| 20521079 |
Recruitment of Pyk2 to SHPS-1 signaling complex is required for IGF-I-dependent mitogenic signaling in vascular smooth muscle cells |
10.1007/s00018-010-0411-x. |
Cell Mol Life Sci |
Recruitment of Pyk2 to SHPS-1 signaling complex is required for IGF-I-dependent mitogenic signaling in vascular smooth muscle cells
Abstract
- In vascular smooth muscle cells, IGF-I stimulates SHPS-1/SHP2/Src complex formation which is required for IGF-I-stimulated cell proliferation. Using SHP2/Src silencing and a Pyk2/Y402F mutant, we showed that Pyk2 was also recruited to the SHPS-1 complex. Pyk2 recruitment to SHPS-1 is mediated via the interaction of Pyk2 Tyr402 and the Src in response to IGF-I. Following Src/Pyk2 association, Src phosphorylates Pyk2 on Tyr881 providing a binding site for Grb2. Cells expressing Pyk2/Y881F showed decreased Grb2 recruitment to SHPS-1 and impaired Shc/Grb2 association. This change led to reduced Erk1/2 (MAP kinase) activation and cell proliferation in response to IGF-I. Our results show that, following its recruitment to the SHPS-1 signaling complex, Pyk2 localizes Grb2 in close proximity to Shc thereby facilitating Shc/Grb2 association which leads to Erk1/2 activation in response to IGF-I. Thus, Pyk2 recruitment to SHPS-1 plays an important role in regulating the IGF-I-stimulated mitogenic response.
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| 20545947 |
Design of potent IGF1-R inhibitors related to bis-azaindoles |
10.1111/j.1747-0285.2010.00991.x. |
Chem Biol Drug Des |
Design of potent IGF1-R inhibitors related to bis-azaindoles
Abstract
- From an azaindole lead, identified in high throughput screen, a series of potent bis-azaindole inhibitors of IGF1-R have been synthesized using rational drug design and SAR based on a in silico binding mode hypothesis. Although the resulting compounds produced the expected improved potency, the model was not validated by the co-crystallization experiments with IGF1-R.
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| 20551055 |
Nuclear alternate estrogen receptor GPR30 mediates 17beta-estradiol-induced gene expression and migration in breast cancer-associated fibroblasts |
10.1158/0008-5472.CAN-10-0408. |
Cancer Res |
Nuclear alternate estrogen receptor GPR30 mediates 17beta-estradiol-induced gene expression and migration in breast cancer-associated fibroblasts
Abstract
- Fibroblasts are the principal cellular component of connective tissue and are associated with cancer cells at all stages of tumor progression. Structural and functional contributions of fibroblasts to the growth, survival, and invasive capacity of cancer cells are beginning to emerge. In breast carcinoma, approximately 80% of stromal fibroblasts termed cancer-associated fibroblasts (CAF) are thought to manifest an activated phenotype that promotes cancer cell proliferation tumor growth at metastatic sites similar to the primary tumor. In this report, we show that CAFs respond to physiologic concentrations of 17beta-estradiol (E2) by rapidly inducing extracellular signal-regulated kinase phosphorylation and immediate early gene expression, including c-fos and connective tissue growth factor, and cyclin D1. Notably, the E2 response is mediated by the alternate estrogen receptor GPR30, which interfaces with the epidermal growth factor receptor (EGFR) signaling pathway. In particular, E2 stimulates a physical interaction between GPR30 and phosphorylated EGFR, recruiting them to the cyclin D1 gene promoter. Nuclear localization induced by E2 was confirmed by cellular immunofluorescence methods. GPR30 was required for CAF proliferation and migration induced by E2. Our results provide important new mechanistic insights into how CAFs are stimulated by estrogen through a GPR30-mediated nuclear signaling pathway. More generally, they define estrogenic GPR30 signaling as a functionally important component of the tumor microenvironment.
