| 10051558 |
Unexpected crucial role of residue 225 in serine proteases |
10.1073/pnas.96.5.1852. |
Proc Natl Acad Sci U S A |
Unexpected crucial role of residue 225 in serine proteases
Abstract
- Residue 225 in serine proteases of the chymotrypsin family is Pro or Tyr in more than 95% of nearly 300 available sequences. Proteases with Y225 (like some blood coagulation and complement factors) are almost exclusively found in vertebrates, whereas proteases with P225 (like degradative enzymes) are present from bacteria to human. Saturation mutagenesis of Y225 in thrombin shows that residue 225 affects ligand recognition up to 60,000-fold. With the exception of Tyr and Phe, all residues are associated with comparable or greatly reduced catalytic activity relative to Pro. The crystal structures of three mutants that differ widely in catalytic activity (Y225F, Y225P, and Y225I) show that although residue 225 makes no contact with substrate, it drastically influences the shape of the water channel around the primary specificity site. The activity profiles obtained for thrombin also suggest that the conversion of Pro to Tyr or Phe documented in the vertebrates occurred through Ser and was driven by a significant gain (up to 50-fold) in catalytic activity. In fact, Ser and Phe are documented in 4% of serine proteases, which together with Pro and Tyr account for almost the entire distribution of residues at position 225. The unexpected crucial role of residue 225 in serine proteases explains the evolutionary selection of residues at this position and shows that the structural determinants of protease activity and specificity are more complex than currently believed. These findings have broad implications in the rational design of enzymes with enhanced catalytic properties.
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| 10052845 |
The biological activity of selected cyclic dipeptides |
10.1111/j.2042-7158.1998.tb03355.x. |
J Pharm Pharmacol |
The biological activity of selected cyclic dipeptides
Abstract
- Cyclic dipeptides are widely used as models for larger peptides because of their simplicity and limited conformational freedom. Some cyclic dipeptides have been shown to be antiviral, antibiotic and anti-tumour. The aim of this study was to determine the biological activity of four cyclic dipeptides synthesized in this laboratory: cyclo(L-phenylalanyl-L-prolyl), cyclo(L-tyrosyl-L-prolyl), cyclo(L-tryptophanyl-L-prolyl) and cyclo(L-tryptophanyl-L-tryptophanyl). The enhancement or inhibition of calcium channels in ventricular myocytes from rats and delayed-rectifier potassium channels in ventricular myocytes from guinea-pigs were determined by use of the whole-cell patch-clamp technique. The induction of differentiation in HT-29 cells was assessed by assaying for an increase in the expression of alkaline phosphatase. Antibiotic properties against Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, Bacillus subtilus and Streptococcus sp. were determined by use of the Kirby-Bauer disc-diffusion assay. Results from these assays indicate that the cyclic dipeptides have biological activity in both prokaryotes and eukaryotes. Three of the dipeptides block cation channels in ventricular myocytes and all increase the expression of alkaline phosphatase. All the dipeptides have concentration-dependent antibacterial properties. These results suggest that with increased solubility the cyclic dipeptides might have potential as muscle relaxants, anti-tumour compounds and antibiotics.
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| 10064732 |
Characterization of a leukotriene C4 export mechanism in human platelets: possible involvement of multidrug resistance-associated protein 1. |
10.1021/bi972332v |
J. Lipid Res. |
Characterization of a leukotriene C4 export mechanism in human platelets: possible involvement of multidrug resistance-associated protein 1.
