| 22882276 |
The inhibitory effects of aureobasidin A on Candida planktonic and biofilm cells |
10.1111/j.1439-0507.2012.02225.x. |
Mycoses |
The inhibitory effects of aureobasidin A on Candida planktonic and biofilm cells
Abstract
- Aureobasidin A (AbA) is a cyclic depsipeptide antifungal compound that inhibits a wide range of pathogenic fungi. In this study, the in vitro susceptibility of 92 clinical isolates of various Candida species against AbA was assessed by determining the planktonic and biofilm MICs of the isolates. The MIC(50) and MIC(90) of the planktonic Candida yeast were 1 and 1 μg ml(-1), respectively, whereas the biofilm MIC(50) and MIC(90) of the isolates were 8 and ≥64 μg ml(-1) respectively. This study demonstrates AbA inhibition on filamentation and biofilm development of C. albicans. The production of short hyphae and a lack of filamentation might have impaired biofilm development of AbA-treated cells. The AbA resistance of mature Candidia biofilms (24 h adherent population) was demonstrated in this study.
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| 22885029 |
Defensin like peptide from Panulirus argus relates structurally with beta defensin from vertebrates |
10.1016/j.fsi.2012.07.013. |
Fish Shellfish Immunol |
Defensin like peptide from Panulirus argus relates structurally with beta defensin from vertebrates
Abstract
- Naturally occurring antimicrobial peptides take place in the first line of host defense against pathogen as part of the humoral innate immune response. β-defensins are among the most abundant antimicrobial peptides in mammals, and thought to be solely found in vertebrates until a recent report describing the cloning and sequencing of defensin like peptides in the spiny lobster Panulirus japonicus. In the current study, we cloned and sequenced two genes from the hemocytes of the spiny lobster Panulirus argus encoding for two isoforms of defensin-like peptides, thus confirming the presence of this protein in the Panulirus genus. The 44 amino acids mature peptides showed the conservation of cysteine pattern characterizing the β-defensins, as well as known amino acids residues critical to exert their antimicrobial activity. They are also amphipathics, hydrophobics, and display an overall positive charge (+1) located at the C-terminus. The tertiary structure obtained by homology modeling indicated that likely conformations of lobster peptides are highly similar to β-defensins from vertebrates. The phylogenetic study carried out by probabilistic methods confirmed the relation with ancestral β-defensin from vertebrates. The finding of a putative defensin-like peptide in the expressed sequence tag (EST) of the lobster Homarus americanus with high homology with those of P. argus described in this study, would indicate the presence of this peptides in Palinuridae family. Taking into account all similarities between these peptides with β-defensins from vertebrates, it is conceivable to further support the finding of a new family of β-defensins in invertebrate.
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| 22888052 |
Kidney podocytes as specific targets for cyclo(RGDfC)-modified nanoparticles |
10.1002/smll.201200733. |
Small |
Kidney podocytes as specific targets for cyclo(RGDfC)-modified nanoparticles
Abstract
- Renal nanoparticle passage opens the door for targeting new cells like podocytes, which constitute the exterior part of the renal filter. When cyclo(RGDfC)-modified Qdots are tested on isolated primary podocytes for selective binding to the αvβ3 integrin receptor a highly cell- and receptor-specific binding can be observed. In displacement experiments with free cyclo(RGDfC) IC(50) values of 150 nM for αvβ3 integrin over-expressing U87-MG cells and 60 nM for podocytes are measured. Confocal microscopy shows a cellular Qdot uptake into vesicle-like structures. Our ex vivo study gives clear evidence that, after renal filtration, nanoparticles can be targeted to podocyte integrin receptors in the future. This could be a highly promising approach for future therapy and diagnostics of podocyte-associated diseases.
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| 22888118 |
Structural flexibility regulates phosphopeptide-binding activity of the tyrosine kinase binding domain of Cbl-c |
10.1093/jb/mvs085. |
J Biochem |
Structural flexibility regulates phosphopeptide-binding activity of the tyrosine kinase binding domain of Cbl-c
Abstract
- Through their ubiquitin ligase activity, Cbl-family proteins suppress signalling mediated by protein-tyrosine kinases (PTKs), but can also function as adaptor proteins to positively regulate signalling. The tyrosine kinase binding (TKB) domain of this family is critical for binding with tyrosine-phosphorylated target proteins. Here, we analysed the crystal structure of the TKB domain of Cbl-c/Cbl-3 (Cbl-c TKB), which is a distinct member of the mammalian Cbl-family. In comparison with Cbl TKB, Cbl-c TKB showed restricted structural flexibility upon phosphopeptide binding. A mutation in Cbl-c TKB augmenting this flexibility enhanced its binding to target phosphoproteins. These results suggest that proteins, post-translational modifications or mutations that alter structural flexibility of the TKB domain of Cbl-family proteins could regulate their binding to target phosphoproteins and thereby, affect PTK-mediated signalling.