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| 20563613 |
Labaditin, a cyclic peptide with rich biotechnological potential: preliminary toxicological studies and structural changes in water and lipid membrane environment |
10.1007/s00726-010-0648-6. |
Amino Acids |
Labaditin, a cyclic peptide with rich biotechnological potential: preliminary toxicological studies and structural changes in water and lipid membrane environment
Abstract
- Cyclic peptides isolated from the plants of the Euphorbiaceae family have been largely studied due to their rigid conformation, which is considered significant for biologic activity. The peptide Labaditin (L(0)) and its open chain analogs (L(1)) were synthesized by the solid-phase peptide synthesis technique (Fmoc/tBu), and purified to elucidate its interaction with membrane models. A shift in λ(max) emission and Stern-Volmer constants values indicate that both tryptophans migrate to a more apolar environment, with L(1) decreasing less than L(0). A circular dichroism (CD) study revealed that L(0) was kept unstructured in aqueous media as much as in the presence of dipalmitoilphosphatidylcholine liposomes. The thermodynamic studies by differential calorimetry (DSC) show a ΔH increase (50 and 18 kcal/mol, for L(0) and L(1), respectively) with peptide concentrations, which is indicative of lipids associating with peptides, resulting in the inability of the lipids to participate in the main transition. Therefore, all CD, DSC, and fluorescence data suggest a greater L(0) membrane insertion. A probable mechanism for Labaditin interaction is based initially on the hydrophobic interaction of the peptide with the lipid membrane, conformational change, peptide adsorption on the lipid surface, and internalization process. Peptide's antibacterial effect was also evaluated and revealed that only L(0) showed reduction in viability in Gram-positive bacteria while no effects to the Gram-negative.
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| 20564007 |
A new "era" for cyclotide sequencing |
10.1002/bip.21400. |
Biopolymers |
A new "era" for cyclotide sequencing
Abstract
- In recent years, the discovery of a large family of macrocyclic peptides, the cyclotides, has revealed Natures ingenuity in molecular drug design. The incorporation of a cyclic peptide backbone and a knotted arrangement of disulfide bridges into their structures confers extraordinary chemical, thermal, and enzymatic stability on these biologically active peptides. However, these structural attributes present challenges in the identification of cyclotides. Until now, the sequencing of cyclotides has been slow and inefficient owing to inherent difficulties in the separation of these hydrophobic peptides from plants, the multiple chemical and enzymatic derivatization steps required to make them amenable to mass spectrometric sequencing, and the lack of software tools to efficiently deal with these circular permutants. The current bottleneck slowing the speed of cyclotide sequencing is the requirement for multiple HPLC purification steps before analysis. Here, we have applied proteomic strategies to fast-track the discovery of known, modified and novel sequences. Using four fractions from a previously well-characterized cyclotide-containing plant species, Viola odorata, 11 new sequences, as well as a plethora of known and modified cyclotides, were uncovered. In addition, the methodology was validated through analysis of crude leaf extracts ofOldenlandia affinis and Arabidopsis thaliana. The unambiguous identification of a suite of cyclotides in the Oldenlandia affinis extract provided the ultimate proof-of-concept for this application. Major advances in methodology include the use of optimized LC-MS/MS conditions and design of a custom-built cyclotide database, in which mature cyclotide sequences are excised, replicated and appended, marking a new "era" for cyclotide sequencing.
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| 20564013 |
Structural and biochemical characteristics of the cyclotide kalata B5 from Oldenlandia affinis |
10.1002/bip.21409. |
Biopolymers |
Structural and biochemical characteristics of the cyclotide kalata B5 from Oldenlandia affinis
Abstract
- Cyclotides are a large family of plant-derived proteins typified by their head-to-tail cyclic backbone and knotted arrangement of three disulfide bonds. Although they display a diverse range of biological activities, their native function is thought to be plant defense. Here we characterized the expression, three-dimensional structure, and hemolytic activity of the cyclotide kalata B5 from the African plant Oldenlandia affinis. Kalata B5 shows an interesting seasonal variation in its expression and can only be isolated during certain times of the year, when the plant is flowering. It displays a typical tightly folded cyclic Scystine knot structure. A range of pH and temperature titrations reveal that a conserved glutamic acid in loop 1 Sof the structure forms a key hydrogen bond network, similar to that reported previously for other cyclotides. However, specific line broadening in the NMR spectra of kalata B5 suggests that the hydrogen bonding network in this peptide is less rigid than in other cyclotides. Notably, the pK9a) of Glu6 of 4.5 is higher than the values for other cyclotides studied so far, which range from 3.0 to 4.0, providing a further indication of a weaker hydrogen bond network. Kalata B5 has only moderate hemolytic activity compared with other highly expressed cyclotides, and this reduced activity probably reflects its more flexible structure. As is the case with other cyclotides, kalata B5 has an exposed hydrophobic region on its surface, supporting suggestions that this hydrophobic patch is a key feature for membrane binding and biological activity of cyclotides.
