| 18547517 |
The structure of a two-disulfide intermediate assists in elucidating the oxidative folding pathway of a cyclic cystine knot protein |
10.1016/j.str.2008.02.023. |
Structure |
The structure of a two-disulfide intermediate assists in elucidating the oxidative folding pathway of a cyclic cystine knot protein
Abstract
- We have determined the three-dimensional structure of a two-disulfide intermediate (Cys(8)-Cys(20), Cys(14)-Cys(26)) on the oxidative folding pathway of the cyclotide MCoTI-II. Cyclotides have a range of bioactivities and, because of their exceptional stability, have been proposed as potential molecular scaffolds for drug design applications. The three-dimensional structure of the stable two-disulfide intermediate shows for the most part identical secondary and tertiary structure to the native state. The only exception is a flexible loop, which is collapsed onto the protein core in the native state, whereas in the intermediate it is more loosely associated with the remainder of the protein. The results suggest that the native fold of the peptide does not represent the free energy minimum in the absence of the Cys(1)-Cys(18) disulfide bridge and that although there is not a large energy barrier, the peptide must transiently adopt an energetically unfavorable state before the final disulfide can form.
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| 18551142 |
Limited value of cyclosporine A for the treatment of patients with uveitis associated with juvenile idiopathic arthritis |
10.1038/eye.2008.174. |
Eye (Lond) |
Limited value of cyclosporine A for the treatment of patients with uveitis associated with juvenile idiopathic arthritis
Abstract
- Juvenile idiopathic arthritis (JIA) is often associated with severe chronic anterior uveitis (CAU), and immunosuppressive therapy may be required. In this study, the value of cyclosporine A (CsA) as monotherapy or as combination therapy for treating uveitis was studied in a large cohort of JIA children.
Multicentre retrospective study including 82 JIA children (girls n=60) suffering from unilateral or bilateral (n=55) CAU. The indication for CsA was active uveitis, although patients were on topical or systemic corticosteroids, MTX, or other immunosuppressive drugs.
Inactivity of uveitis during the entire treatment period (mean 3.9 years) was obtained with CsA monotherapy in 6 of 25 (24%) patients, but more often when CsA was combined with the immunosuppressives (35/72 patients; 48.6%, P=0.037), or MTX (18/37 patients, 48.6%, P=0.065), which had already been given. With CsA (mean dosage 2.9 mg/kg), systemic immunosuppressive drugs and steroids could be reduced by >or=50% (n=19) or topical steroids reduced to <or=2 drops/eye/day (n=40) in selected patients. Pre-existing cystoid macular oedema did not resolve under CsA treatment in any of the patients. In nine patients (11%), CsA was discontinued because of systemic hypertension (n=1), elevated creatinine levels (n=3), or other adverse effects (n=5).
These observations suggest that CsA has limited value as a second-line immunosuppressive drug for the treatment of JIA-associated CAU. The efficacy was better as the combination therapy in patients not responding to other immunosuppressives (eg, MTX) than the systemic monotherapy.
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| 18552180 |
Isolation and characterization of carnocyclin a, a novel circular bacteriocin produced by Carnobacterium maltaromaticum UAL307 |
10.1128/AEM.00817-08. |
Appl Environ Microbiol |
Isolation and characterization of carnocyclin a, a novel circular bacteriocin produced by Carnobacterium maltaromaticum UAL307
Abstract
- Carnobacterium maltaromaticum UAL307, isolated from fresh pork, exhibits potent activity against a number of gram-positive organisms, including numerous Listeria species. Three bacteriocins were isolated from culture supernatant, and using matrix-assisted laser desorption ionization-time of flight mass spectrometry and Edman sequencing, two of these bacteriocins were identified as piscicolin 126 and carnobacteriocin BM1, both of which have previously been described. The remaining bacteriocin, with a molecular mass of 5,862 Da, could not be sequenced by traditional methods, suggesting that the peptide was either cyclic or N-terminally blocked. This bacteriocin showed remarkable stability over a wide temperature and pH range and was unaffected by a variety of proteases. After digestion with trypsin and alpha-chymotrypsin, the peptide was de novo sequenced by tandem mass spectrometry and a linear sequence deduced, consisting of 60 amino acids. Based on this sequence, the molecular mass was predicted to be 5,880 Da, 18 units higher than the observed molecular mass, which suggested that the peptide has a cyclic structure. Identification of the genetic sequence revealed that this peptide is circular, formed by a covalent linkage between the N and C termini following cleavage of a 4-residue peptide leader sequence. The results of structural studies suggest that the peptide is highly structured in aqueous conditions. This bacteriocin, named carnocyclin A, is the first reported example of a circular bacteriocin produced by Carnobacterium spp.