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| 22890611 |
Characterizing circular peptides in mixtures: sequence fragment assembly of cyclotides from a violet plant by MALDI-TOF/TOF mass spectrometry |
10.1007/s00726-012-1376-x. |
Amino Acids |
Characterizing circular peptides in mixtures: sequence fragment assembly of cyclotides from a violet plant by MALDI-TOF/TOF mass spectrometry
Abstract
- Cyclotides are a very abundant class of plant peptides that display significant sequence variability around a conserved cystine-knot motif and a head-to-tail cyclized backbone conferring them with remarkable stability. Their intrinsic bioactivities combined with tools of peptide engineering make cyclotides an interesting template for the design of novel agrochemicals and pharmaceuticals. However, laborious isolation and purification prior to de novo sequencing limits their discovery and hence their use as scaffolds for peptide-based drug development. Here we extend the knowledge about their sequence diversity by analysing the cyclotide content of a violet species native to Western Asia and the Caucasus region. Using an experimental approach, which was named sequence fragment assembly by MALDI-TOF/TOF, it was possible to characterize 13 cyclotides from Viola ignobilis, whereof ten (vigno 1-10) display previously unknown sequences. Amino acid sequencing of various enzymatic digests of cyclotides allowed the accurate assembly and alignment of smaller fragments to elucidate their primary structure, even when analysing mixtures containing multiple peptides. As a model to further dissect the combinatorial nature of the cyclotide scaffold, we employed in vitro oxidative refolding of representative vigno cyclotides and confirmed the high dependency of folding yield on the inter-cysteine loop sequences. Overall this work highlights the immense structural diversity and plasticity of the unique cyclotide framework. The presented approach for the sequence analysis of peptide mixtures facilitates and accelerates the discovery of novel plant cyclotides.
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| 22890978 |
Application of fragment screening and merging to the discovery of inhibitors of the Mycobacterium tuberculosis cytochrome P450 CYP121. |
10.1002/anie.201202544 |
Angew. Chem. Int. Ed. Engl. |
Application of fragment screening and merging to the discovery of inhibitors of the Mycobacterium tuberculosis cytochrome P450 CYP121.
Abstract
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| 22904072 |
Rhythmic binding of Topoisomerase I impacts on the transcription of Bmal1 and circadian period. |
10.1093/nar/gks779 |
Nucleic Acids Res. |
Rhythmic binding of Topoisomerase I impacts on the transcription of Bmal1 and circadian period.
Abstract
- The Bmal1 gene is essential for the circadian system, and its promoter has a unique open chromatin structure. We examined the mechanism of topoisomerase I (Top1) to understand the role of the unique chromatin structure in Bmal1 gene regulation. Camptothecin, a Top1 inhibitor, and Top1 small interfering RNA (siRNA) enhanced Baml1 transcription and lengthened its circadian period. Top1 is located at an intermediate region between two ROREs that are critical cis-elements of circadian transcription and the profile of Top1 binding indicated anti-phase circadian oscillation of Bmal1 transcription. Promoter assays showed that the Top1-binding site is required for transcriptional suppression and that it functions cooperatively with the distal RORE, supporting that Bmal1 transcription is upregulated by Top1 inhibition. A DNA fragment between the ROREs, where the Top1-binding site is located, behaved like a right-handed superhelical twist, and modulation of Top1 activity by camptothecin and Top1 siRNA altered the footprint profile, indicating modulation of the chromatin structure. These data indicate that Top1 modulates the chromatin structure of the Bmal1 promoter, regulates Bmal1 transcription and influences the circadian period.
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| 22917879 |
Antimicrobial peptides from the skin of the Asian frog, Odorrana jingdongensis: de novo sequencing and analysis of tandem mass spectrometry data |
10.1016/j.jprot.2012.08.004. |
J Proteomics |
Antimicrobial peptides from the skin of the Asian frog, Odorrana jingdongensis: de novo sequencing and analysis of tandem mass spectrometry data
Abstract
- Eight intact antimicrobial peptides were identified from the skin of Odorrana jingdongensis by de novo sequencing following low energy ESI CID Q-TOF MS/MS in positive-mode with the help of Edman degradation and structural similarity analysis. We devised exact mass measurements to discriminate the K/Q amino acid residue in the peptides between 2.0 kDa to 3.8 kDa. Moreover, the cleavage at the CS bond at the side chain of Met was observed in all the spectra of the peptides containing Met residue. And we found unusual cleavages within the intramolecular disulfide loop with high frequency. Our data revealed that the cleavage pathways are significantly different from those reported previously which are similar to the cycle peptide cleavage mode followed by the secondary cleavage at the CS bond on oxidized Cys. Thus, our results highly suggest that ion series generated from the cleavages within the intramolecular disulfide loop should be considered in both the top-down sequencing and the disulfide bridge location with the presence of a relatively high intensity of MH(+)-28 ion marker. Furthermore, our activity data implied that different AMPs may use different strategies to kill microbes.