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| 20564018 |
Cyclotides are a component of the innate defense of Oldenlandia affinis |
10.1002/bip.21419. |
Biopolymers |
Cyclotides are a component of the innate defense of Oldenlandia affinis
Abstract
- Cyclotides are small cysteine-rich plant peptides similar in size and processing to the defensins. Long-term growth of the Rubiaceae family plant Oldenlandia affinis under different conditions reveals a diverse cyclotide gene and peptide expression profile, including tissue specificity, suggesting that different cyclotides are regulated differently both spatially and in response to the environment. To determine whether cyclotide precursor gene regulation was dynamic we exposed O. affinis to a range of abiotic, biotic, and hormonal stimuli and monitored Oak1-4 expression over a 48-h period. Unlike some defensins, the genes for cyclotide precursor proteins Oak1-4 did not display dynamic change, indicating that they contribute to the basal defense of O. affinis. Despite this lack of dynamism, the cyclotide profile of plants grown on plates differed markedly from field-grown plants and so prompted attempts to discover novel cyclotides and precursor genes. The two most abundant cyclotides from plate-grown O. affinis were sequenced and one was found to be an unusual linear cyclotide derivative, kalata B20-lin. Degenerate PCR of plate-grown O. affinis obtained five novel cyclotide genes including Oak9 which encodes for kalata B20-lin and appears to have arisen by the presence of a premature stop codon.
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| 20575512 |
Isolation, characterization, and bioactivity of cyclotides from the Micronesian plant Psychotria leptothyrsa |
10.1021/np9007365. |
J Nat Prod |
Isolation, characterization, and bioactivity of cyclotides from the Micronesian plant Psychotria leptothyrsa
Abstract
- Cyclotides, the largest known family of head-to-tail cyclic peptides, have approximately 30 amino acid residues with a complex structure containing a circular peptide backbone and a cystine knot. They are found in plants from the Violaceae and Rubiaceae families and are speculated to function in plant protection. In addition to their insecticidal properties, cyclotides display cytotoxic, anti-HIV, antimicrobial, and inhibition of neurotensin binding activities. Although cyclotides are present in all violaceous species hitherto screened, their distribution and expression in Rubiaceae are not fully understood. In this study, we show that Psychotria leptothyrsa var. longicarpa (Rubiaceae) contains a suite of different cyclotides. The cyclotide fractions were isolated by RP-HPLC, and sequences of six new peptides, named psyles A-F, were determined by MS/MS sequencing. One of these, psyle C, is the first rubiaceous linear variant known. Psyles A, C, and E were analyzed in a fluorometric microculture assay to determine cytotoxicity toward the human lymphoma cell line U937-GTB. The IC(50) values of psyles A, C, and E were 26, 3.50, and 0.76 muM, respectively. This study expands the number of known rubiaceous cyclotides and shows that the linear cyclotide maintains cytotoxicity.
|
| 20580652 |
Isolation and characterization of cytotoxic cyclotides from Viola tricolor |
10.1016/j.peptides.2010.05.004. |
Peptides |
Isolation and characterization of cytotoxic cyclotides from Viola tricolor
Abstract
- Many plants of the Violaceae plant family have been used in traditional remedies, and these plants often contain cyclotides, a particular type of plant cyclopeptide that is distinguished by a cyclic cystine knot motif. In general, bioactive plant cyclopeptides are interesting candidates for drug development. In the current study, a suite of 14 cyclotides, which includes seven novel cyclotides [vitri B, C, D, E, F, varv Hm, and He], together with seven known cyclotides [varv A, D, E, F, H, vitri A, and cycloviolacin O2], was isolated from Viola tricolor, a common flower. A chromatography-based method was used to isolate the cyclotides, which were characterized using tandem mass spectrometry and NMR spectroscopy. Several of the cyclotides showed cytotoxic activities against five cancer cell lines, U251, MDA-MB-231, A549, DU145, and BEL-7402. Three cyclotides, vitri A, vitri F, and cycloviolacin O2, were the most cytotoxic. The cytotoxic activity of the cyclotides did not correlate well with their hemolytic activity, indicating that different interactions, most likely with membranes, are involved for cytotoxic and hemolytic activities. Homology modeling of the structures was used in deriving structure-activity relationships.
