FIP200 inhibits ß-catenin-mediated transcription by promoting APC-independent ß-catenin ubiquitination.

Focal adhesion kinase-family-interacting protein of 200 kDa (FIP200) has been shown to regulate multiple cellular functions, including cell adhesion, autophagy, development and proliferation. Furthermore, FIP200 is considered to have tumor-suppressive activity, which may be correlated with its inactivation in human breast cancers, in addition to its role as an important signal transduction node. Herein, we report that FIP200 interacts with the oncoprotein ß-catenin. Moreover, FIP200 promotes destabilization of wild-type ß-catenin, but not a cancer-causing form of ß-catenin, and as a result represses the ß-catenin-mediated transcription. FIP200-induced degradation of ß-catenin is independent of adenomatous polyposis coli (APC) of the well-established ß-catenin destruction complex (glycogen synthase kinase-3ß/axin/APC), in a component of ß-catenin E3 ubiquitin ligase, ß-TrCP-dependent manner. Thus, the APC-independent ß-catenin degradation by FIP200 suggests a role for FIP200 in tumor suppression in the presence of APC dysfunction. These findings reveal a new and important function of FIP200 in regulation of the Wnt/ß-catenin pathway.

Microarray expression profiling of tissues from mice with uniparental duplications of chromosomes 7 and 11 to identify imprinted genes.

Microarray analysis allows the screening of thousands of identifiable genes in a single experiment. The challenge of this approach is to combine the new technology with established genetic tools to associate genes with specific biological function. In this study we have designed a screen to identify imprinted genes from mice with uniparental duplications of proximal Chromosomes (Chrs) 7 and 11, using microarray analysis. By comparing the expression patterns in embryonic and newborn tissues of maternally versus paternally inherited proximal Chrs 7 and 11, we have correctly identified four out of five known imprinted genes represented on a microarray. We have additionally identified two novel imprinted candidate genes as well as a differentially expressed clone that is a potential downstream target. Interpretation of the microarray data requires careful preparation of age- and strain-matched samples and attention to detail in tissue dissection technique.

Roles of histidine residues in tobacco acetolactate synthase.

Acetolactate synthase (ALS) catalyzes the first common step in the biosynthesis of valine, leucine, and isoleucine in plants and microorganisms. ALS is the target of several structurally diverse classes of herbicides, including sulfonylureas, imidazolinones, and triazolopyrimidines. The roles of three well-conserved histidine residues (H351, H392, and H487) in tobacco ALS were determined using site-directed mutagenesis. Both H487F and H487L mutations abolished the enzymatic activity as well as the binding affinity for the cofactor FAD. Nevertheless, the mutation of H487F did not affect the secondary structure of the ALS. The K(m) values of H351M, H351Q, and H351F are approximately 18-, 60-, and fivefold higher than that of the wild-type ALS, respectively. Moreover, the K(c) value of H351Q for FAD is about 137-fold higher than that of wALS. Mutants H351M and H351Q showed very strong resistance to Londax (a sulfonylurea) and Cadre (an imidazolinone), whereas mutant H351F was weakly resistant to them. However, the secondary structures of mutants H351M and H351Q appeared to be different from that of wALS. The mutation of H392M did not have any significant effect on the kinetic parameters nor the resistance to ALS-inhibiting herbicides. These results suggest that the His487 residue is located at the active site of the enzyme and is likely involved in the binding of cofactor FAD in tobacco ALS. Mutational analyses of the His351 residue imply that the active site of the ALS is probably close to its binding site of the herbicides, Londax and Cadre.

Crystallization of Clonorchis sinensis 26 kDa glutathione S-transferase and its fusion proteins with peptides of different lengths.

A Clonorchis sinensis 26 kDa glutathione S-transferase (CsGST) and its fusion proteins containing 14 and 48 amino-acid peptides at the N-terminus have been crystallized using polyethylene glycol monomethylether 550 as a precipitant. Crystals of the three proteins show very similar crystal properties: they diffract to at least 2.3 A resolution and belong to the orthorhombic space group P2(1)2(1)2(1). The unit-cell parameters of CsGST crystals were a = 66.64 (1), b = 68.91 (1), c = 123.41 (2) A, which are very close to those of the crystals of the two fusion proteins. In addition, CsGST fusion proteins containing varying extents of N-terminal-extended peptides are incorporated into a crystal, indicating that the extended peptides have little effect on crystal packing. These results suggest that the crystallization system of CsGST/peptide fusion protein may be generally applicable to obtain crystals of small peptides.

