A novel sterol-binding protein reveals heterogeneous cholesterol distribution in neurite outgrowth and in late endosomes/lysosomes.

We identified a mushroom-derived protein, maistero-2 that specifically binds 3-hydroxy sterol including cholesterol (Chol). Maistero-2 bound lipid mixture in Chol-dependent manner with a binding threshold of around 30%. Changing lipid composition did not significantly affect the threshold concentration. EGFP-maistero-2 labeled cell surface and intracellular organelle Chol with higher sensitivity than that of well-established Chol probe, D4 fragment of perfringolysin O. EGFP-maistero-2 revealed increase of cell surface Chol during neurite outgrowth and heterogeneous Chol distribution between CD63-positive and LAMP1-positive late endosomes/lysosomes. The absence of strictly conserved Thr-Leu pair present in Chol-dependent cytolysins suggests a distinct Chol-binding mechanism for maistero-2.

De novo Sequencing of Novel Mycoviruses From Fusarium sambucinum: An Attempt on Direct RNA Sequencing of Viral dsRNAs.

An increasing number of viruses are continuously being found in a wide range of organisms, including fungi. Recent studies have revealed a wide viral diversity in microbes and a potential importance of these viruses in the natural environment. Although virus exploration has been accelerated by short-read, high-throughput sequencing (HTS), and viral de novo sequencing is still challenging because of several biological/molecular features such as micro-diversity and secondary structure of RNA genomes. This study conducted de novo sequencing of multiple double-stranded (ds) RNA (dsRNA) elements that were obtained from fungal viruses infecting two Fusarium sambucinum strains, FA1837 and FA2242, using conventional HTS and long-read direct RNA sequencing (DRS). De novo assembly of the read data from both technologies generated near-entire genomic sequence of the viruses, and the sequence homology search and phylogenetic analysis suggested that these represented novel species of the Hypoviridae, Totiviridae, and Mitoviridae families. However, the DRS-based consensus sequences contained numerous indel errors that differed from the HTS consensus sequences, and these errors hampered accurate open reading frame (ORF) prediction. Although with its present performance, the use of DRS is premature to determine viral genome sequences, the DRS-mediated sequencing shows great potential as a user-friendly platform for a one-shot, whole-genome sequencing of RNA viruses due to its long-reading ability and relative structure-tolerant nature.

Photoswitchable phospholipid FRET acceptor: Detergent free intermembrane transfer assay of fluorescent lipid analogs.

We have developed and characterized a novel photoswitchable phospholipid analog termed N-nitroBIPS-DPPG. The fluorescence can be switched on and off repeatedly with minimal photobleaching by UV or visible light exposure, respectively. The rather large photochromic head group is inserted deeply into the interfacial membrane region conferring a conical overall lipid shape, preference for a positive curvature and only minimal intermembrane transfer. Utilizing the switchable NBD fluorescence quenching ability of N-nitroBIPS-DPPG, a detergent free intermembrane transfer assay system for NBD modified lipids was demonstrated and validated. As NBD quenching can be turned off, total NBD associated sample fluorescence can be determined without the need of detergents. This not only reduces detergent associated systematic errors, but also simplifies assay handling and allows assay extension to detergent insoluble lipid species.

A novel sphingomyelin/cholesterol domain-specific probe reveals the dynamics of the membrane domains during virus release and in Niemann-Pick type C.

