Affinity labeling of the proteasome by a belactosin A derived inhibitor

Belactosin A is a potent proteasome inhibitor isolated from Streptomyces metabolites. Here we show that a hydrophobic belactosin A derivative, dansyl-KF33955, can covalently, and specifically, affinity label the catalytic subunits of the 26S proteasome, which consists of the 20S protein degrading core particle and the 19S regulatory particles. The labeling of catalytic subunits proceeds faster in intact proteasomes in vivo than in isolated 20S core particles. These data suggest that the 19S regulatory particle may facilitate entry of the inhibitor into the 20S core particle. This cell-permeable chemical probe is an excellent tool with which to study the interactions of this proteasome inhibitor with proteasomes in intact cells.     

Fungal Infection of Mantis Shrimp (<Emphasis Type="Italic">Oratosquilla oratoria</Emphasis>) Caused by Two Anamorphic Fungi Found in Japan

Two fungal pathogens of the mantis shrimp (Oratosquilla oratoria) in Yamaguchi and Aichi Prefectures, Japan are described as the new species Plectosporium oratosquillae and Acremonium sp. (a member of the Emericellopsis marine clade). Both fungi infect the gills of the mantis shrimp, which become brown or black due to melanization. The former species is characterized by its slow growth on artificial seawater yeast extract peptone glucose (PYGS) agar, pale yellow to pale orange or grayish yellow colonies, short cylindrical solitary phialides with a wavy tip, and one-celled ellipsoidal conidia. Although lacking the two-celled conidia demonstrated by the type species Plectosporium tabacinum, the taxonomic placement of the new species was confirmed by DNA sequence analysis of the internal transcribed spacer (ITS) region of ribosomal DNA (ITS1, 5.8S rDNA and ITS2). Acremonium sp., the other causal pathogen, differs from P. oratosquillae by its fast growth on PYGS agar, pale orange to salmon-colored colonies, long, slender conidiophores consisting of solitary phialides with tips lacking an undulate outline, and typically cylindrical conidia. Analysis of ITS and β-tubulin gene sequences placed this fungus within the phylogenetically distinct Emericellopsis (anam. Acremonium) marine clade. Various physiological characteristics of both pathogens were also investigated. This is the first report of a fungal infection found on the mantis shrimp in Japan.     

Characterization of a sHsp of Schizosaccharomyces pombe, SpHsp15.8, and the implication of its functional mechanism by comparison with another sHsp, SpHsp16.0

There exist two small heat shock proteins (sHsps) in the fission yeast, Schizosaccharomyces pombe (S. pombe), whose expressions are highly induced by heat stress. We have previously expressed, purified, and characterized one of the sHsps, SpHsp16.0. In this study, we examined the other sHsp, SpHsp15.8. It suppressed the thermal aggregation of citrate synthase (CS) from porcine heart and dithiothreitol-induced aggregation of insulin from bovine pancreas with very high efficiency. Almost one SpHsp15.8 subunit was sufficient to protect one protein molecule from aggregation. Like SpHsp16.0, SpHsp15.8 dissociated into small oligomers and then interacted with denatured substrate proteins. SpHsp16.0 exhibited a clear enthalpy change for denaturation occurring over 60 degrees C in differential scanning calorimetry (DSC). However, we could not observe any significant enthalpy change in the DSC of SpHsp15.8. The difference is likely to be caused by the adhesive characteristics of SpHsp15.8. The oligomer dissociation of SpHsp15.8 and SpHsp16.0 and their interactions with denatured substrate proteins were studied by fluorescence polarization analysis (FPA). Both sHsps exhibited a temperature-dependent decrease of fluorescence polarization, which correlates with the dissociation of large oligomers to small oligomers. The dissociation of the SpHsp15.8 oligomer began at about 35 degrees C and proceeded gradually. On the contrary, the SpHsp16.0 oligomer was stable up to approximately 45 degrees C, but then dissociated into small oligomers abruptly at this temperature. Interestingly, SpHsp16.0 is likely to interact with denatured CS in the dissociated state, while SpHsp15.8 is likely to interact with CS in a large complex. These results suggest that S. pombe utilizes two sHsps that function in different manners, probably to cope with a wide range of temperatures and various denatured proteins.     

