| 12149243 |
Sumoylation of topoisomerase I is involved in its partitioning between nucleoli and nucleoplasm and its clearing from nucleoli in response to camptothecin. |
10.1074/jbc.m200388200 |
J. Biol. Chem. |
Sumoylation of topoisomerase I is involved in its partitioning between nucleoli and nucleoplasm and its clearing from nucleoli in response to camptothecin.
Abstract
- Previous studies identified a small fraction of putatively sumoylated topoisomerase I (TOP1) under basal conditions ( approximately 1%), and anticancer camptothecins that trap the TOP1-DNA covalent intermediate markedly increase the sumoylation of TOP1 (<or=10%). To study the role of the sumoylation of TOP1, we mutated sites on green fluorescent protein (GFP)-TOP1 corresponding to the consensus sequence for protein sumoylation (PsiKXE, where Psi is a hydrophobic residue) and assayed the mutants for basal and camptothecin-induced sumoylation. Only one of the eight mutants, K117R, located in the highly charged NH2-terminal region, showed a substantial reduction ( approximately 5-fold) in basal and camptothecin-induced sumoylation; thus, Lys-117 appears to be the major sumoylation site. A triple mutant having the PsiKXE sequences flanking K117R additionally mutated (K103R/K117R/K153R) showed little if any sumoylation, but was degraded like wild-type GFP-TOP1 during camptothecin treatment. However, K103R/K117R/K153R-GFP-TOP1 was markedly concentrated within nucleoli, depleted from the remainder of nucleus, and failed to be cleared from nucleoli in response to camptothecin treatment. These data are consistent with a model wherein basal transient sumoylation of the NH2-terminal, highly charged, disordered region prevents TOP1 binding to sites in nucleoli, thus driving it to bind in the nucleoplasm; and camptothecin treatment, which increases TOP1 sumoylation, further shifts the binding resulting in delocalization of TOP1 from nucleoli to nucleoplasm.
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| 12169606 |
Identification by heterologous expression and gene disruption of VisA as L-lysine 2-aminotransferase essential for virginiamycin S biosynthesis in Streptomyces virginiae |
10.1128/JB.184.17.4811-4818.2002. |
J Bacteriol |
Identification by heterologous expression and gene disruption of VisA as L-lysine 2-aminotransferase essential for virginiamycin S biosynthesis in Streptomyces virginiae
Abstract
- The visA gene of Streptomyces virginiae has been thought to be a part of the virginiamycin S (VS) biosynthetic gene cluster based on its location in the middle of genes that encode enzymes highly similar to those participating in the biosynthesis of streptogramin-type antibiotics. Heterologous expression of the visA gene was achieved in Escherichia coli by an N-terminal fusion with thioredoxin (TrxA), and the intact recombinant VisA protein (rVisA) was purified after cleavage with enterokinase to remove the TrxA moiety. The purified rVisA showed clear L-lysine 2-aminotransferase activity with an optimum pH of around 8.0 and an optimum temperature at 35 degrees C, with 2-oxohexanoate as the best amino acceptor, indicating that VisA converts L-lysine into Delta(1)-piperidine 2-carboxylic acid. A visA deletion mutant of S. virginiae was created by homologous recombination, and the in vivo function of the visA gene was studied by phenotypic comparison between the wild type and the visA deletion mutant. No differences in growth in liquid media or in morphological behavior on solid media were observed, indicating that visA is not involved in primary metabolism or morphological differentiation. However, the visA mutant failed to produce VS while maintaining the production of virginiamycin M(1) at a level comparable to that of the parental wild-type strain, demonstrating that visA is essential to VS biosynthesis. These results, together with the observed recovery of the defect in VS production by the external addition of 3-hydroxypicolinic acid (3-HPA), a starter molecule in VS biosynthesis, suggest that VisA is the first enzyme of the VS biosynthetic pathway and that it supplies 3-HPA from L-lysine.