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| 18558743 |
Aerucyclamides A and B: isolation and synthesis of toxic ribosomal heterocyclic peptides from the cyanobacterium Microcystis aeruginosa PCC 7806 |
10.1021/np800118g. |
J Nat Prod |
Aerucyclamides A and B: isolation and synthesis of toxic ribosomal heterocyclic peptides from the cyanobacterium Microcystis aeruginosa PCC 7806
Abstract
- Two new modified hexacyclopeptides, aerucyclamides A and B, were isolated from the toxic freshwater cyanobacterium Microcystis aeruginosa PCC 7806. The constitution was assigned by spectroscopic methods, and the configuration determined by chemical degradation and analysis by Marfey's method combined with chemical synthesis. Synthetic aerucyclamide B was obtained through oxidation of aerucyclamide A (MnO2, benzene). The aerucyclamides were found to be toxic to the freshwater crustacean Thamnocephalus platyurus, exhibiting LC50 values for congeners A and B of 30.5 and 33.8 microM, respectively.
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| 18558744 |
Structural and conformational analysis of hydroxycyclochlorotine and cyclochlorotine, chlorinated cyclic peptides from Penicillium islandicum |
10.1021/np800150m. |
J Nat Prod |
Structural and conformational analysis of hydroxycyclochlorotine and cyclochlorotine, chlorinated cyclic peptides from Penicillium islandicum
Abstract
- A new chlorinated cyclic pentapeptide, hydroxycyclochlorotine (1), has been isolated from Penicillium islandicum, and the structure including absolute stereochemistry of 1 and conformational properties of 1 and cyclochlorotine (2) in DMSO-d6 were elucidated by using extensive 2D NMR and chemical means. Hydroxycyclochlorotine (1) and astin B (3) from Aster tataricus, each containing an allo threonine at residue 2, have a cis proline configuration, whereas cyclochlorotine (2) has two conformational states in solution, which may be produced from cis-trans isomerization of the proline amide bond. The presence of an intramolecular hydrogen bond between Ser (3)-NH and a hydroxyl oxygen atom of alloThr (2) may serve to maintain the backbone conformation with a cis proline amide bond.
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| 18559263 |
Molecular genetic mining of the Aspergillus secondary metabolome: discovery of the emericellamide biosynthetic pathway |
10.1016/j.chembiol.2008.05.010. |
Chem Biol |
Molecular genetic mining of the Aspergillus secondary metabolome: discovery of the emericellamide biosynthetic pathway
Abstract
- The recently sequenced genomes of several Aspergillus species have revealed that these organisms have the potential to produce a surprisingly large range of natural products, many of which are currently unknown. We have found that A. nidulans produces emericellamide A, an antibiotic compound of mixed origins with polyketide and amino acid building blocks. Additionally, we describe the discovery of four previously unidentified, related compounds that we designate emericellamide C-F. Using recently developed gene targeting techniques, we have identified the genes involved in emericellamide biosynthesis. The emericellamide gene cluster contains one polyketide synthase and one nonribosomal peptide synthetase. From the sequences of the genes, we are able to deduce a biosynthetic pathway for the emericellamides. The identification of this biosynthetic pathway opens the door to engineering novel analogs of this structurally complex metabolite.
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| 18559951 |
Identification of a new transmembrane adaptor protein that constitutively binds Grb2 in B cells |
10.1189/jlb.0208087. |
J Leukoc Biol |
Identification of a new transmembrane adaptor protein that constitutively binds Grb2 in B cells
Abstract
- Transmembrane adaptor proteins couple antigen receptor engagement to downstream signaling cascades in lymphocytes. One example of these proteins is the linker for activation of T cells (LAT), which plays an indispensable role in T cell activation and development. Here, we report identification of a new transmembrane adaptor molecule, namely growth factor receptor-bound protein 2 (Grb2)-binding adaptor protein, transmembrane (GAPT), which is expressed in B cells and myeloid cells. Similar to LAT, GAPT has an extracellular domain, a transmembrane domain, and a cytoplasmic tail with multiple Grb2-binding motifs. In contrast to other transmembrane adaptor proteins, GAPT is not phosphorylated upon BCR ligation but associates with Grb2 constitutively through its proline-rich region. Targeted disruption of the gapt gene in mice affects neither B cell development nor a nitrophenylacetyl-specific antibody response. However, in the absence of GAPT, B cell proliferation after BCR cross-linking is enhanced. In aged GAPT(-/-) mice, the number of marginal zone (MZ) B cells is increased, and other B cell subsets are normal. The serum concentrations of IgM, IgG2b, and IgG3 are also elevated in these mice. These data indicate that GAPT might play an important role in control of B cell activation and proper maintenance of MZ B cells.