Abstract
- Platelets express leukotriene (LT) C4 synthase and can thus participate in the formation of bioactive LTC4. To further elucidate the relevance of this capability, we have now determined the capacity of human platelets to export LTC4. Endogenously formed LTC4 was efficiently released from human platelets after incubation with LTA4 at 37 degrees C, whereas only 15% of produced LTC4 was exported when the cells were incubated at 0 degrees C. The activation energy of the process was calculated to 49.9 +/- 7.7 kJ/mol, indicating carrier-mediated LTC4 export. This was also supported by the finding that the transport was saturable, reaching a maximal export rate of 470 +/- 147 pmol LTC4/min x 10(9) platelets. Furthermore, markedly suppressed LTC4 transport was induced by a combination of the metabolic inhibitors antimycin A and 2-deoxyglucose, suggesting energy-dependent export. The presence in platelets of multidrug resistance-associated protein 1 (MRP1), a protein described to be an energy-dependent LTC4 transporter in various cell types, was demonstrated at the mRNA and protein level. Additional support for a role of MRP1 in platelet LTC4 export was obtained by the findings that the process was inhibited by probenecid and the 5-lipoxygenase-activating protein (FLAP) inhibitor, MK-886. The present findings further support the physiological relevance of platelet LTC4 production.
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| 10074060 |
Surface motility of serratia liquefaciens MG1 |
10.1128/JB.181.6.1703-1712.1999. |
J Bacteriol |
Surface motility of serratia liquefaciens MG1
Abstract
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| 10075760 |
Seven novel macrocyclic polypeptides from Viola arvensis |
10.1021/np9803878. |
J Nat Prod |
Seven novel macrocyclic polypeptides from Viola arvensis
Abstract
- Seven novel macrocyclic polypeptides, designated as varv peptides B-H, have been isolated from the aerial parts of Viola arvensis. Their primary structures have been elucidated by automated Edman degradation and mass spectrometry. They all consist of 29 or 30 amino acid residues, covalently cyclized via the amide backbone and by three internal disulfide bridges. Their amino acid sequences are as follows: varv peptide B, cyclo-(TCFGGTCNTPGCSCDPWPMCSRNGLPVCGE); varv peptide C, cyclo-(TCVGGTCNTPGCSCSWPVCTRNGVPICGE); varv peptide D, cyclo-(TCVGGSCNTPGCSCSWPVCTRNGLPICGE); varv peptide E, cyclo-(TCVGGTCNTPGCSCSWPVCTRNGLPICGE); varv peptide F, cyclo-(TCTLGTCYTAGCSCSWPVCTRNGVPICGE); varv peptide G, cyclo-(TCFGGTCNTPGCSCDPWPVCSRNGVPVCGE); and varv peptide H, cyclo-(TCFGGTCNTPGCSCETWPVCSRNGLPVCGE). The varv peptides B-H exhibited high degrees of homology with the hitherto known macrocyclic peptides varv peptide A, kalata B1, violapeptide I, circulins A and B, and cyclopsychotride A.
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| 10075778 |
New jaspamide derivatives from the marine sponge Jaspis splendans collected in Vanuatu |
10.1021/np9803225. |
J Nat Prod |
New jaspamide derivatives from the marine sponge Jaspis splendans collected in Vanuatu
Abstract
- Two new jaspamide derivatives (1 and 2) along with jaspamide have been isolated from the marine sponge Jaspis splendans collected in Vanuatu. Their chemical structures were determined from 1D and 2D NMR studies and MS data. These two compounds inhibited the in vitro growth of the NSCLC-N6 human tumor cell lines with IC50 values in the microg/mL range.
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| 10086340 |
cbl-b inhibits epidermal growth factor receptor signaling |
10.1038/sj.onc.1202499. |
Oncogene |
cbl-b inhibits epidermal growth factor receptor signaling
Abstract
- The role of cbl-b in signaling by the epidermal growth factor receptor (EGFR) was studied and compared with c-cbl. We demonstrate in vivo, that cbl-b, like c-cbl, is phosphorylated and recruited to the EGFR upon EGF stimulation and both cbl proteins can bind to the Grb2 adaptor protein. To investigate the functional role of cbl proteins in EGFR signaling, we transfected cbl-b or c-cbl into 32D cells overexpressing the EGFR (32D/EGFR). This cell line is absolutely dependent on exogenous IL-3 or EGF for sustained growth. 32D/EGFR cells overexpressing cbl-b showed markedly inhibited growth in EGF compared to c-cbl transfectants and vector controls. This growth inhibition by cbl-b was the result of a dramatic increase in the number of cells undergoing apoptosis. Consistent with this finding, cbl-b overexpression markedly decreased the amplitude and duration of AKT activation upon EGF stimulation compared to either vector controls or c-cbl overexpressing cells. In addition, the duration of EGF mediated MAP kinase and Jun kinase activation in cells overexpressing cbl-b is shortened. These data demonstrate that cbl-b inhibits EGF-induced cell growth and that cbl-b and c-cbl have distinct roles in EGF mediated signaling.