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| 12169771 |
Inhibitory control of growth hormone secretion by somatostatin in rat pituitary GC cells: sst(2) but not sst(1) receptors are coupled to inhibition of single-cell intracellular free calcium concentrations |
10.1159/000064424. |
Neuroendocrinology |
Inhibitory control of growth hormone secretion by somatostatin in rat pituitary GC cells: sst(2) but not sst(1) receptors are coupled to inhibition of single-cell intracellular free calcium concentrations
Abstract
- Rat pituitary tumor cells (GC cells) exhibit spontaneous oscillations of intracellular free calcium concentration ([Ca(2+)](i)) that allow continuous release of growth hormone (GH). Of the somatostatin (SRIH) receptor subtypes (sst receptors) mediating SRIH action, sst(1) and sst(2) receptors are highly expressed by GC cell membranes. In the present study, the effects of sst(1) or sst(2) receptor activation on single-cell [Ca(2+)](i) were investigated in GC cells by confocal fluorescence microscopy. In addition, the effects of sst(1) or sst(2) receptor activation on GH secretion were also studied. Our results demonstrate that SRIH decreases [Ca(2+)](i) baseline and almost completely blocks Ca(2+) transients through activation of sst(2) but not of sst(1) receptors. In contrast, SRIH effectively inhibits GH secretion through activation of both sst(1) and sst(2) receptors. Blocking Ca(2+) transients is less efficient than SRIH to inhibit GH release. The cyclic octapeptide, CYN-154806, antagonizes sst(2) receptors at [Ca(2+)](i) since it abolishes the sst(2) receptor-mediated inhibition of [Ca(2+)](i) without affecting single-cell Ca(2+) signals. On the other hand, CYN-154806 alone potently inhibits GH secretion through the involvement of pertussis toxin-sensitive G proteins. In conclusion, the present results demonstrate that SRIH inhibition of GH release in GC cells involves mechanisms either dependent or independent on SRIH modulation of [Ca(2+)](i). The implications of CYN-154806 inhibition of GH secretion are discussed.
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| 12171928 |
SKAP55 recruits to lipid rafts and positively mediates the MAPK pathway upon T cell receptor activation |
10.1074/jbc.M206023200. |
J Biol Chem |
SKAP55 recruits to lipid rafts and positively mediates the MAPK pathway upon T cell receptor activation
Abstract
- T cell receptor (TCR) engagement triggers a series of events including protein tyrosine kinase activation, tyrosine phosphorylation of adapter proteins, and multiple protein-protein interactions. We observed that adapter protein SKAP55, the Src kinase-associated phosphoprotein, formed homodimers through its SH3 domain and SK region. SKAP55 as a substrate interacted with Fyn kinase in vivo. In Jurkat cells, interaction between SKAP55 and Fyn kinase depended on TCR activation. Stable overexpression of SKAP55 in Jurkat cells caused mitogen-activated protein kinase activation following TCR engagement. Anti-CD3 stimulation also promoted the interaction of SKAP55 with Grb-2 in T cells. Mutational analysis revealed that tyrosine 271 in SKAP55 played a pivotal role for interaction with both Fyn kinase and adapter protein Grb-2, indicating that the Fyn-phosphorylated SKAP55 transiently associates with adapter Grb-2 to mediate mitogen-activated protein kinase activation. Intriguingly, T cell receptor engagement dramatically induced the translocation of endogenous SKAP55 to lipid rafts where SKAP55 was found to interact with Fyn kinase, suggesting that the positive function of SKAP55 via its association with Fyn and other signaling components may have been involved in raft-mediated T cell activation.
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| 12176973 |
The transcriptional repressor Sp3 is associated with CK2-phosphorylated histone deacetylase 2. |
10.1074/jbc.c200378200 |
J. Biol. Chem. |
The transcriptional repressor Sp3 is associated with CK2-phosphorylated histone deacetylase 2.
