The Tumor Suppressor BCL7B Functions in the Wnt Signaling Pathway

Human BCL7 gene family consists of BCL7A, BCL7B, and BCL7C. A number of clinical studies have reported that BCL7 family is involved in cancer incidence, progression, and development. Among them, BCL7B, located on chromosome 7q11.23, is one of the deleted genes in patients with Williams-Beuren syndrome. Although several studies have suggested that malignant diseases occurring in patients with Williams-Beuren syndrome are associated with aberrations in BCL7B, little is known regarding the function of this gene at the cellular level. In this study, we focused on bcl-7, which is the only homolog of BCL7 gene family in Caenorhabditis elegans, and analyzed bcl-7 deletion mutants. As a result, we found that bcl-7 is required for the asymmetric differentiation of epithelial seam cells, which have self-renewal properties as stem cells and divide asymmetrically through the WNT pathway. Distal tip cell development, which is regulated by the WNT pathway in Caenorhabditis elegans, was also affected in bcl-7-knockout mutants. Interestingly, bcl-7 mutants exhibited nuclear enlargement, reminiscent of the anaplastic features of malignant cells. Furthermore, in KATOIII human gastric cancer cells, BCL7B knockdown induced nuclear enlargement, promoted the multinuclei phenotype and suppressed cell death. In addition, this study showed that BCL7B negatively regulates the Wnt-signaling pathway and positively regulates the apoptotic pathway. Taken together, our data indicate that BCL7B/BCL-7 has some roles in maintaining the structure of nuclei and is involved in the modulation of multiple pathways, including Wnt and apoptosis. This study may implicate a risk of malignancies with BCL7B-deficiency, such as Williams-Beuren syndrome.     

Megazol and its bioisostere 4H-1,2,4-triazole: comparing the trypanocidal,cytotoxic and genotoxic activities and their in vitro and in silico interactions with the Trypanosoma brucei nitroreductase enzyme

Megazol (7) is a 5-nitroimidazole that is highly active against Trypanosoma cruzi and Trypanosoma brucei, as well as drug-resistant forms of trypanosomiasis. Compound 7 is not used clinically due to its mutagenic and genotoxic properties, but has been largely used as a lead compound. Here, we compared the activity of 7 with its 4H-1,2,4-triazole bioisostere (8) in bloodstream forms of T. brucei and T. cruzi and evaluated their activation by T. brucei type I nitroreductase (TbNTR) enzyme. We also analysed the cytotoxic and genotoxic effects of these compounds in whole human blood using Comet and fluorescein diacetate/ethidium bromide assays. Although the only difference between 7 and 8 is the substitution of sulphur (in the thiadiazole in 7) for nitrogen (in the triazole in 8), the results indicated that 8 had poorer antiparasitic activity than 7 and was not genotoxic, whereas 7 presented this effect. The determination of Vmax indicated that although 8 was metabolised more rapidly than 7, it bounds to the TbNTR with better affinity, resulting in equivalent kcat/KM values. Docking assays of 7 and 8 performed within the active site of a homology model of the TbNTR indicating that 8 had greater affinity than 7.     

Antifungal activity of 1′-homo-N-1,2,3-triazol-bicyclic carbonucleosides: A novel type of compound afforded by azide-enolate (3+2) cycloaddition

The first report of 1′-homo-N-1,2,3-triazol-bicyclic carbonucleosides (7a and 7b) is described herein. Azide-enolate (3+2) cycloaddition afforded the synthesis of this novel type of compound. Antifungal activity was evaluated in vitro against four filamentous fungi (Aspergillus fumigatus, Trichosporon cutaneum, Rhizopus oryzae and Mucor hiemalis) as well as nine species of Candida spp. as yeast specimens. These pre-clinical studies suggest that compounds 7a and 7b are promising candidates for complementary biological studies due to their good activity against Candida spp.     

The microbiological transformation of some trachylobane diterpenoids by Gibberella fujikuroi

The microbiological transformation of ent-trachylobane, ent-7α-hydroxytrachylobane and ent-19-hydroxytrachylobane into trachylobagibberellins A₇, A₉, A₁₃, A₂₅, A₄₀ and A₄₇ by Gibberella fujikuroi is described. Whereas 7β-hydroxy- and 7β,18-dihydroxytrachylobanolides were obtained from ent-trachylobane and ent-trachyloban- 19-ol, the presence of a 7β-hydroxyl group directed metabolism exclusively into the gibberellin pathway. An 18-hydroxyl group as in ent-7α,18-dihydroxytrachylobane inhibited oxidation at C-6 affording ent-7α,18,19-trihydroxytrachylobane as the major metabolite.     