We identified a novel, nontoxic mushroom protein that specifically binds to a complex of sphingomyelin (SM), a major sphingolipid in mammalian cells, and cholesterol (Chol). The purified protein, termed nakanori, labeled cell surface domains in an SM- and Chol-dependent manner and decorated specific lipid domains that colocalized with inner leaflet small GTPase H-Ras, but not K-Ras. The use of nakanori as a lipid-domain-specific probe revealed altered distribution and dynamics of SM/Chol on the cell surface of Niemann-Pick type C fibroblasts, possibly explaining some of the disease phenotype. In addition, that nakanori treatment of epithelial cells after influenza virus infection potently inhibited virus release demonstrates the therapeutic value of targeting specific lipid domains for anti-viral treatment.-Makino, A., Abe, M., Ishitsuka, R., Murate, M., Kishimoto, T., Sakai, S., Hullin-Matsuda, F., Shimada, Y., Inaba, T., Miyatake, H., Tanaka, H., Kurahashi, A., Pack, C.-G., Kasai, R. S., Kubo, S., Schieber, N. L., Dohmae, N., Tochio, N., Hagiwara, K., Sasaki, Y., Aida, Y., Fujimori, F., Kigawa, T., Nishibori, K., Parton, R. G., Kusumi, A., Sako, Y., Anderluh, G., Yamashita, M., Kobayashi, T., Greimel, P., Kobayashi, T. A novel sphingomyelin/cholesterol domain-specific probe reveals the dynamics of the membrane domains during virus release and in Niemann-Pick type C.

Reprint of "Sequence and phylogenetic analyses of novel totivirus-like double-stranded RNAs from field-collected powdery mildew fungi".

The identification of mycoviruses contributes greatly to understanding of the diversity and evolutionary aspects of viruses. Powdery mildew fungi are important and widely studied obligate phytopathogenic agents, but there has been no report on mycoviruses infecting these fungi. In this study, we used a deep sequencing approach to analyze the double-stranded RNA (dsRNA) segments isolated from field-collected samples of powdery mildew fungus-infected red clover plants in Japan. Database searches identified the presence of at least ten totivirus (genus Totivirus)-like sequences, termed red clover powdery mildew-associated totiviruses (RPaTVs). The majority of these sequences shared moderate amino acid sequence identity with each other (<44%) and with other known totiviruses (<59%). Nine of these identified sequences (RPaTV1a, 1b and 2-8) resembled the genome of the prototype totivirus, Saccharomyces cerevisiae virus-L-A (ScV-L-A) in that they contained two overlapping open reading frames (ORFs) encoding a putative coat protein (CP) and an RNA dependent RNA polymerase (RdRp), while one sequence (RPaTV9) showed similarity to another totivirus, Ustilago maydis virus H1 (UmV-H1) that encodes a single polyprotein (CP-RdRp fusion). Similar to yeast totiviruses, each ScV-L-A-like RPaTV contains a -1 ribosomal frameshift site downstream of a predicted pseudoknot structure in the overlapping region of these ORFs, suggesting that the RdRp is translated as a CP-RdRp fusion. Moreover, several ScV-L-A-like sequences were also found by searches of the transcriptome shotgun assembly (TSA) libraries from rust fungi, plants and insects. Phylogenetic analyses show that nine ScV-L-A-like RPaTVs along with ScV-L-A-like sequences derived from TSA libraries are clustered with most established members of the genus Totivirus, while one RPaTV forms a new distinct clade with UmV-H1, possibly establishing an additional genus in the family. Taken together, our results indicate the presence of diverse, novel totiviruses in the powdery mildew fungus populations infecting red clover plants in the field.

Prolyl isomerase Pin1 regulates doxorubicin-inducible P-glycoprotein level by reducing Foxo3 stability.

It has been known that the phosphoSer/Thr-Pro-specific peptidyl prolyl cis/trans isomerase Pin1 regulates a variety of intracellular signaling pathways, including the response to the genotoxic drug doxorubicin. Pin1 binds phosphorylated p53 and stabilizes p53 to cause cell cycle arrest and apoptosis quickly in response to doxorubicin. Here we show another mechanism of Pin1 to maintain cell sensitivity to genotoxic stress, irrespective of whether p53 is present or not. In response to the genotoxic drug, Pin1 binds and decreases levels of the phosphorylated Foxo3, the positive transcription factor of P-glycoprotein (P-gp) gene. Through this mechanism of action, Pin1 decreases the level of P-gp and signals the cell to pump the genotoxic drugs out. This shows that Pin1 is implemented in maintaining the susceptibility to the genotoxic drugs by controlling P-gp level as well as p53-dependent apoptosis and cell cycle signaling pathways.

Sequence and phylogenetic analyses of novel totivirus-like double-stranded RNAs from field-collected powdery mildew fungi.