Abstract
- Sp1 and Sp3 are ubiquitously expressed mammalian transcription factors that function as activators or repressors. Although both transcription factors share a common domain involved in forming multimers, we demonstrate that Sp1 and Sp3 form separate complexes in estrogen-dependent human breast cancer cells. Sp1 and Sp3 complexes associate with histone deacetylases (HDACs) 1 and 2. Although most HDAC2 is not phosphorylated in the breast cancer cells, HDAC2 bound to Sp1 and Sp3 and cross-linked to chromatin in situ is highly enriched in a phosphorylated form that has a reduced mobility in SDS-polyacrylamide gels. We show that protein kinase CK2 is associated with and phosphorylates HDAC2. Alkaline phosphatase treatment of HDAC2 and Sp1 and Sp3 complexes reduced the associated HDAC activity. Protein kinase CK2 is up-regulated in several cancers including breast cancer, and Sp1 and Sp3 have key roles in estrogen-induced proliferation and gene expression in estrogen-dependent breast cancer cells. CK2 phosphorylation of HDAC2 recruited by Sp1 or Sp3 could regulate HDAC activity and alter the balance of histone deacetylase and histone acetyltransferase activities and dynamic chromatin remodeling of estrogen-regulated genes.
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| 12193600 |
Structure of a novel P-superfamily spasmodic conotoxin reveals an inhibitory cystine knot motif |
10.1074/jbc.M206690200. |
J Biol Chem |
Structure of a novel P-superfamily spasmodic conotoxin reveals an inhibitory cystine knot motif
Abstract
- Conotoxin gm9a, a putative 27-residue polypeptide encoded by Conus gloriamaris, was recently identified as a homologue of the "spasmodic peptide", tx9a, isolated from the venom of the mollusk-hunting cone shell Conus textile (Lirazan, M. B., Hooper, D., Corpuz, G. P., Ramilo, C. A., Bandyopadhyay, P., Cruz, L. J., and Olivera, B. M. (2000) Biochemistry 39, 1583-1588). The C. gloriamaris spasmodic peptide has been synthesized, and the refolded polypeptide was shown to be biologically active using a mouse bioassay. The chemically synthesized gm9a elicited the same symptomatology described previously for natively folded tx9a, and gm9a and tx9a were of similar potency, implying that neither the two gamma-carboxyglutamate (Gla) residues found in tx9a (Ser(8) and Ala(13) in gm9a) nor Gly(1) (Ser(1) in gm9a) are crucial for biological activity. We have determined the three-dimensional structure of gm9a in aqueous solution and demonstrated that the molecule adopts the well known inhibitory cystine knot motif constrained by three disulfide bonds involving Cys(2)-Cys(16), Cys(6)-Cys(18) and Cys(12)-Cys(23). Based on the gm9a structure, the sites of Gla substitution in tx9a are in loops located on one surface of the molecule, which is unlikely to be involved directly in receptor binding. Because this is the first structure reported for a member of the newly defined P-superfamily conotoxins, a comparison has been made with structurally related conotoxins. This shows that the structural scaffold that characterizes the P-conotoxins has the greatest potential for exhibiting structural diversity among the robust inhibitory cystine knot-containing conotoxins, a finding that has implications for functional epitope mimicry and protein engineering.