A chemosystematic study of the phenolics of Sonchus

Thirty-three Sonchus, one Embergeria, one Babcockia and five Taeckholmia species were surveyed for their phenolic constituents. The coumarins scopoletin and aesculetin were found as major constituents of Embergeria, Babcockia and Taeckholmia species, and in lesser amount in some Sonchus species. Six flavone glycosides were identified: apigenin 7-glucuronide, apigenin 7-rutinoside, luteolin 7-glucoside, luteolin 7-glucuronide, luteolin 7-rutinoside and luteolin 7-glucosylglucuronide and the systematic significance of their distribution is discussed.     

Leaf flavonoid and other phenolic glycosides as indicators of parentage in six ornamental Fuchsia species and their hybrids

In a leaf flavonoid analysis of six Fuchsia species and seven Fuchsia hybrids, flavonols were found to be abundant in all taxa except F. procumbens. Flavone glycosides were found in only three species: luteolin 7-glucoside in F. splendens; and luteolin and apigenin 7-glucuronides and 7-glucuronidesulphates, tricin 7-glucuronidesulphate and diosmetin 7-glucuronide from one or both of the New Zealand species F. procumbens and F. excorticata. Luteolin 7- glucuronidesulphate is reported for the first time. Other less common phenolics identified include the flavanone, eriodictyol 7-glucoside from F. excorticata, a galloylglucose from F. triphylla, and a galloylglucosesulphate present in all taxa. Eight of the flavonoid glycosides proved useful as marker substances for particular Fuchsia species: quercetin 3- rhamnoside, 3-glucuronide and 3-rutinoside for F.fulgens; quercetin and kaempferol 3-galactosides for F. boliviana var. luxurians; diosmetin 7-glucuronide for F. excorticata and apigenin 7-glucuronide and 7-glucuronidesulphate for F. procumbens. The chemical results on the hybrids support the view that the cultivar ‘Mary’ is a hybrid of F. boliviana var. luxurians and F. triphylla and that both F.fulgens and F. triphylla are involved as parents of the cultivars ‘Koralle’ and ‘Traudchen Bondstedt’.     

Regulation of APCCdh1 E3 ligase activity by the Fbw7/cyclin E signaling axis contributes to the tumor suppressor function of Fbw7

Fbw7 and Cdh1 are substrate-recognition subunits of the SCF- and APC-type E3 ubiquitin ligases, respectively. There is emerging evidence suggesting that both Fbw7 and Cdh1 function as tumor suppressors by targeting oncoproteins for destruction. Loss of Fbw7, but not Cdh1, is frequently observed in various human tumors. However, it remains largely unknown how Fbw7 mechanistically functions as a tumor suppressor and whether there is a signaling crosstalk between Fbw7 and Cdh1. Here, we report that Fbw7-deficient cells not only display elevated expression levels of SCF^Fbw7 substrates, including cyclin E, but also have increased expression of various APC^Cdh1 substrates. We further defined cyclin E as the critical signaling link by which Fbw7 governs APC^Cdh1 activity, as depletion of cyclin E in Fbw7-deficient cells results in decreased expression of APC^Cdh1 substrates to levels comparable to those in wild-type (WT) cells. Conversely, ectopic expression of cyclin E recapitulates the aberrant APC^Cdh1 substrate expression observed in Fbw7-deficient cells. More importantly, 4A-Cdh1 that is resistant to Cdk2/cyclin E-mediated phosphorylation, but not WT-Cdh1, reversed the elevated expression of various APC^Cdh1 substrates in Fbw7-deficient cells. Overexpression of 4A-Cdh1 also resulted in retarded cell growth and decreased anchorage-independent colony formation. Altogether, we have identified a novel regulatory mechanism by which Fbw7 governs Cdh1 activity in a cyclin E-dependent manner. As a result, loss of Fbw7 can lead to aberrant increase in the expression of both SCF^Fbw7 and APC^Cdh1 substrates. Our study provides a better understanding of the tumor suppressor function of Fbw7, and suggests that Cdk2/cyclin E inhibitors could serve as effective therapeutic agents for treating Fbw7-deficient tumors.     