Atg26-Mediated Pexophagy Is Required for Host Invasion by the Plant Pathogenic Fungus Colletotrichum orbiculare

The number of peroxisomes in a cell can change rapidly in response to changing environmental and physiological conditions. Pexophagy, a type of selective autophagy, is involved in peroxisome degradation, but its physiological role remains to be clarified. Here, we report that cells of the cucumber anthracnose fungus Colletotrichum orbiculare undergo peroxisome degradation as they infect host plants. We performed a random insertional mutagenesis screen to identify genes involved in cucumber pathogenesis by C. orbiculare. In this screen, we isolated a homolog of Pichia pastoris ATG26, which encodes a sterol glucosyltransferase that enhances pexophagy in this methylotrophic yeast. The C. orbiculare atg26 mutant developed appressoria but exhibited a specific defect in the subsequent host invasion step, implying a relationship between pexophagy and fungal phytopathogenicity. Consistent with this, its peroxisomes are degraded inside vacuoles, accompanied by the formation of autophagosomes during infection-related morphogenesis. The autophagic degradation of peroxisomes was significantly delayed in the appressoria of the atg26 mutant. Functional domain analysis of Atg26 suggested that both the phosphoinositide binding domain and the catalytic domain are required for pexophagy and pathogenicity. In contrast with the atg26 mutant, which is able to form appressoria, the atg8 mutant, which is defective in the entire autophagic pathway, cannot form normal appressoria in the earlier steps of morphogenesis. These results indicate a specific function for Atg26-enhanced pexophagy during host invasion by C. orbiculare.     

Overexpression and purification of rat peroxisomal membrane protein 22, PMP22, in Pichia pastoris

Peroxisomal membrane protein 22, PMP22, is an integral membrane protein that has four putative transmembrane-spanning regions. First reported as a major component of rat liver peroxisomal membranes and suggested to be involved in the metabolism of reactive oxygen species, its function and structure are still unknown owing to a lack of biochemical and structural experiments. Here we report the overproduction and purification of rat PMP22 (rPMP22) with the use of a methylotrophic yeast, Pichia pastoris, as a host. rPMP22 was localized not to peroxisomal membranes but to membrane compartments, such as the nuclear envelope. Highly pure rPMP22 was obtained in two steps. Several physicochemical assays indicated that the purified preparation should retain its functional structure. Furthermore, fed-batch fermentation yielded 90 mg of rPMP22 protein from 4L of culture. This is the first report to demonstrate the overproduction of a recombinant rPMP22 in the membrane compartments of P. pastoris.     

Mycobacteria Exploit Host Hyaluronan for Efficient Extracellular Replication

In spite of the importance of hyaluronan in host protection against infectious organisms in the alveolar spaces, its role in mycobacterial infection is unknown. In a previous study, we found that mycobacteria interact with hyaluronan on lung epithelial cells. Here, we have analyzed the role of hyaluronan after mycobacterial infection was established and found that pathogenic mycobacteria can grow by utilizing hyaluronan as a carbon source. Both mouse and human possess 3 kinds of hyaluronan synthases (HAS), designated HAS1, HAS2, and HAS3. Utilizing individual HAS-transfected cells, we show that HAS1 and HAS3 but not HAS2 support growth of mycobacteria. We found that the major hyaluronan synthase expressed in the lung is HAS1, and that its expression was increased after infection with Mycobacterium tuberculosis. Histochemical analysis demonstrated that hyaluronan profoundly accumulated in the granulomatous legion of the lungs in M. tuberculosis-infected mice and rhesus monkeys that died from tuberculosis. We detected hyaluronidase activity in the lysate of mycobacteria and showed that it was critical for hyaluronan-dependent extracellular growth. Finally, we showed that L-Ascorbic acid 6-hexadecanoate, a hyaluronidase inhibitor, suppressed growth of mycobacteria in vivo. Taken together, our data show that pathogenic mycobacteria exploit an intrinsic host-protective molecule, hyaluronan, to grow in the respiratory tract and demonstrate the potential usefulness of hyaluronidase inhibitors against mycobacterial diseases.     

Tiny keratotic brown lesions on the interdigital web between the toes of a healthy man caused by Curvularia species infection and a review of cutaneous Curvularia infections

A 74-year-old healthy Japanese man had noticed an itchy lesion with two tiny thick brown scales on the fourth interdigital web of his right foot 3 months earlier. The brown fungus isolated from the scales had demonstrated brown, ellipsoidal, obovoid or clavate, 3-septate conidia. Morphologically, the fungus was identified as Curvularia sp., and was differentiated from other human pathogenic species of the genus, such as C. lunata, C. pallescens, C. trifolii, C. clavata, and C. brachyspora, by molecular analysis based on the DNA sequence data. The fungus grows well on keratotic materials (hairs, nails, and callus), which indicates that it might have the ability to infect the skin surface.