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| 18566513 |
Direct interaction between a human digestive protease and the mucoadhesive poly(acrylic acid) |
10.1107/S0907444908013474. |
Acta Crystallogr D Biol Crystallogr |
Direct interaction between a human digestive protease and the mucoadhesive poly(acrylic acid)
Abstract
- Carboxypeptidase A1 has been the subject of extensive research in the last 30 y and is one of the most widely studied zinc metalloenzymes. However, the three-dimensional structure of the human form of the enzyme is not yet available. This report describes the three-dimensional structure of human carboxypeptidase A1 (hCPA1) derived from crystals that belong to the tetragonal space group P4(3)2(1)2 and diffract to 1.6 angstroms resolution. A description of the ternary complex hCPA1-Zn2+-poly(acrylic acid) is included as a model of the interaction of mucoadhesive polymers with proteases in the gastrointestinal tract. The direct mode of interaction between poly(acrylic acid) and the active site of the target protease was confirmed by in vitro inhibition assays. The structure was further analyzed in silico through the optimal docking-area method. The characterization of binding sites on the surface of hCPA1 and a comparison with other available carboxypeptidase structures provided further insights into the formation of multiprotein complexes and the activation mechanisms of carboxypeptidase zymogens. The high-resolution structure of hCPA1 provides an excellent template for the modelling of physiologically relevant carboxypeptidases and could also contribute to the design of specific agents for biomedical purposes.
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| 18566589 |
Small-molecule inhibition and activation-loop trans-phosphorylation of the IGF1 receptor |
10.1038/emboj.2008.116. |
EMBO J |
Small-molecule inhibition and activation-loop trans-phosphorylation of the IGF1 receptor
Abstract
- The insulin-like growth factor-1 receptor (IGF1R) is a receptor tyrosine kinase (RTK) that has a critical role in mitogenic signalling during embryogenesis and an antiapoptotic role in the survival and progression of many human tumours. Here, we present the crystal structure of the tyrosine kinase domain of IGF1R (IGF1RK), in its unphosphorylated state, in complex with a novel compound, cis-3-[3-(4-methyl-piperazin-l-yl)-cyclobutyl]-1-(2-phenyl-quinolin-7-yl)-imidazo[1,5-a]pyrazin-8-ylamine (PQIP), which we show is a potent inhibitor of both the unphosphorylated (basal) and phosphorylated (activated) states of the kinase. PQIP interacts with residues in the ATP-binding pocket and in the activation loop, which confers specificity for IGF1RK and the highly related insulin receptor (IR) kinase. In this crystal structure, the IGF1RK active site is occupied by Tyr1135 from the activation loop of an symmetry (two-fold)-related molecule. This dimeric arrangement affords, for the first time, a visualization of the initial trans-phosphorylation event in the activation loop of an RTK, and provides a molecular rationale for a naturally occurring mutation in the activation loop of the IR that causes type II diabetes mellitus.