Interdependence of the Peroxisome-targeting Receptors in Arabidopsis thaliana: PEX7 Facilitates PEX5 Accumulation and Import of PTS1 Cargo into Peroxisomes

Peroxisomes compartmentalize certain metabolic reactions critical to plant and animal development. The import of proteins from the cytosol into the organelle matrix depends on more than a dozen peroxin (PEX) proteins, with PEX5 and PEX7 serving as receptors that shuttle proteins bearing one of two peroxisome-targeting signals (PTSs) into the organelle. PEX5 is the PTS1 receptor; PEX7 is the PTS2 receptor. In plants and mammals, PEX7 depends on PEX5 binding to deliver PTS2 cargo into the peroxisome. In this study, we characterized a pex7 missense mutation, pex7-2, that disrupts both PEX7 cargo binding and PEX7-PEX5 interactions in yeast, as well as PEX7 protein accumulation in plants. We examined localization of peroxisomally targeted green fluorescent protein derivatives in light-grown pex7 mutants and observed not only the expected defects in PTS2 protein import but also defects in PTS1 import. These PTS1 import defects were accompanied by reduced PEX5 accumulation in light-grown pex7 seedlings. Our data suggest that PEX5 and PTS1 import depend on the PTS2 receptor PEX7 in Arabidopsis and that the environment may influence this dependence. These data advance our understanding of the biogenesis of these essential organelles and provide a possible rationale for the retention of the PTS2 pathway in some organisms.     

Regioisomerism-dependent TLR7 agonism and antagonism in an imidazoquinoline

Chronic immune activation is a hallmark of progressive HIV infection. Recent reports point to the engagement of toll-like receptor 7 (TLR7) and -9 by viral RNA as contributing to the activation of innate immune responses, which drive viral replication leading to immune exhaustion. The only known class of TLR7 antagonists is single-stranded phosphorothioate oligonucleotides, which has been demonstrated to inhibit immune activation in human and Rhesus macaque in vitro models. The availability of a selective and potent small-molecule TLR7 antagonist should allow the evaluation of potential benefits of suppression of TLR7-mediated immune activation in HIV/AIDS. Gardiquimod is a known N¹-substituted 1H-imidazoquinoline TLR7 agonist, the synthesis of which has not been published. We show that the 3H regioisomer is completely inactive as a TLR7 agonist and is weakly antagonistic. A des-amino precursor of the 3H regioisomer is more potent as a TLR7 antagonist, with an IC₅₀ value of 7.5 μM. This class of compound may serve as a starting point for the development of small-molecule inhibitors of TLR7.     

Mcp7, a meiosis-specific coiled-coil protein of fission yeast, associates with Meu13 and is required for meiotic recombination

We previously showed that Meu13 of Schizosaccharomyces pombe functions in homologous pairing and recombination at meiosis I. Here we show that a meiosis-specific gene encodes a coiled-coil protein that complexes with Meu13 during meiosis in vivo. This gene denoted as mcp7⁺ (after meiotic coiled-coil protein) is an ortholog of Mnd1 of Saccharomyces cerevisiae. Mcp7 proteins are detected on meiotic chromatin. The phenotypes of mcp7Δ cells are similar to those of meu13Δ cells as they show reduced recombination rates and spore viability and produce spores with abnormal morphology. However, a delay in initiation of meiosis I chromosome segregation of mcp7Δ cells is not so conspicuous as meu13Δ cells, and no meiotic delay is observed in mcp7Δmeu13Δ cells. Mcp7 and Meu13 proteins depend on each other differently; Mcp7 becomes more stable in meu13Δ cells, whereas Meu13 becomes less stable in mcp7Δ cells. Genetic analysis shows that Mcp7 acts in the downstream of Dmc1, homologs of Escherichia coli RecA protein, for both recombination and subsequent sporulation. Taken together, we conclude that Mcp7 associates with Meu13 and together they play a key role in meiotic recombination.     