The identification of mycoviruses contributes greatly to understanding of the diversity and evolutionary aspects of viruses. Powdery mildew fungi are important and widely studied obligate phytopathogenic agents, but there has been no report on mycoviruses infecting these fungi. In this study, we used a deep sequencing approach to analyze the double-stranded RNA (dsRNA) segments isolated from field-collected samples of powdery mildew fungus-infected red clover plants in Japan. Database searches identified the presence of at least ten totivirus (genus Totivirus)-like sequences, termed red clover powdery mildew-associated totiviruses (RPaTVs). The majority of these sequences shared moderate amino acid sequence identity with each other (<44%) and with other known totiviruses (<59%). Nine of these identified sequences (RPaTV1a, 1b and 2-8) resembled the genome of the prototype totivirus, Saccharomyces cerevisiae virus-L-A (ScV-L-A) in that they contained two overlapping open reading frames (ORFs) encoding a putative coat protein (CP) and an RNA dependent RNA polymerase (RdRp), while one sequence (RPaTV9) showed similarity to another totivirus, Ustilago maydis virus H1 (UmV-H1) that encodes a single polyprotein (CP-RdRp fusion). Similar to yeast totiviruses, each ScV-L-A-like RPaTV contains a -1 ribosomal frameshift site downstream of a predicted pseudoknot structure in the overlapping region of these ORFs, suggesting that the RdRp is translated as a CP-RdRp fusion. Moreover, several ScV-L-A-like sequences were also found by searches of the transcriptome shotgun assembly (TSA) libraries from rust fungi, plants and insects. Phylogenetic analyses show that nine ScV-L-A-like RPaTVs along with ScV-L-A-like sequences derived from TSA libraries are clustered with most established members of the genus Totivirus, while one RPaTV forms a new distinct clade with UmV-H1, possibly establishing an additional genus in the family. Taken together, our results indicate the presence of diverse, novel totiviruses in the powdery mildew fungus populations infecting red clover plants in the field.

Evaluation of aegerolysins as novel tools to detect and visualize ceramide phosphoethanolamine, a major sphingolipid in invertebrates.

Ceramide phosphoethanolamine (CPE), a sphingomyelin analog, is a major sphingolipid in invertebrates and parasites, whereas only trace amounts are present in mammalian cells. In this study, mushroom-derived proteins of the aegerolysin family­pleurotolysin A2 (PlyA2; K(D) = 12 nM), ostreolysin (Oly; K(D) = 1.3 nM), and erylysin A (EryA; K(D) = 1.3 nM)­strongly associated with CPE/cholesterol (Chol)-containing membranes, whereas their low affinity to sphingomyelin/Chol precluded establishment of the binding kinetics. Binding specificity was determined by multilamellar liposome binding assays, supported bilayer assays, and solid-phase studies against a series of neutral and negatively charged lipid classes mixed 1:1 with Chol or phosphatidylcholine. No cross-reactivity was detected with phosphatidylethanolamine. Only PlyA2 also associated with CPE, independent of Chol content (K(D) = 41 µM), rendering it a suitable tool for visualizing CPE in lipid-blotting experiments and biologic samples from sterol auxotrophic organisms. Visualization of CPE enrichment in the CNS of Drosophila larvae (by PlyA2) and in the bloodstream form of the parasite Trypanosoma brucei (by EryA) by fluorescence imaging demonstrated the versatility of aegerolysin family proteins as efficient tools for detecting and visualizing CPE.

Binding of a pleurotolysin ortholog from Pleurotus eryngii to sphingomyelin and cholesterol-rich membrane domains.