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| 20588300 |
AS1387392, a novel immunosuppressive cyclic tetrapeptide compound with inhibitory activity against mammalian histone deacetylase |
10.1038/ja.2010.51. |
J Antibiot (Tokyo) |
AS1387392, a novel immunosuppressive cyclic tetrapeptide compound with inhibitory activity against mammalian histone deacetylase
Abstract
- The novel immunosuppressant AS1387392 has been isolated from Acremonium sp. No. 27082. This compound showed a strong inhibitory effect against mammalian histone deacetylase and T-cell proliferation. Further, AS1387392 showed a good oral absorption, and its plasma concentration was higher than that of FR235222, an analog of AS1387392 that inhibited histone deacetylase previously reported. Given these findings, AS1387392 may represent an important new lead in developing immunosuppressant.
|
| 20596523 |
Development and validation of a method for profiling post-translational modification activities using protein microarrays |
10.1371/journal.pone.0011332. |
PLoS One |
Development and validation of a method for profiling post-translational modification activities using protein microarrays
Abstract
- Background:
Post-translational modifications (PTMs) impact on the stability, cellular location, and function of a protein thereby achieving a greater functional diversity of the proteome. To fully appreciate how PTMs modulate signaling networks, proteome-wide studies are necessary. However, the evaluation of PTMs on a proteome-wide scale has proven to be technically difficult. To facilitate these analyses we have developed a protein microarray-based assay that is capable of profiling PTM activities in complex biological mixtures such as whole-cell extracts and pathological specimens.
Methodology/principal findings:
In our assay, protein microarrays serve as a substrate platform for in vitro enzymatic reactions in which a recombinant ligase, or extracts prepared from whole cells or a pathological specimen is overlaid. The reactions include labeled modifiers (e.g., ubiquitin, SUMO1, or NEDD8), ATP regenerating system, and other required components (depending on the assay) that support the conjugation of the modifier. In this report, we apply this methodology to profile three molecularly complex PTMs (ubiquitylation, SUMOylation, and NEDDylation) using purified ligase enzymes and extracts prepared from cultured cell lines and pathological specimens. We further validate this approach by confirming the in vivo modification of several novel PTM substrates identified by our assay.
Conclusions/significance:
This methodology offers several advantages over currently used PTM detection methods including ease of use, rapidity, scale, and sample source diversity. Furthermore, by allowing for the intrinsic enzymatic activities of cell populations or pathological states to be directly compared, this methodology could have widespread applications for the study of PTMs in human diseases and has the potential to be directly applied to most, if not all, basic PTM research.
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| 20649464 |
Improving the stability of α-conotoxin AuIB through N-to-C cyclization: the effect of linker length on stability and activity at nicotinic acetylcholine receptors |
10.1089/ars.2010.3458. |
Antioxid Redox Signal |
Improving the stability of α-conotoxin AuIB through N-to-C cyclization: the effect of linker length on stability and activity at nicotinic acetylcholine receptors
Abstract
- Modification of α-conotoxin frameworks through cyclization via an oligopeptide linker has previously been shown as an effective strategy for improving in vivo stability. We have extended this strategy by investigating cyclic analogs of α-conotoxin AuIB, a selective α(3)β(4) nicotinic acetylcholine receptor (nAChR) antagonist, to examine a range of oligopeptide linker lengths on the oxidative formation of disulfide bonds, activity at nAChRs, and stability to degradation by chymotrypsin. Upon nondirected random oxidation, the ribbon isomer formed preferentially with the globular isomer occurring as a minor by-product. Therefore, a regioselective disulfide bond forming strategy was used to prepare the cAuIB-2 globular isomer in high yield and purity. The cAuIB-2 globular isomer exhibited a threefold decrease in activity for the α(3)β(4) nAChR compared to wild-type-AuIB, although it was selective for α(3)β(4) over α(7) and α(4)β(2) subtypes. On the other hand, the cAuIB-2 ribbon isomer was shown to be inactive at all three nAChR subtypes. theless, all of the cyclic analogs were found to be significantly more stable to degradation by chymotrypsin than wild-type AuIB. As such, the cAuIB-2 globular isomer could constitute a useful probe for studying the role of the α(3)β(4) nAChR in a range of in vivo experimental paradigms.
