in a human mast cell line, HMC-1, which does not express FcεRI. Gal-9 induced apoptosis and inhibited PMA/ionomycin-mediated degranulation of HMC-1 cells. On the other hand, Gal-9 induced cytokine and/or chemokine production by HMC-1 cells, dependent on activation of ERK1/2 but not p38 MAPK. In addition, the lectin activity of Gal-9 was required for Gal-9-mediated cytokine secretion by HMC-1 cells. These observations suggest that Gal-9 has dual properties as both a regulator and an activator of mast cells.

Silica and Double-Stranded RNA Synergistically Induce Bronchial Epithelial Apoptosis and Airway Inflammation

Silica crystals (silica), which are the main mineral component of volcanic ash and desert dust, can activate the caspase-1-activating inflammasome in phagocytic cells to secrete IL-1β. Although inhalation of silica-containing dust is known to exacerbate chronic respiratory diseases, probably through inflammasome activation, its direct effects on bronchial epithelial cells remain unclear. Here, we show that silica and double-stranded RNA (dsRNA) synergistically induces caspase-9-dependent apoptosis, but not inflammasome activation, of bronchial epithelial cells. Intranasal administration of silica and dsRNA to mice synergistically enhanced neutrophil infiltration in the airway without IL-1β release in the bronchoalveolar lavage fluid. Histopathological analysis revealed that silica or dsRNA alone induced slight airway inflammation, whereas combined administration significantly enhanced airway inflammation and epithelial damage. These novel findings suggest that inhalation of silica-containing dust may cause inflammasome-independent airway inflammation, possibly by damaging the epithelial barrier, especially at the time of viral infection. These responses may also be involved in acute lung injury caused by inhaled silica-containing dust.