Anti-elastase and anti-hyaluronidase of phenolic substance from Areca catechu as a new anti-ageing agent.

We have previously screened 150 medicinal plants for the inhibition of elastase and found significant inhibitory effects of the extracts of Areca catechu L. on the ageing and inflammation of skin tissues. To isolate and identify the compounds having biological activity, they were further purified by each fraction of solvents, silica gel column chromatography, preparative TLC and reversed-phase HPLC. The peak in HPLC, which coincided with the inhibitory activity against elastase, was identified as a phenolic substance by using various colorimetric methods, UV and IR. IC(50) values of this phenolic substance were 26.9 mug mL(-1) for porcine pancreatic elastase (PPE) and 60.8 mug mL(-1) for human neutrophil elastase (HNE). This phenolic substance showed more potent activity than that of reference compounds, oleanolic acid (76.5 mug mL(-1) for PPE, 219.2 mug mL(-1) for HNE) and ursolic acid (31.0 mug mL(-1) for PPE, 118.6 mug mL(-1) for HNE). According to the Lineweaver-Burk plots, the inhibition against both PPE and HNE by this phenolic substance was competitive inhibition with the substrate. The phenolic substance from A. catechu effectively inhibited hyaluronidase activity (IC(50) : 210 mug mL(-1) ). These results suggest that the phenolic substance purified from A. catechu has an anti-ageing effect by protecting connective tissue proteins.

Amino acid residues conferring herbicide tolerance in tobacco acetolactate synthase.

Acetolactate synthase (ALS) is the common enzyme in the biosynthetic pathways leading to valine, leucine, and isoleucine in plants and microorganisms. ALS is the target site of several classes of structurally unrelated herbicides including sulfonylureas, imidazolinones, and triazolopyrimidines. To identify the residues conferring herbicide tolerance in tobacco ALS, site-directed mutagenesis for three residues, Ala121, Pro187 and Ser652, was performed. Mutant A121T showed strong resistance to Londax (a sulfonylurea) and Cadre (an imidazolinone), while mutant S652T was resistant only to Cadre. The S652N mutation abolished the binding affinity of FAD, and inactivated the enzyme. Double mutation of Ala121 and Ser652 with Thr yielded a mutant highly tolerant to Londax, Cadre, and TP (a triazolopyrimidine sulfonamide), but has enzymatic properties similar to those of wild-type. Substitution of Pro187 with Ser resulted in the enzyme highly susceptible to oxidation and fragmentation. These results suggest that two residues Ala121 and Ser652 are potent residues conferring herbicide resistance in tobacco ALS, and that double mutation of Ala121 and Ser652 by Thr can confer stronger tolerance to Londax, Cadre, and TP.

cis-fumagillin, a new methionine aminopeptidase (type 2) inhibitor produced by Penicillium sp. F2757.

Selective inhibition against the yeast MetAP2 (methionine aminopeptidase type 2) was detected in the fermentation broth of a fungus F2757 that was later identified as Penicillium janczewskii. A new compound cis-fumagillin methyl ester (1) was isolated from the diazomethane treated fermentation extracts together with the known compound fumagillin methyl ester (2). The cis-fumagillin methyl ester, a stereoisomer of fumagillin methyl ester at the C2'-C3' position of the aliphatic side chain, selectively inhibited growth of the map1 mutant yeast strain (MetAP1 deletion strain) at a concentration as low as 1 ng. However, the wild type yeast w303 and the mutant map2 (MetAP2 deleted) strains were resistant up to 10 microg of the compound. In enzyme experiments, compound 1 inhibited the MetAP2 with an IC50 value of 6.3 nM, but it did not inhibit the MetAP1 (IC50 >200 microM). Compound 2 also inhibited the MetAP2 with an IC50 value of 9.2 nM and 105 microM against MetAP1.

8-O-Methylsclerotiorinamine, antagonist of the Grb2-SH2 domain, isolated from Penicillium multicolor.