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| 10090780 |
Structural and conformational requirements for high-affinity binding to the SH2 domain of Grb2(1) |
10.1021/jm9811007. |
J Med Chem |
Structural and conformational requirements for high-affinity binding to the SH2 domain of Grb2(1)
Abstract
- Following earlier work on cystine-bridged peptides, cyclic phosphopeptides containing nonreducible mimics of cystine were synthesized that show high affinity and specificity toward the Src homology (SH2) domain of the growth factor receptor-binding protein (Grb2). Replacement of the cystine in the cyclic heptapeptide cyclo(CYVNVPC) by D-alpha-acetylthialysine or D-alpha-lysine gave cyclo(YVNVP(D-alpha-acetyl-thiaK)) (22) and cyclo(YVNVP(D-alpha-acetyl-K)) (30), which showed improved binding 10-fold relative to that of the control peptide KPFYVNVEF (1). NMR spectroscopy and molecular modeling experiments indicate that a beta-turn conformation centered around YVNV is essential for high-affinity binding. X-ray structure analyses show that the linear peptide 1 and the cyclic compound 21 adopt a similar binding mode with a beta-turn conformation. Our data confirm the unique structural requirements of the ligand binding site of the SH2 domain of Grb2. Moreover, the potency of our cyclic lactams can be explained by the stabilization of the beta-turn conformation by three intramolecular hydrogen bonds (one mediated by an H2O molecule). These stable and easily accessible cyclic peptides can serve as templates for the evaluation of phosphotyrosine surrogates and further chemical elaboration.
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| 10092189 |
Beauveriolides, specific inhibitors of lipid droplet formation in mouse macrophages, produced by Beauveria sp. FO-6979 |
10.7164/antibiotics.52.1. |
J Antibiot (Tokyo) |
Beauveriolides, specific inhibitors of lipid droplet formation in mouse macrophages, produced by Beauveria sp. FO-6979
Abstract
- Beauveria sp. FO-6979, a soil isolate, was found to produce inhibitors of lipid droplet formation in mouse peritoneal macrophages. A new compound beauveriolide III was isolated along with a known compound beauveriolide I from the fermentation broth of the producing strain by solvent extraction, ODS column chromatography, silica gel column chromatography and preparative HPLC. Beauveriolides I and III caused a reduction in the number and size of cytosolic lipid droplets in macrophages at 10 microM without any cytotoxic effect on macrophages.
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| 10092190 |
Structure elucidation of fungal beauveriolide III, a novel inhibitor of lipid droplet formation in mouse macrophages |
10.7164/antibiotics.52.7. |
J Antibiot (Tokyo) |
Structure elucidation of fungal beauveriolide III, a novel inhibitor of lipid droplet formation in mouse macrophages
Abstract
- The structure of fungal beauveriolide III, an inhibitor of lipid droplet formation in mouse macrophages, was elucidated to be cyclo-(3S,4S)-3-hydroxy-4-methyloctanoyl-L-phenylalanyl-L-alanyl- D-allo-isoleucyl by spectral analyses and chemical degradation.