|
| 20660740 |
CXCR4/YY1 inhibition impairs VEGF network and angiogenesis during malignancy |
10.1073/pnas.1008256107. |
Proc Natl Acad Sci U S A |
CXCR4/YY1 inhibition impairs VEGF network and angiogenesis during malignancy
Abstract
- Tumor growth requires neoangiogenesis. VEGF is the most potent proangiogenic factor. Dysregulation of hypoxia-inducible factor (HIF) or cytokine stimuli such as those involving the chemokine receptor 4/stromal-derived cell factor 1 (CXCR4/SDF-1) axis are the major cause of ectopic overexpression of VEGF in tumors. Although the CXCR4/SDF-1 pathway is well characterized, the transcription factors executing the effector function of this signaling are poorly understood. The multifunctional Yin Yang 1 (YY1) protein is highly expressed in different types of cancers and may regulate some cancer-related genes. The network involving CXCR4/YY1 and neoangiogenesis could play a major role in cancer progression. In this study we have shown that YY1 forms an active complex with HIF-1alpha at VEGF gene promoters and increases VEGF transcription and expression observed by RT-PCR, ELISA, and Western blot using two different antibodies against VEGFB. Long-term treatment with T22 peptide (a CXCR4/SDF-1 inhibitor) and YY1 silencing can reduce in vivo systemic neoangiogenesis (P < 0.01 and P < 0.05 vs. control, respectively) during metastasis. Moreover, using an in vitro angiogenesis assay, we observed that YY1 silencing led to a 60% reduction in branches (P < 0.01) and tube length (P < 0.02) and a 75% reduction in tube area (P < 0.001) compared with control cells. A similar reduction was observed using T22 peptide. We demonstrated that T22 peptide determines YY1 cytoplasmic accumulation by reducing its phosphorylation via down-regulation of AKT, identifying a crosstalk mechanism involving CXCR4/YY1. Thus, YY1 may represent a crucial molecular target for antiangiogenic therapy during cancer progression.
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| 20664604 |
Bioconversion of AS1387392: screening and characterization of actinomycetes that convert AS1387392 to AS1429716 |
10.1038/ja.2010.89. |
J Antibiot (Tokyo) |
Bioconversion of AS1387392: screening and characterization of actinomycetes that convert AS1387392 to AS1429716
Abstract
- AS1387392 was a novel and powerful histone deacetylase inhibitor with an excellent oral absorption profile, but this compound was lacking in active moieties, which are essential to synthesize more derivatives. In our screening program to identify actinomycetes capable of converting AS1387392 to AS1429716, which has an active moiety to synthesize more derivatives, we identified 12 strains capable of efficient hydroxylation. Results of phylogenetic analysis of 16S rDNA sequences suggested that these strains belonged to the genera Lentzea, Saccharopolyspora, Sphaerisporangium and Amycolatopsis. Morphological and chemical characteristics as well as results of phylogenetic analysis suggested that strain No. 7980 was a new species belonging to the genus Amycolatopsis, according to the FASTA search result of 16S rDNA gene sequence. Using these strains, we can easily produce AS1429716 as a chemical template for further chemical modifications, which may provide more effective and safer immunosuppressant.
|
| 20665759 |
High-resolution crystal structure of a lasso Peptide |
10.1002/cmdc.201000264. |
ChemMedChem |
High-resolution crystal structure of a lasso Peptide
Abstract
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