A new secondary metabolite, 8-O-methylsclerotiorinamine (1), was isolated from a strain of Penicillium multicolor, and its structure was established using NMR spectroscopy and chemical evidence. The metabolite inhibited significantly the binding between the Grb2-SH2 domain and the phosphopeptide derived from the Shc protein and also blocked the protein-protein interactions of Grb2-Shc in cell-based experiments, with IC(50) values of 5.3 and 50 microM, respectively.

Natural and synthetic analogues of actinomycin D as Grb2-SH2 domain blockers.

Natural analogues (D, C2, and VII) of actinomycin inhibit Grb2 SH2 domain binding with phosphopeptide-derived from Shc in vitro and in intracellular system. To study structure-activity relationships, 13 actinomycin analogues were synthesized and we found that the inhibition activity depended on the substituents of cyclic peptide groups in actinomycin and two analogues with Tyr residue are the most potent inhibitors with IC50 value of 0.5 and 0.8 microM, respectively.

Structural and functional role of cysteinyl residues in tobacco acetolactate synthase.

Acetolactate synthase (ALS) is the common enzyme in the biosynthesis of valine, leucine, and isoleucine. The role of four cysteinyl residues in tobacco ALS was determined using site-directed mutagenesis and cysteine-specific cleavage. The C411A mutation abolished the enzymatic activity, as well as the binding affinity for the cofactor FAD. The activation constant of C411S for FAD is approximately 50-fold higher than that of wALS. The C607S mutation did not significantly affect the kinetic parameters. The IC(50) values of C411S and C607S for ALS-inhibiting herbicides are not much different from those of wALS. Two mutants, C163S and C309S, are labile and readily degraded to peptide fragments. The treatment of wALS with 2-nitro-5-thiocyanobenzoic acid, specific for cleavage of the N-terminal side of cysteine, yielded three peptides of 37.0, 22. 0, and 7.0 kDa. This fragmentation pattern is consistent with that deduced from the amino acid sequence of tobacco ALS, assuming the disulfide bond between Cys163 and Cys309. These results suggest that Cys411 is involved in the binding of FAD and that the intrachain disulfide bond between Cys163 and Cys309 plays a key role in maintaining the correct conformation of tobacco ALS.

Actinomycin D as a novel SH2 domain ligand inhibits Shc/Grb2 interaction in B104-1-1 (neu*-transformed NIH3T3) and SAA (hEGFR-overexpressed NIH3T3) cells.

Actinomycins, a family of bicyclic chromopeptide lactones with strong antineoplastic activity, were screened as inhibitors of Shc/Grb2 interaction in in vitro assay systems. To investigate the effects of actinomycin D on Shc/Grb2 interaction in cell-based experiments, we used SAA (normal hEGFR-overexpressed NIH3T3) cells and B104-1-1 (neu*-transformed NIH3T3) cells, because a large number of the Shc/Grb2 complexes were detected. Associated protein complexes containing Shc were immunoprecipitated from actinomycin D-treated cell lysates with polyclonal anti-Shc antibody. Then the association with Grb2 was assessed by immunoblotting with monoclonal anti-Grb2 antibody. The result of the immunoblotting experiment revealed that actinomycin D inhibited Shc/Grb2 interaction in a dose-dependent manner in both B104-1-1 and EGF-stimulated SAA cells. The inhibition of Shc/Grb2 interaction by actinomycin D in B104-1-1 cells also reduced tyrosine phosphorylation of MAP kinase (Erk1/Erk2), one of the major components in the Ras-MAP kinase signaling pathway. These results suggest that actinomycin D could be a non-phosphorylated natural and cellular membrane-permeable SH2 domain antagonist.

Role of tryptophanyl residues in tobacco acetolactate synthase.

Acetolactate synthase (ALS) catalyzes the first common step in the biosynthesis of valine, leucine, and isoleucine. ALS is the target of three classes of herbicides, the sulfonylureas, the imidazolinones, and the triazolopyrimidines. Five mutants (W266F, W439F, W490F, W503F, and W573F) of the ALS gene from Nicotiana tabacum were constructed and expressed in Escherichia coli, and the enzymes were purified. The W490F mutation abolished the binding affinity for cofactor FAD and inactivated the enzyme. The replacement of Trp573 by Phe yielded a mutant ALS resistant to the three classes of herbicides. The other three mutations, W266F, W439F, and W503F, did not significantly affect the enzymatic properties and the sensitivity to the herbicides. These results indicate that the Trp490 residue is essential for the binding of FAD and that Trp573 is located at the herbicide binding site. The data also suggest that the three classes of herbicides bind ALS competitively.