A mixture of sphingomyelin (SM) and cholesterol (Chol) exhibits a characteristic lipid raft domain of the cell membranes that provides a platform to which various signal molecules as well as virus and bacterial proteins are recruited. Several proteins capable of specifically binding either SM or Chol have been reported. However, proteins that selectively bind to SM/Chol mixtures are less well characterized. In our screening for proteins specifically binding to SM/Chol liposomes, we identified a novel ortholog of Pleurotus ostreatus, pleurotolysin (Ply)A, from the extract of edible mushroom Pleurotus eryngii, named PlyA2. Enhanced green fluorescent protein (EGFP)-conjugated PlyA2 bound to SM/Chol but not to phosphatidylcholine/Chol liposomes. Cell surface labeling of PlyA2-EGFP was abolished after sphingomyelinase as well as methyl-ß-cyclodextrin treatment, removing SM and Chol, respectively, indicating that PlyA2-EGFP specifically binds cell surface SM/Chol rafts. Tryptophan to alanine point mutation of PlyA2 revealed the importance of C-terminal tryptophan residues for SM/Chol binding. Our results indicate that PlyA2-EGFP is a novel protein probe to label SM/Chol lipid domains both in cell and model membranes.

Prolyl isomerase Pin1 regulates mouse embryonic fibroblast differentiation into adipose cells.

BACKGROUND: A peptidyl prolyl cis/trans isomerase, Pin1, regulates insulin signal transduction. Pin1 reduces responses to insulin stimulation by binding CRTC2 (CREB-regulated transcriptional co-activator 2) and PPARγ (peroxisome prolifereator- activated receptor γ), but conversely enhances insulin signaling by binding IRS-1 (insulin receptor substrate-1), Akt kinase, and Smad3. Therefore, it is still unclear whether Pin1 inhibits or enhances adipose cell differentiation. METHODOLOGY/PRINCIPAL FINDINGS: Pin1(-/-) and wild-type mice were fed with high fat diets and adipose tissue weight was measured. Compared to wild-type mice, Pin1(-/-) mice had lower adipose tissue weight, while the weight of other tissues was similar. Mouse embryo fibroblasts (MEFs), prepared from both groups of mice, were induced to differentiate into adipose cells by stimulation with insulin. However, the rate of differentiation of MEFs from Pin1(-/-) mice was less than that of MEFs from wild-type mice. The rate of insulin-induced MEF cell differentiation in Pin1(-/-) mice was restored by increasing expression of Pin1. We found that Pin1 binds to phosphoThr172- and phosphoSer271-Pro sites in CREB suppress the activity in COS-7 cells. CONCLUSION AND SIGNIFICANCE: Pin1 enhanced the uptake of triglycerides and the differentiation of MEF cells into adipose cells in response to insulin stimulation. Results of this study suggest that Pin1 down-regulation could be a potential approach in obesity-related dysfunctions, such as high blood pressure, diabetes, non-alcoholic steatohepatitis.

Marianins A and B, prenylated phenylpropanoids from Mariannaea camptospora.

Marianins A (1) and B (2), two new prenylated phenylpropanoids, were isolated from the culture extract of the fungus Mariannaea camptospora. Structures of marianins were elucidated by interpretation of NMR and other spectroscopic data. 1 is a 5-methylcoumarin bearing two prenyloxy groups, while 2 is an orcinol derivative substituted with a 3,3-dimethyl-4-pentenoyl chain. 2 is possibly derived from 1 through a Claisen rearrangement of the prenyl group, followed by lactone hydrolysis and decarboxylation. These compounds showed weak antibacterial activity against Micrococcus luteus.

Global analysis of the expression patterns of transcriptional regulatory factors in formation of embryoid bodies using sensitive oligonucleotide microarray systems.

We manufactured a highly sensitive oligonucleotide microarray system comprised entirely of transcription regulatory factors (a TF oligo microarray) in order to comprehensively analyze the expression profiles of transcription factors in mice. We compared the expression profiles of transcription regulatory factors in mouse embryonic stem (ES) cells and ES-differentiated cells by using this TF oligo microarray, a cDNA microarray, a GeneChip system, and quantitative RT-PCR. The TF oligo microarray was able to comprehensively analyze the expression profile of transcription regulatory factors. In addition, we used the manufactured TF oligo microarray to analyze the expression patterns of transcriptional regulatory factors during the formation of embryoid bodies. The TF array was able to reveal the chronologic expression profile of transcription regulatory factors involved in embryogenesis or the maintenance of pluripotency in ES cells.

Global analysis of the regulatory network structure of gene expression in Saccharomyces cerevisiae.