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| 12199716 |
Effects of a tryptophanyl substitution on the structure and antimicrobial activity of C-terminally truncated gaegurin 4 |
10.1046/j.1432-1033.2002.03139.x. |
Eur J Biochem |
Effects of a tryptophanyl substitution on the structure and antimicrobial activity of C-terminally truncated gaegurin 4
Abstract
- Gaegurin 4 (GGN4), a 37-residue antimicrobial peptide, consists of two amphipathic alpha helices (residues 2-10 and 16-32) connected by a flexible loop region (residues 11-15). As part of an effort to develop new peptide antibiotics with low molecular mass, the activities of C-terminally truncated GGN4 analogues were tested. Delta24-37 GGN4, a peptide analogue with 14 residues truncated from the C-terminus of GGN4, showed a complete loss of antimicrobial activity. However, the single substitution of aspartic acid 16 by tryptophan (D16W) in the Delta24-37 GGN4 completely restored the antimicrobial activity, without any significant hemolytic activity. In contrast, neither the D16F nor K15W substitution of the Delta24-37 GGN4 allowed such a dramatic recovery of activity. In addition, the D16W substitution of the native GGN4 significantly enhanced the hemolytic activity as well as the antimicrobial activity. The structural effect of the D16W substitution in the Delta24-37 GGN4 was investigated by CD, NMR, and fluorescence spectroscopy. The results showed that the single tryptophanyl substitution at position 16 of the Delta24-37 GGN4 induced an alpha helical conformation in the previously flexible loop region in intact GGN4, thereby forming an entirely amphipathic alpha helix. In addition, the substituted tryptophan itself plays an important role in the membrane-interaction of the peptide.
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| 12200282 |
Lactobacillus plantarum MiLAB 393 produces the antifungal cyclic dipeptides cyclo(L-Phe-L-Pro) and cyclo(L-Phe-trans-4-OH-L-Pro) and 3-phenyllactic acid |
10.1128/AEM.68.9.4322-4327.2002. |
Appl Environ Microbiol |
Lactobacillus plantarum MiLAB 393 produces the antifungal cyclic dipeptides cyclo(L-Phe-L-Pro) and cyclo(L-Phe-trans-4-OH-L-Pro) and 3-phenyllactic acid
Abstract
- We have isolated a Lactobacillus plantarum strain (MiLAB 393) from grass silage that produces broad-spectrum antifungal compounds, active against food- and feed-borne filamentous fungi and yeasts in a dual-culture agar plate assay. Fusarium sporotrichioides and Aspergillus fumigatus were the most sensitive among the molds, and Kluyveromyces marxianus was the most sensitive yeast species. No inhibitory activity could be detected against the mold Penicillium roqueforti or the yeast Zygosaccharomyces bailii. An isolation procedure, employing a microtiter well spore germination bioassay, was devised to isolate active compounds from culture filtrate. Cell-free supernatant was fractionated on a C(18) SPE column, and the 95% aqueous acetonitrile fraction was further separated on a preparative HPLC C(18) column. Fractions active in the bioassay were then fractionated on a porous graphitic carbon column. The structures of the antifungal compounds cyclo(L-Phe-L-Pro), cyclo(L-Phe-trans-4-OH-L-Pro) and 3-phenyllactic acid (L/D isomer ratio, 9:1), were determined by nuclear magnetic resonance spectroscopy, mass spectrometry, and gas chromatography. MIC values against A. fumigatus and P. roqueforti were 20 mg ml(-1) for cyclo(L-Phe-L-Pro) and 7.5 mg ml(-1) for phenyllactic acid. Combinations of the antifungal compounds revealed weak synergistic effects. The production of the antifungal cyclic dipeptides cyclo(L-Phe-L-Pro) and cyclo(L-Phe-trans-4-OH-L-Pro) by lactic acid bacteria is reported here for the first time.
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| 12201711 |
Nagahamide A, an antibacterial depsipeptide from the marine sponge Theonella swinhoei |
10.1021/ol0262791. |
Org Lett |
Nagahamide A, an antibacterial depsipeptide from the marine sponge Theonella swinhoei
Abstract
- [structure: see text] An antibacterial depsipeptide, nagahamide A (1), has been isolated from the marine sponge Theonella swinhoei. Its structure was determined on the basis of spectral and chemical methods. Absolute configuration of amino acid residues was determined by Marfey's analysis, and relative stereochemistry of the polyketide moiety was assigned by comparison of NMR data with those of a model compound.