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| 18571156 |
Protective effect of JBP485 on concanavalin A-induced hepatocyte toxicity in primary cultured rat hepatocytes |
10.1016/j.ejphar.2008.04.066. |
Eur J Pharmacol |
Protective effect of JBP485 on concanavalin A-induced hepatocyte toxicity in primary cultured rat hepatocytes
Abstract
- Cyclo-trans-4-L-hydroxyprolyl-L-serine (JBP485) is a dipeptide isolated from Laennec, and Laennec is a hydrolyzate of human placenta. Evidence has indicated that JBP485 exhibits potent anti-hepatitis activity. In this study, we investigated the protective effect and possible mechanisms of action of JBP485 in Concanavalin A (Con A)-induced hepatotoxicity in vitro. Two in vitro models were established. Model I: primary cultured female rat hepatocytes were only incubated with Con A (50 microg/ml); model II: co-culture system of hepatocytes and autologous splenic lymphocytes, both were stimulated with Con A (20 microg/ml). JBP485 (25 microM) was pre-incubated with the two models. Our results showed that JBP485 reduced cellular aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and tumor necrosis factor alpha (TNF-alpha) leakage following the application of Con A in both of the models. Potential protective mechanisms were elucidated by measuring DNA fragmentations, immunocytochemistry and RT-PCR. We showed that DNA fragmentations in hepatocytes were attenuated in the JBP485 pre-incubated groups, and at the same time, immunocytochemistry and RT-PCR indicated that expression levels of caspase-3 protein and mRNA in the JBP485 treated groups were decreased compared with those in the untreated groups. Moreover, intercellular adhesion molecule-1 (ICAM-1) was also down-regulated by this dipeptide. The results indicate that JBP485 exhibits hepatoprotective effect through inhibition of hepatocyte apoptosis and ICAM-1 expression.
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| 18585815 |
Characteristics of signalling properties mediated by long-acting insulin analogue glargine and detemir in target cells of insulin |
10.1016/j.diabres.2008.05.007. |
Diabetes Res Clin Pract |
Characteristics of signalling properties mediated by long-acting insulin analogue glargine and detemir in target cells of insulin
Abstract
- Glargine and detemir are long-acting human insulin analogues with a smooth peakless profile of action. Although their binding affinities to the insulin receptor have been studied, little is known about the subsequent signalling properties activated after the binding. We directly compared intracellular signalling properties of them in various cultured cells. Regarding the metabolic signalling, glargine and insulin-induced comparable dose-dependent phosphorylation of insulin receptor, IRS-1, Akt, and GSK3, whereas detemir-induced kinetics were markedly lower in 3T3-L1 adipocytes and L6 myocytes. A similar pattern of phosphorylation induction was observed in primary hepatocytes and vascular smooth muscle cells (VSMCs). Because of the binding of detemir to albumin with high affinity, the phosphorylation kinetics and glucose uptake of detemir, but not glargine, decreased with increasing concentrations of BSA. Concerning the mitogenic properties, glargine and insulin-induced comparable dose-dependent phosphorylation of MAP kinase (MAPK) and 5-bromo-2'-deoxyuridine (BrdU) incorporation. Detemir-induced phosphorylation of MAPK was apparently reduced, whereas it stimulated BrdU incorporation with relatively similar dose-dependent manner in VSMCs. These results indicate that glargine has comparable properties to human insulin in metabolic and mitogenic signalling and action. In contrast, detemir-induced metabolic signaling is less potent in all cell types studied, and is reduced further by increasing concentrations of albumin.
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| 18590275 |
Solid-support based total synthesis and stereochemical correction of brunsvicamide A |
10.1021/ol801064d. |
Org Lett |
Solid-support based total synthesis and stereochemical correction of brunsvicamide A
Abstract
- A total synthesis of the cyanobacterial metabolite brunsvicamide A and the correction of its originally assigned stereochemistry are reported. Key elements were the construction of a urea building block, peptide elongation on solid phase, and on-resin cyclization of the peptide chain, with good overall yield. Detailed structural investigations uncovered that brunsvicamide A features a previously undetected d-lysine residue in its backbone, setting the foundation for all further investigations in this compound class.
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| 18599825 |
Colutellin A, an immunosuppressive peptide from Colletotrichum dematium |
10.1099/mic.0.2008/017954-0. |
Microbiology (Reading) |
Colutellin A, an immunosuppressive peptide from Colletotrichum dematium
Abstract
- Colletotrichum dematium is an endophytic fungus recovered from a Pteromischum sp. growing in a tropical forest in Costa Rica. This fungus makes a novel peptide antimycotic, colutellin A, with a MIC of 3.6 microg ml(-1) (48 h) against Botrytis cinerea and Sclerotinia sclerotiorum. Collutellin A has a mass of 1127.7 Da and contains residues of Ile, Val, Ser, N-methyl-Val and beta-aminoisobutryic acid in nominal molar ratios of 3 : 2 : 1 : 1 : 1, respectively. Independent lines of evidence suggest that the peptide is cyclic and sequences of Val-Ile-Ser-Ile and Ile-Pro-Val have been deduced by MS/MS as well as Edman degradation methods. Colutellin A inhibited CD4(+) T-cell activation of interleukin 2 (IL-2) production with an IC(50) of 167.3+/-0.38 nM, whereas cyclosporin A in the same test yielded a value of 61.8 nM. Inhibition of IL-2 production by collutellin A at such a low concentration indicates the potential immunosuppressive activity of this compound. In repeated experiments, cyclosporin A at or above 8 microg ml(-1) exhibited high levels of cytotoxicity on human peripheral blood mononuclear cells, whereas collutellin A or DMSO (carrier) alone, after 24 and 48 h of culture, exhibited no toxicity. Because of these properties collutellin A has potential as a novel immunosuppressive drug.