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| 10092609 |
Insect immunity. Isolation from the lepidopteran Heliothis virescens of a novel insect defensin with potent antifungal activity |
10.1074/jbc.274.14.9320. |
J Biol Chem |
Insect immunity. Isolation from the lepidopteran Heliothis virescens of a novel insect defensin with potent antifungal activity
Abstract
- Lepidoptera have been reported to produce several antibacterial peptides in response to septic injury. However, in marked contrast to other insect groups, no inducible antifungal molecules had been described so far in this insect order. Surprisingly, also cysteine-rich antimicrobial peptides, which predominate in the antimicrobial defense of other insects, had not been discovered in Lepidoptera. Here we report the isolation from the hemolymph of immune induced larvae of the lepidopteran Heliothis virescens of a cysteine-rich molecule with exclusive antifungal activity. We have fully characterized this antifungal molecule, which has significant homology with the insect defensins, a large family of antibacterial peptides directed against Gram-positive strains. Interestingly, the novel peptide shows also similarities with the antifungal peptide drosomycin from Drosophila. Thus, Lepidoptera appear to have built their humoral immune response against bacteria on cecropins and attacins. In addition, we report that Lepidoptera have conferred antifungal properties to the well conserved structure of antibacterial insect defensins through amino acid replacements.
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| 10092860 |
The cyclic structure of microcin J25, a 21-residue peptide antibiotic from Escherichia coli |
10.1046/j.1432-1327.1999.00085.x. |
Eur J Biochem |
The cyclic structure of microcin J25, a 21-residue peptide antibiotic from Escherichia coli
Abstract
- Microcin J25 (MccJ25) is the single representative of the immunity group J of the microcin group of peptide antibiotics produced by Enterobacteriaceae. It induces bacterial filamentation in susceptible cells in a non-SOS-dependent pathway R. A. Salomon and R. Farias (1992) J. Bacteriol. 174, 7428-7435. MccJ25 was purified to homogeneity from the growth medium of a microcin-overproducing Escherichia coli strain by reverse-phase HPLC. Based on amino acid composition and absolute configuration determination, liquid secondary ion and electrospray mass spectrometry, extensive two-dimensional NMR, enzymatic and chemical degradations studies, the structure of MccJ25 was elucidated as a 21-residue peptide, cyclo(-Val1-Gly-Ile-Gly-Thr- Pro-Ile-Ser-Phe-Tyr-Gly-Gly-Gly-Ala-Gly-His-Val-Pro-Glu-Tyr-Phe21- ). Although MccJ25 showed high resistance to most of endoproteases, linearization by thermolysin occurred from cleavage at the Phe21-Val1 bond and led to a single peptide, MccJ25-L. While MccJ25 exhibited remarkable antibiotic activity towards Salmonella newport and several E. coli strains (minimal inhibitory concentrations ranging between 0.01 and 0.2 microgram.mL-1), the thermolysin-linearized microcin showed a dramatic decrease of the activity, indicating that the cyclic structure is essential for the MccJ25 biological properties. As MccJ25 is ribosomally synthesized as a larger peptide precursor endowed with an N-terminal extremity, the present study shows that removal of this extension and head-tail cyclization of the resulting propeptide are the only post-translational modifications involved in the maturation of MccJ25, that appears as the first cyclic microcin.
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| 10096847 |
Antineoplastic agents. 400. Synthesis of the Indian Ocean marine sponge cyclic heptapeptide phakellistatin 2 |
10.1021/np980168m. |
J Nat Prod |
Antineoplastic agents. 400. Synthesis of the Indian Ocean marine sponge cyclic heptapeptide phakellistatin 2
Abstract
- Solution-phase synthesis of the marine sponge constituent phakellistatin 2 (1), cyclo(Tyr-Pro-Phe-Pro-Ile-Ile-Pro), was completed using a combination of stepwise coupling and (4 + 3) segment condensation. Use of diethyl phosphorocyanidate for the peptide bond formations gave the linear heptapeptide in 54% yield. Cyclization was achieved in high yields utilizing TBTU (2), BOP-C1 (3), PyBroP (4), and HOAt (5), resulting in 50-65% yields of phakellistatin 2 (1) depending on the method employed. The synthetic cyclic peptide was chemically but not biologically identical with the natural product.