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| 12203574 |
Total synthesis of the nematicidal cyclododecapeptide omphalotin A by using racemization-free triphosgene-mediated couplings in the solid phase |
10.1002/1521-3773(20020703)41:13<2307::AID-ANIE2307>3.0.CO;2-Y. |
Angew Chem Int Ed Engl |
Total synthesis of the nematicidal cyclododecapeptide omphalotin A by using racemization-free triphosgene-mediated couplings in the solid phase
Abstract
|
| 12203956 |
[ADMAdda5]-microcystins in Planktothrix agardhii strain PH-123 (cyanobacteria)--importance for monitoring of microcystins in the environment |
10.1002/tox.10042. |
Environ Toxicol |
[ADMAdda5]-microcystins in Planktothrix agardhii strain PH-123 (cyanobacteria)--importance for monitoring of microcystins in the environment
Abstract
- Two major and two minor microcystins (MCYST) were isolated from a hepatotoxic Danish strain of Planktothrix agardhii (Gomont) Anagnostidis et Komárek by reversed-phase high-performance liquid chromatography. The microcystins were characterized by UV spectroscopy, amino acid analysis, fast atom bombardment mass spectrometry (FABMS), and high-resolution FABMS. The major microcystins were further analysed by collisionally induced tandem electrospray ionization MS. The microcystins were found to be demethylated variants of MCYST-HtyR (homotyrosine-arginine) and MCYST-LR (leucine-arginine). The two major microcystins contained an acetyl-demethyl variant (ADMAdda) of 3-amino-9-acetoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (Adda). This is the first report of [ADMAdda5]-microcystins in Planktothrix. The two [ADMAdda5]-microcystins inhibited protein phosphatase activity but showed low cross-reactivity with antibodies of an enzyme-linked immunosorbent assay (ELISA), emphasizing the potential underestimation of the toxicity of natural blooms dominated by Planktothrix when microcystin content is quantified using only an ELISA.
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| 12209008 |
8-Oxoguanine rearranges the active site of human topoisomerase I |
10.1073/pnas.192282699. |
Proc Natl Acad Sci U S A |
8-Oxoguanine rearranges the active site of human topoisomerase I
Abstract
- 7,8-Dihydro-8-oxoguanine (8-oxoG) is the most common form of oxidative DNA damage in human cells. Biochemical studies have shown that 8-oxoG decreases the DNA cleavage activity of human topoisomerase I, an enzyme vital to DNA metabolism and stability. We present the 3.1-A crystal structure of human topoisomerase I in noncovalent complex with a DNA oligonucleotide containing 8-oxoG at the +1 position in the scissile strand. We find that 8-oxoG reorganizes the active site of human topoisomerase I into an inactive conformation relative to the structures of topoisomerase I-DNA complexes elucidated previously. The catalytic Tyr-723-Phe rotates away from the DNA cleavage site and packs into the body of the molecule. A second active-site residue, Arg-590, becomes disordered and is not observed in the structure. The docked, inactive conformation of Tyr-723-Phe is reminiscent of the related tyrosine recombinase family of integrases and recombinases, suggesting a common regulatory mechanism. We propose that human topoisomerase I binds to DNA first in an inactive conformation and then rearranges its active site for catalysis. 8-OxoG appears to impact topoisomerase I by stabilizing the inactive, DNA-bound state.
|
| 12218175 |
Crystal structure of calcineurin-cyclophilin-cyclosporin shows common but distinct recognition of immunophilin-drug complexes. |
10.1073/pnas.192206699 |
Proc. Natl. Acad. Sci. U.S.A. |
Crystal structure of calcineurin-cyclophilin-cyclosporin shows common but distinct recognition of immunophilin-drug complexes.