Gene expression in eukaryotic cells is controlled by the concerted action of various transcription factors. To help clarify these complex mechanisms, we attempted to develop a method for extracting maximal information regarding the transcriptional control pathways. To this end, we first analyzed the expression profiles of numerous transcription factors in yeast cells, under the assumption that the expression levels of these factors would be elevated under conditions in which the factors were active in the cells. Based on the results, we successfully categorized about 400 transcription factors into three groups based on their expression profiles. We then analyzed the effect of the loss of function of various induced transcription factors on the global expression profile to investigate the above-mentioned assumption of a correlation between transcription elevation and functional activity. By comparing the expression profiles of wild-type with those of disruption mutants using microarrays, we were able to detect a substantial number of relations between transcription factors and the genes they regulate. The results of these experiments suggested that our approach is useful for understanding the global transcriptional networks of eukaryotic cells, in which most genes are regulated in a temporal and conditional manner.

Dbp9p, a member of the DEAD box protein family, exhibits DNA helicase activity.

The yeast Dbp9p is a member of the DEAD box family of RNA helicases, which are thought to be involved in RNA metabolism. Dbp9p seems to function in ribosomal RNA biogenesis, but it has not been biochemically characterized. To analyze the enzymatic characteristics of the protein, we expressed a recombinant Dbp9p in Escherichia coli and purified it to homogeneity. The purified protein exhibited RNA unwinding and binding activity in the absence of NTP, and this activity was abolished by a mutation in the RNA-binding domain. We then characterized the ATPase activity of Dbp9p with respect to cofactor specificity; the activity was found to be severely inhibited by yeast total RNA and moderately inhibited by poly(U), poly(A), and poly(C) but to be stimulated by yeast genomic DNA and salmon sperm DNA. In addition, Dbp9p exhibited DNA-DNA and DNA-RNA helicase activity in the presence of ATP. These results indicate that Dbp9p has biochemical characteristics unique among DEAD box proteins.

Pin1 and Par14 peptidyl prolyl isomerase inhibitors block cell proliferation.

Disruption of the parvulin family peptidyl prolyl isomerase (PPIase) Pin1 gene delays reentry into the cell cycle when quiescent primary mouse embryo fibroblasts are stimulated with serum. Since Pin1 regulates cell cycle progression, a Pin1 inhibitor would be expected to block cell proliferation. To identify such inhibitors, we screened a chemical compound library for molecules that inhibited human Pin1 PPIase activity in vitro. We found a set of compounds that inhibited Pin1 PPIase activity in vitro with low microM IC50s and inhibited the growth of several cancer lines. Among the inhibitors, PiB, diethyl-1,3,6,8-tetrahydro-1,3,6,8-tetraoxobenzo[lmn] phenanthroline-2,7-diacetate ethyl 1,3,6,8-tetrahydro-1,3,6,8-tetraoxo-benzo[lmn] phenanthroline-(2H,7H)-diacetate, had the least nonspecific toxicity. These results suggest that Pin1 inhibitors could be used as a novel type of anticancer drug that acts by blocking cell cycle progression.

Uridine phosphorylase is a potential prognostic factor in patients with oral squamous cell carcinoma.

BACKGROUND: Uridine phosphorylase (UPase) catalyzes the reversible phosphorolysis of uridine to uracil. The expression levels and the enzymatic activity of UPase are reported to be higher in human solid tumors than in adjacent normal tissues. However, to the authors' knowledge the clinical significance of UPase expression as determined by immunohistochemical analysis has not been demonstrated in human malignancies. METHODS: The authors prepared the antibody against UPase, examined the staining of UPase in 72 cases of oral squamous cell carcinoma (SCC) with immunohistochemical analysis using the antibody, and analyzed its relation to clinical and pathologic factors. RESULTS: UPase stained mainly within the invasive edges of tumors and macrophages. UPase-positive staining was observed in 56 of the 72 tumors (77.8%). High staining of UPase (defined as > 50% of cells in a tumor biopsy that are positive for UPase) in primary tumors frequently was associated with the presence of metastasis to lymph nodes (P = 0.007 by the Fischer exact test) and with lower overall survival (P = 0.03 by the log-rank test). CONCLUSIONS: The high rate of UPase staining in biopsies from patients with oral SCC may be a prognostic marker for these individuals.