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| 10190031 |
Effects of beauverolide L and cyclosporin A on humoral and cellular immune response of the greater wax moth, Galleria mellonella |
10.1016/s0742-8413(98)10082-8. |
Comp Biochem Physiol C Pharmacol Toxicol Endocrinol |
Effects of beauverolide L and cyclosporin A on humoral and cellular immune response of the greater wax moth, Galleria mellonella
Abstract
- The effects of beauverolide L and cyclosporin A, cyclic peptidic metabolites, produced by several genera of entomopathogenic fungi on immune responses of last instar larvae of the greater wax moth Galleria mellonella have been examined. Intrahemocoelic injection of either metabolite-coated silica particles or dissolved metabolites in a concentrations ranging between 10 and 30 micrograms per larvae caused no mortality but activated humoral responses in G. mellonella larvae. The challenge induced a significant release of lysozyme and cecropin-like activity into the hemolymph, suggesting stimulatory activity on humoral immune responses. Injected metabolite-coated particles were rapidly surrounded by hemocytes which subsequently accomplished formation of melanized nodules, which increased in size and number compared with controls. In vitro assays with dissolved metabolites indicated no adverse effects of beauverolide L or cyclosporin A on attachment or spreading of isolated plasmatocytes but dose-dependent inhibition of their phagocytic activity. Isolated plasmatocytes incubated with cyclosporin A or beauverolide L exhibited cytoskeleton alterations that differed from those observed in plasmatocytes from infected G. mellonella larvae or reported from other fungal secondary metabolites. The experiments provided further data to elucidate the role of fungal secondary metabolites in development of mycoses in insects.
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| 10193193 |
Conformational change of ascidiacyclamide caused by asymmetric modification for an isoleucine residue: structural analyses of Gly, Leu, and Pheascidiacyclamides by x-ray diffraction and NMR spectroscopy |
10.1002/(SICI)1097-0282(199905)49:6<459::AID-BIP4>3.0.CO;2-G. |
Biopolymers |
Conformational change of ascidiacyclamide caused by asymmetric modification for an isoleucine residue: structural analyses of Gly, Leu, and Pheascidiacyclamides by x-ray diffraction and NMR spectroscopy
Abstract
- Ascidiacyclamide, a cytotoxic cyclic peptide from tunicate, is composed of unusual amino acids and has a repeated sequence, c-thiazole-D-Val-oxazoline-L-Ile-2 (IleASC). The symmetric chemical structure has been assumed to be correlated with the cytotoxicity, and it is reasonable to consider that the disturbance of its structure from the C2 symmetry results in the changes of conformation and activity. In order to quantitatively estimate the molecular conformation-activity relationship, an isoleucine residue was substituted by Gly, Leu, or Phe to disturb the C2 symmetry. The conformations of three derivatives were examined by nmr spectroscopy and the crystal structure of LeuASC was also analyzed by x-ray diffraction method. The 1H-nmr experiments and the constrained molecular dynamics simulations showed the twisted "figure 8" conformers for Gly and PheASCs and the "square" conformer for LeuASC in the DMSO solution. The x-ray crystal analysis of LeuASC also revealed the square form similar to the solution structure. On the other hand, their cytotoxic activities were measured using L1210 leukemia cells and were related with the bulkiness and/or hydrophobicity of the side chain of the substituted amino acid; Phe > or = Ile > Leu >> GlyASCs. As an attempt to consider the correlation between the activity and conformer, the accessible surface area (ASA) was calculated for each derivative to estimate the size or bulkiness of its conformation. Although the ASAs of nmr structures were not directly related to the type of conformer (figure 8 or square form), it was an important probe to consider the cytotoxicity of each derivative.
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