Abstract
- Calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, is the common target for two immunophilin-immunosuppressant complexes, cyclophilin A-cyclosporin A (CyPA-CsA) and FKBP-FK506. How the two structurally distinct immunophilin-drug complexes bind the same target has remained unknown. We report the crystal structure of calcineurin (CN) in complex with CyPA-CsA at 2.8-A resolution. The CyPA-CsA complex binds to a composite surface formed by the catalytic and regulatory subunits of CN, where the complex of FK506 and its binding protein FKBP also binds. While the majority of the CN residues involved in the binding are common for both immunophilin-immunosuppressant complexes, a significant number of the residues are distinct. Unlike FKBP-FK506, CyPA-CsA interacts with Arg-122 at the active site of CN, implying direct involvement of CyPA-CsA in the regulation of CN catalysis. The simultaneous interaction of CyPA with both the composite surface and the active site of CN suggests that the composite surface may serve as a substrate recognition site responsible for the narrow substrate specificity of CN. The comparison of CyPA-CsA-CN with FKBP-FK506-CN significantly contributes to understanding the molecular basis of regulation of CN activity by the immunophilin-immunosuppressant.
|
| 12226088 |
Binding of the concave surface of the Sds22 superhelix to the alpha 4/alpha 5/alpha 6-triangle of protein phosphatase-1. |
10.1074/jbc.m206838200 |
J. Biol. Chem. |
Binding of the concave surface of the Sds22 superhelix to the alpha 4/alpha 5/alpha 6-triangle of protein phosphatase-1.
Abstract
- Functional studies of the protein phosphatase-1 (PP1) regulator Sds22 suggest that it is indirectly and/or directly involved in one of the most ancient functions of PP1, i.e. reversing phosphorylation by the Aurora-related protein kinases. We predict that the conserved portion of Sds22 folds into a curved superhelix and demonstrate that mutation to alanine of any of eight residues (Asp(148), Phe(170), Glu(192), Phe(214), Asp(280), Glu(300), Trp(302), or Tyr(327)) at the concave surface of this superhelix thwarts the interaction with PP1. Furthermore, we show that all mammalian isoforms of PP1 have the potential to bind Sds22. Interaction studies with truncated versions of PP1 and with chimeric proteins comprising fragments of PP1 and the yeast PP1-like protein phosphatase Ppz1 suggest that the site(s) required for the binding of Sds22 reside between residues 43 and 173 of PP1gamma(1). Within this region, a major interaction site was mapped to a triangular region delineated by the alpha4-, alpha5-, and alpha6-helices. Our data also show that well known regulatory binding sites of PP1, such as the RVXF-binding channel, the beta12/beta13-loop, and the acidic groove, are not essential for the interaction with Sds22.
|
| 12235857 |
[New antifungal antibiotics, bacillopeptins and fusaricidins] |
10.1248/yakushi.122.651. |
Yakugaku Zasshi |
[New antifungal antibiotics, bacillopeptins and fusaricidins]
Abstract
- We isolated four strains of bacteria producing antifungal antibiotics from the rhizosphere of garlic with basal rot caused by the plant pathogenic fungal strain Fusarium oxysporum. Among them, Bacillus subtilis FR-2 was found to produce new antifungal antibiotics, named bacillopeptins A, B, and C. Their structures have been determined by 1D and 2D NMR and MS experiments, and amino acid analysis coupled with chiral HPLC, to be cyclic lipopeptides each containing a long-chain beta-amino acid. Another bacterial strain, Bacillus polymyxa KT-8, was shown to produce new antifungal antibiotics named fusaricidins A, B, C, and D which are more potent than bacillopeptins in their antimicrobial activity. The structures of the fusaricidins have been elucidated similarly as bacillopeptins to be cyclic hexadepsipeptides all containing 15-guanidino-3-hydroxypentadecanoic acid as a side chain. Fusaricidins strongly inhibit the growth of various kinds of fungi and moreover surprisingly show strong inhibitory activity against Gram-positive bacteria such as Staphylococcus aureus or Micrococcus luteus.
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