Synthesis of the quinoline-linked triazolopyrimidine analogues and their interactions with the recombinant tobacco acetolactate synthase.

Acetolactate synthase (ALS) is the first common enzyme in the biosynthesis of L-leucine, L-isoleucine, and L-valine. Triazolopyrimidine sulfonamide (TP) is a mixed-type inhibitor of ALS with respect to both pyruvate and thiamine pyrophosphate. In this study, we synthesized new substituted quinoline-linked TP analogues and several TP analogues which contained either unsubstituted aminoquinolines or amino isoquinolines. In addition, we examined the interactions of both the wild-type and the sulfonylurea-resistant recombinant tobacco ALS enzymes in a highly pure and active form with the quinoline-linked TP analogues, respectively. The wild-type tobacco ALS was extremely sensitive to inhibition by the quinoline-linked TP analogues. In contrast, the mutant tobacco ALS was insensitive to both the quinoline-linked triazolopyrimidine and the sulfonylurea herbicides. The results indicate that the ability of the quinoline-linked TP analogues to inhibit ALS is highly sensitive to substitution at the ortho position (C-7) and to the position of the ring nitrogen around the sulfonamide functionality (C-8).

The effects of areca catechu L extract on anti-inflammation and anti-melanogenesis.

Ethanolic extract (CC-516) from Areca catechu L was prepared and its various biological activities were evaluated, CC-516 showed potent anti-oxidative, free radical scavenging, and anti-hyaluronidase activity. Anti-oxidative effect of CC-516 (IC _ 50: 45.4 mug/ml) was lower than butylated hydroxytoluene (IC _ 50: 5mug ml), but similar to tocopherol and higher than ascorbic acid. Especially, CC-516 exhibited relatively high free radical scavenging activity (IC _ 50: 10.2 mug/ml) compared to control. CC-516 inhibited effectively hyaluronidase activity (IC _ 50: 416 mug/ml), showed inhibition in vivo on delayed hypersensitivity as well as croton-oil induced ear edema in mice when it was topically applied. These results strongly suggest that CC-516 may reduce immunoregulatory/inflammatory skin trouble. Also, from the results, we have elucidated that CC-516 showed anti-allergic and anti-cytotoxicity activity. The whitening effect of CC-516 shown by the inhibition of mushroom tyrosinase activity with IC _ 50 of 0.48 mg/ml and of melanin synthesis in B16 melanoma cells. This study indicates that CC-516 is effective on anti-inflammatory/anti-melanogenesis, and can be used as a new agent for cosmetics.

The effects of areca catechu L extract on anti-aging.

The anti-aging effects of Areca catechu L extract (CC-516) on skin were investigate both in vitro and in vivo. The CC-516 has a high proportion of proline (13%) of free amino acid content. The inhibitory effect of CC-516 on the elastase exhibited 37 to 90% inhibition by 10 to 250 mug/ml concentration; the IC _ 50 values with 40.8 mug/ml for porcine pancreatic elastase (PPE) and 48.1 mug/ml for human leukocyte elastase (HLE), respectively. One of the effects of elastase is that it is known to reduce the number of elastin fibers at the level of the enzyme deposition. The number of elastin fibers was increased when we drift from the deposit number of elastase with 100 mg/ml of CC-516. CC-516 showed protection of elastic fiber against degradation by the enzyme in an ex vivo assay. The CC-516 increased proliferation of human fibroblast cell by 85% at 10 ; -4 concentration, compared with control, whereas the increase by ascorbic acid was 50%. The collagen synthesis was increased by 40% at 10 ; -4% concentration of CC-516. The treatment with CC-516 improved skin hydration, the skin elasticity, and skin wrinkles. From this study, we suggest that CC-516 can be used as a new anti-aging component for cosmetics.

Inhibitory Effects of 150 Plant Extracts on Elastase Activity, and Their Anti-inflammatory Effects.