Novel Biotransformations of 7-Ethoxycoumarin by Streptomyces griseus

Biotransformation of 7-ethoxycoumarin by Streptomyces griseus resulted in the accumulation of two metabolites which were isolated and identified as 7-hydroxycoumarin and 7-hydroxy-6-methoxycoumarin. A novel series of biotransformation reactions is implicated in the conversion of the ethoxycoumarin substrate to these products, including O-deethylation, 6-hydroxylation to form a 6,7-dihydroxycoumarin catechol, and subsequent O-methylation. Either 7-hydroxycoumarin or 6,7-dihydroxycoumarin was biotransformed to 7-hydroxy-6-methoxycoumarin by S. griseus. Trace amounts of the isomeric 6-hydroxy-7-methoxycoumarin were detected when 6,7-dihydroxycoumarin was used as the substrate. Efforts to obtain a cell-free catechol-O-methyltransferase enzyme system from S. griseus were unsuccessful. However, [methyl-¹⁴C]methionine was used with cultures of S. griseus to form 7-hydroxy-6-[¹⁴C]methoxycoumarin.     

Discrimination between aminoacyl groups on su+ 7 tRNA by elongation factor Tu

The su⁺7 nonsense suppressor of Escherichia coli is a mutant tRNA^Trp that can be aminoacylated with either tryptophan or glutamine. We have compared the ternary complexes of glutaminyl and tryptophanyl-su⁺7 tRNA with elongation factor Tu and GTP. Glutaminyl-su⁺7 tRNA binds more strongly than tryptophanyl-su⁺7 tRNA to EF Tu · GTP. The greatest distinction between the two species of the tRNA is seen in their dissociation rates from the complex, which differ by as much as fivefold. The distinction is affected by pH values around neutrality. These results show that EF Tu can distinguish between two aminoacyl-tRNAs which differ only in the aminoacyl group. The implications for the unusual amino acid specificity of su⁺7 tRNA are discussed.     

aldh7a1 Regulates Eye and Limb Development in Zebrafish

Uveal coloboma is a potentially blinding congenital ocular malformation caused by failure of the optic fissure to close during development. Although mutations in numerous genes have been described, these account for a minority of cases, complicating molecular diagnosis and genetic counseling. Here we describe a key role of aldh7a1 as a gene necessary for normal eye development. We show that morpholino knockdown of aldh7a1 in zebrafish causes uveal coloboma and misregulation of nlz1, another known contributor to the coloboma phenotype, as well as skeletal abnormalities. Knockdown of aldh7a1 leads to reduced cell proliferation in the optic cup of zebrafish, delaying the approximation of the edges of the optic fissure. The aldh7a1 morphant phenotype is partially rescued by co-injection of nlz1 mRNA suggesting that nlz1 is functionally downstream of aldh7a1 in regulating cell proliferation in the optic cup. These results support a role of aldh7a1 in ocular development and skeletal abnormalities in zebrafish.     

Arabidopsis thaliana PGR7 Encodes a Conserved Chloroplast Protein That Is Necessary for Efficient Photosynthetic Electron Transport

A significant fraction of a plant''s nuclear genome encodes chloroplast-targeted proteins, many of which are devoted to the assembly and function of the photosynthetic apparatus. Using digital video imaging of chlorophyll fluorescence, we isolated proton gradient regulation 7 (pgr7) as an Arabidopsis thaliana mutant with low nonphotochemical quenching of chlorophyll fluorescence (NPQ). In pgr7, the xanthophyll cycle and the PSBS gene product, previously identified NPQ factors, were still functional, but the efficiency of photosynthetic electron transport was lower than in the wild type. The pgr7 mutant was also smaller in size and had lower chlorophyll content than the wild type in optimal growth conditions. Positional cloning located the pgr7 mutation in the At3g21200 (PGR7) gene, which was predicted to encode a chloroplast protein of unknown function. Chloroplast targeting of PGR7 was confirmed by transient expression of a GFP fusion protein and by stable expression and subcellular localization of an epitope-tagged version of PGR7. Bioinformatic analyses revealed that the PGR7 protein has two domains that are conserved in plants, algae, and bacteria, and the N-terminal domain is predicted to bind a cofactor such as FMN. Thus, we identified PGR7 as a novel, conserved nuclear gene that is necessary for efficient photosynthetic electron transport in chloroplasts of Arabidopsis.     