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| 18601958 |
Disulfide-containing peptides from the glandular skin secretions of froglets of the genus Crinia: structure, activity and evolutionary trends |
10.1016/j.regpep.2008.06.004. |
Regul Pept |
Disulfide-containing peptides from the glandular skin secretions of froglets of the genus Crinia: structure, activity and evolutionary trends
Abstract
- The skin secretions of Crinia signifera, C. riparia and C. deserticola contain bioactive disulfide-containing peptides. Signiferin 1 (RLCIPYIIPC-OH) from C. signifera and C. deserticola) contracts smooth muscle at a concentration of 10(-9) M, and effects proliferation of lymphocytes at 10(-6) M. In contrast, riparin 1.1 (RLCIPVIFC-OH) and riparin 1.2 (FLPPCAYKGTC-OH) from C. riparia show lymphocyte activity but do not contract smooth muscle. The lymphocyte and smooth muscle activities involve CCK2R. 3D structures of signiferin 1 and riparin 1.1 have been established using 2D NMR methods: these studies show significant differences in the shapes of the disulfide rings and with the orientations of the N-terminal residues. cDNA cloning establishes that the pre sections of the precursor pre-pro-riparin 1.4-1.6 peptides are different from the conserved pre regions of disulfide-containing antimicrobial peptides from species of the genus Rana found in the northern hemisphere and caerin antimicrobial peptides isolated from Australian tree frogs of the genus Litoria. This suggests that (i) either that riparins 1 have converged to similar structure and function to the ranid and hyloid prepropeptides which were lost initially from the myobatrachid lineage, or (ii) the prepropeptides in all three groups were derived from a single ancestral form that has remained relatively conserved in the hyloid and ranoid lineages but has undergone substantial divergent evolution in the myobatrachids.
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| 18602463 |
Ataxin-2 associates with the endocytosis complex and affects EGF receptor trafficking |
10.1016/j.cellsig.2008.05.018. |
Cell Signal |
Ataxin-2 associates with the endocytosis complex and affects EGF receptor trafficking
Abstract
- Ataxin-2 is a novel protein, where the unstable expansion of an internal polyglutamine domain can cause the neurodegenerative disease Spinocerebellar Ataxia type 2 (SCA2). To elucidate its cellular function, we have used full-length ataxin-2 as bait in a yeast two-hybrid screen of human adult brain cDNA. As binding partners we found endophilin A1 and A3, two brain-expressed members of the endophilin A family involved in synaptic vesicle endocytosis. Co-immunoprecipitation studies confirmed the binding of these proteins as an endogenous complex in mouse brain. In vitro binding experiments narrowed the binding interfaces down to two proline-rich domains on ataxin-2, which interacted with the SH3 domain of endophilin A1/A3. Ataxin-2 and endophilin associated at the endoplasmic reticulum as well as at the plasma membrane as determined by immunofluorescence microscopy of transfected cell lines, and by centrifugation fractionation studies of mouse brain. Importantly, the pattern observed in transfected cells was conserved in rat hippocampal neurons. In the mouse brain, an association of ataxin-2 with endocytic proteins such as the adaptor CIN85 and the ubiquitin ligase c-Cbl was also demonstrated. GST pull-down assays showed ataxin-2 to directly interact with the SH3 domains A and C of CIN85 and with the SH3 domain of Src, a kinase activated after receptor stimulation. Functional studies demonstrated that ataxin-2 affects endocytic trafficking of the epidermal growth factor receptor (EGFR). Taken together, these data implicate ataxin-2 to play a role in endocytic receptor cycling.
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