The inhibitory effects of 150 medicinal plants on elastase activity were investigated. Among the 150 plants, six plant extracts (final concentration 1 mg/ml in methanol) exhibited more than 65% of inhibition of elastase activity. The inhibitory effects of six active plants, including Areca catechu (IC50, 42.4 mug/ml), Cinnamonum cassia (IC50, 208.7 mug/ml), Myristica fragrans (IC50, 284.1 mug/ml), Curcuma longa (IC50, 398.4 mug/ml), Alpinia katsumadai (IC50, 465.7 mug/ml) and Dryopteris cassirrhizoma (IC50, 714.4 mug/ml) on the activity of human leukocyte elastase, hyaluronidase and lipid peroxidation were examined. In the lipid peroxidation assay, using the TBA method, three of the six plants, including Curcuma longa (IC50, 45.5 mug/ml), Areca catechu (IC50, 51.0 mug/ml) and Alpinia katsumadai (IC50, 116.3 mug/ml) exhibited more than 70% inhibition at the concentration of 1 mug/ml, but only one plant, Areca catechu (IC50, 563 mug/ml) showed high inhibitory effect on hyaluronidase activity. The results suggest that medicinal plants showing several biological activities may be potent inhibitors of the anti-ageing process in skin. This property might be useful for application in cosmetics.

A novel a-factor-related peptide of Saccharomyces cerevisiae that exits the cell by a Ste6p-independent mechanism.

Many secreted signaling molecules are synthesized as precursors that undergo multiple maturation steps to generate their mature forms. The Saccharomyces cerevisiae mating pheromone a-factor is a C-terminally isoprenylated and carboxylmethylated dodecapeptide that is initially synthesized as a larger precursor containing 36 or 38 amino acids. We have previously shown that the maturation of a-factor occurs by an ordered biogenesis pathway involving 1) three C-terminal modification steps, 2) two N-terminal proteolytic processing events, and 3) a nonclassical export mechanism mediated by the ATP-binding-cassette (ABC) transporter Ste6p. In the present study, we demonstrate that an unexpected and abundant a-factor-related peptide (AFRP) exists in the culture fluid of MATa cells and that its biogenesis is integrally related to that of mature a-factor itself. We show by purification followed by mass spectrometry that AFRP corresponds to the C-terminal 7 amino acids (VFWDPAC) of mature a-factor (YIIKGVFWDPAC), including both the farnesyl- and carboxylmethylcysteine modifications. The formation and export of AFRP displays three striking features. First, we show that AFRP is produced intracellularly and that mutants (ste24 and axl1) that cannot produce mature a-factor due to an N-terminal processing defect are nevertheless normal for AFRP production. Thus, AFRP is not derived from mature a-factor but, instead, from the P1 form of the a-factor precursor. Second, fusion constructs with foreign amino acids substituted for authentic a-factor residues still yield AFRP-sized molecules; however, the composition of these corresponds to the altered residues instead of to AFRP residues. Thus, AFRP may be generated by a sequence-dependent but length-specific proteolytic activity. Third, a-factor and AFRP use distinct cellular machinery for their secretion. Whereas a-factor export is Ste6p-dependent, AFRP is secreted normally even in a ste6 deletion mutant. Thus, AFRP may exit the cell by another ATP-binding-cassette transporter, a different type of transporter altogether, or possibly by diffusion. Taken together, these studies indicate that the biogenesis of AFRP involves novel mechanisms and machinery, distinct from those used to generate mature a-factor. Because AFRP neither stimulates nor inhibits mating or a-factor halo activity, its function remains an intriguing question.

Soluble overexpression in Escherichia coli, and purification and characterization of wild-type recombinant tobacco acetolactate synthase.

Acetolactate synthase (ALS) is the first common enzyme in the biosynthesis of L-leucine, L-isoleucine, and L-valine. The wild-type ALS gene from Nicotiana tabacum was cloned into the bacterial expression vector pGEX-2T. The resulting recombinant plasmid pGEX-ALS2 was used to transform Escherichia coli strain XL1-Blue, and the wild-type tobacco ALS (wALS) was expressed in the bacteria as a protein fused with glutathione S-transferase (GST). The fusion product GST-wALS was purified in a single step on a glutathione-Sepharose column. The purified GST-wALS was sensitive to a sulfonylurea herbicide, and was lost its sensitivity to end products, L-valine, L-leucine and L-isoleucine. These results suggest that the purified recombinant tobacco ALS was functionally active, and that the sulfonylureas may not bind to the feedback regulatory site on the plant ALS.