Saccharomyces cerevisiae Env7 Is a Novel Serine/Threonine Kinase 16-Related Protein Kinase and Negatively Regulates Organelle Fusion at the Lysosomal Vacuole

Membrane fusion depends on conserved components and is responsible for organelle biogenesis and vesicular trafficking. Yeast vacuoles are dynamic structures analogous to mammalian lysosomes. We report here that yeast Env7 is a novel palmitoylated protein kinase ortholog that negatively regulates vacuolar membrane fusion. Microscopic and biochemical studies confirmed the localization of tagged Env7 at the vacuolar membrane and implicated membrane association via the palmitoylation of its N-terminal Cys13 to -15. In vitro kinase assays established Env7 as a protein kinase. Site-directed mutagenesis of the Env7 alanine-proline-glutamic acid (APE) motif Glu269 to alanine results in an unstable kinase-dead allele that is stabilized and redistributed to the detergent-resistant fraction by interruption of the proteasome system in vivo. Palmitoylation-deficient Env7C13-15S is also kinase dead and mislocalizes to the cytoplasm. Microscopy studies established that env7Δ is defective in maintaining fragmented vacuoles during hyperosmotic response and in buds. ENV7 function is not redundant with a similar role of vacuolar membrane kinase Yck3, as the two do not share a substrate, and ENV7 is not a suppressor of yck3Δ. Bayesian phylogenetic analyses strongly support ENV7 as an ortholog of the gene encoding human STK16, a Golgi apparatus protein kinase with undefined function. We propose that Env7 function in fusion/fission dynamics may be conserved within the endomembrane system.     

Corrigendum

FBW7 (F-box and WD repeat domain containing 7), also known as FBXW7 or hCDC4, is a tumor suppressor gene mutated in a broad spectrum of cancer cell types. As a component of the SCF E3 ubiquitin ligase, FBW7 is responsible for specifically recognizing phosphorylated substrates, many important for tumor progression, and targeting them for ubiquitin-mediated degradation. Although the role of FBW7 as a tumor suppressor is well established, less well studied is how FBW7-mutated cancer cells might be targeted for selective killing. To explore this further, we undertook a genome-wide RNAi screen using WT and FBW7 knockout colorectal cell lines and identified the spindle assembly checkpoint (SAC) protein BUBR1, as a candidate synthetic lethal target. We show here that asynchronous FBW7 knockout cells have increased levels of mitotic APC/C substrates and are sensitive to knockdown of not just BUBR1 but BUB1 and MPS1, other known SAC components, suggesting a dependence of these cells on the mitotic checkpoint. Consistent with this dependence, knockdown of BUBR1 in cells lacking FBW7 results in significant cell aneuploidy and increases in p53 levels. The FBW7 substrate cyclin E was necessary for the genetic interaction with BUBR1. In contrast, the establishment of this dependence on the SAC requires the deregulation of multiple substrates of FBW7. Our work suggests that FBW7 knockout cells are vulnerable in their dependence on the mitotic checkpoint and that this may be a good potential target to exploit in FBW7-mutated cancer cells.     

Trpm7 Protein Contributes to Intercellular Junction Formation in Mouse Urothelium

Trpm7 is a divalent cation-permeable channel that has been reported to be involved in magnesium homeostasis as well as cellular adhesion and migration. We generated urothelium-specific Trpm7 knock-out (KO) mice to reveal the function of Trpm7 in vivo. A Trpm7 KO was induced by tamoxifen and was confirmed by genomic PCR and immunohistochemistry. By using patch clamp recordings in primary urothelial cells, we observed that Mg²⁺-inhibitable cation currents as well as acid-inducible currents were significantly smaller in Trpm7 KO urothelial cells than in cells from control mice. Assessment of voiding behavior indicated a significantly smaller voided volume in Trpm7 KO mice (mean voided volume 0.28 ± 0.08 g in KO mice and 0.36 ± 0.04 g in control mice, p < 0.05, n = 6–8). Histological analysis showed partial but substantial edema in the submucosal layer of Trpm7 KO mice, most likely due to inflammation. The expression of proinflammatory cytokines TNF-α and IL-1β was significantly higher in Trpm7 KO bladders than in controls. In transmission electron microscopic analysis, immature intercellular junctions were observed in Trpm7 KO urothelium but not in control mice. These results suggest that Trpm7 is involved in the formation of intercellular junctions in mouse urothelium. Immature intercellular junctions in Trpm7 knock-out mice might lead to a disruption of barrier function resulting in inflammation and hypersensitive bladder afferent nerves that may affect voiding behavior in vivo.