Isolation and proteomic characterization of human Parvulin-associating preribosomal ribonucleoprotein complexes.

Human parvulin (hParvulin; Par14/EPVH) belongs to the third family of peptidylprolyl cis-trans isomerases that exhibit an enzymatic activity of interconverting the cis-trans conformation of the prolyl peptide bond, and shows sequence similarity to the regulator enzyme for cell cycle transitions, human Pin1. However, the cellular function of hParvulin is entirely unknown. Here, we demonstrate that hParvulin associates with the preribosomal ribonucleoprotein (pre-rRNP) complexes, which contain preribosomal RNAs, at least 26 ribosomal proteins, and 26 trans-acting factors involved in rRNA processing and assembly at an early stage of ribosome biogenesis. Since an amino-terminal domain of hParvulin, which is proposed to be a putative DNA-binding domain, was alone sufficient to associate in principle with the pre-rRNP complexes, the association is probably through protein-RNA interaction. In addition, hParvulin co-precipitated at least 10 proteins not previously known to be involved in ribosome biogenesis. Coincidentally, most of these proteins are implicated in regulation of microtubule assembly or nucleolar reformation during the mitotic phase of the cell. Thus, these results, coupled with the preferential nuclear localization of hParvulin, suggest that hParvulin may be involved in ribosome biogenesis and/or nucleolar re-assembly of mammalian cells.

Identification of genes coding enzymes for ascomycin tetra-hydropyranose ring formation.

Macrocyclic polyketides have generated great interest in biosynthetic chemistry because of the structural complexity and medicinal activities. The synthetic genes consist of the number and type of active sites of modular polyketide synthases. The cosmid library - prepared with the ascomycin (an antibiotics with immunosuppressive activity) - producer, Streptomyces sp. AA6554 genome was screened with an ascomycin ketosynthase gene probe, and one and a half modules were isolated. Database analysis shows that one of the modules consists of the genes coding a series of enzymes for the tetra-hydropyranose ring synthesis.

Loss of Pin1 function in the mouse causes phenotypes resembling cyclin D1-null phenotypes.

Phosphorylation of proteins on serine/threonine residues preceding proline is a key signaling mechanism. The conformation and function of a subset of these phosphorylated proteins is regulated by the prolyl isomerase Pin1 through isomerization of phosphorylated Ser/Thr-Pro bonds. Although young Pin1(-/-) mice have been previously shown to develop normally, we show here that they displayed a range of cell-proliferative abnormalities, including decreased body weight and testicular and retinal atrophies. Furthermore, in Pin1(-/-) adult females, the breast epithelial compartment failed to undergo the massive proliferative changes associated with pregnancy. Interestingly, many of these Pin1-deficient phenotypes such as retinal hypoplasia and mammary gland impairment are also the characteristic of cyclin D1-deficient mice. Cyclin D1 levels were significantly reduced in many tissues in Pin1-deficient mice, including retina and breast epithelial cells from pregnant mice. Moreover, Pin1 directly bound to cyclin D1 phosphorylated on Thr-286-Pro increased cyclin D1 in the nucleus and stabilized cyclin D1. These results indicate that Pin1 positively regulates cyclin D1 function at the transcriptional level, as demonstrated previously, and also through posttranslational stabilization, which together explain why Pin1 loss-of-function phenotypes in the mouse resemble cyclin D1-null phenotypes. Our results provide genetic evidence for an essential role of Pin1 in maintaining cell proliferation and regulating cyclin D1 function.

Application of peptide nucleic acid (PNA) for detection of transcription factors binding probes.

We designed novel PNA monomer units that could post-synthetically and quantitatively introduce photo-functionalized molecules into PNA oligomers. Designed photo-functionalized PNAs were applicable to wide fields such as genome based analysis. In this study, we applied a photo-functionalized PNA oligomers for the detection of transcription factors binding probes.