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| 22924493 |
Viequeamide A, a cytotoxic member of the kulolide superfamily of cyclic depsipeptides from a marine button cyanobacterium |
10.1021/np300321b. |
J Nat Prod |
Viequeamide A, a cytotoxic member of the kulolide superfamily of cyclic depsipeptides from a marine button cyanobacterium
Abstract
- The viequeamides, a family of 2,2-dimethyl-3-hydroxy-7-octynoic acid (Dhoya)-containing cyclic depsipeptides, were isolated from a shallow subtidal collection of a "button" cyanobacterium (Rivularia sp.) from near the island of Vieques, Puerto Rico. Planar structures of the two major compounds, viequeamide A (1) and viequeamide B (2), were elucidated by 2D-NMR spectroscopy and mass spectrometry, whereas absolute configurations were determined by traditional hydrolysis, derivative formation, and chromatography in comparison with standards. In addition, a series of related minor metabolites, viequeamides C-F (3-6), were characterized by HRMS fragmentation methods. Viequeamide A was found to be highly toxic to H460 human lung cancer cells (IC(50) = 60 ± 10 nM), whereas the mixture of B-F was inactive. From a broader perspective, the viequeamides help to define a "superfamily" of related cyanobacterial natural products, the first of which to be discovered was kulolide. Within the kulolide superfamily, a wide variation in biological properties is observed, and the reported producing strains are also highly divergent, giving rise to several intriguing questions about structure-activity relationships and the evolutionary origins of this metabolite class.
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| 22926524 |
RBB, a novel transcription repressor, represses the transcription of HDM2 oncogene |
10.1038/onc.2012.386. |
Oncogene |
RBB, a novel transcription repressor, represses the transcription of HDM2 oncogene
Abstract
- The p53 tumor suppressor is important in many aspects of cell biology. Tight regulation of p53 is thus imperative for maintaining cell homeostasis and preventing tumorigenesis. The stabilization and activity of p53 is primarily regulated by MDM2, which is encoded for by HDM2. However, how the expression and activity of MDM2 is regulated remains largely unknown. Here, we report a novel BTB and BEN domains-containing protein, RBB. We demonstrated that RBB is a novel transcriptional repressor binding specific DNA motif via a homodimer and interacting with the nucleosome remodeling and deacetylase (NuRD) complex. Genome wide transcription target analysis by ChIP sequencing revealed that RBB represses the transcription of a series of functionally important genes including HDM2. We showed that RBB recruits the NuRD complex to the internal promoter of HDM2 and inhibits the expression of MDM2 protein, leading to subsequent stabilization of tumor suppressor p53. Significantly, we showed that RBB suppresses cell proliferation and sensitizes cells to DNA damage-induced apoptosis. Our data indicate that RBB is a novel transcriptional repressor and an important regulator of p53 pathway.
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| 22932785 |
SSTR2 is the functionally dominant somatostatin receptor in human pancreatic beta- and alpha-cells. |
10.1152/ajpendo.00207.2012 |
Am. J. Physiol. |
SSTR2 is the functionally dominant somatostatin receptor in human pancreatic beta- and alpha-cells.
Abstract
- Somatostatin-14 (SST) inhibits insulin and glucagon secretion by activating G protein-coupled somatostatin receptors (SSTRs), of which five isoforms exist (SSTR1-5). In mice, the effects on pancreatic β-cells are mediated by SSTR5, whereas α-cells express SSTR2. In both cell types, SSTR activation
Results in membrane hyperpolarization and suppression of exocytosis. Here, we examined the mechanisms by which SST inhibits secretion from human β- and α-cells and the SSTR isoforms mediating these effects. Quantitative PCR revealed high expression of SSTR2, with lower levels of SSTR1, SSTR3, and SSTR5, in human islets. Immunohistochemistry showed expression of SSTR2 in both β- and α-cells. SST application hyperpolarized human β-cells and inhibited action potential firing. The membrane hyperpolarization was unaffected by tolbutamide but antagonized by tertiapin-Q, a blocker of G protein-gated inwardly rectifying K⁺ channels (GIRK). The effect of SST was mimicked by an SSTR2-selective agonist, whereas a SSTR5 agonist was marginally effective. SST strongly (>70%) reduced depolarization-evoked exocytosis in both β- and α-cells. A slightly weaker inhibition was observed in both cell types after SSTR2 activation. SSTR3- and SSTR1-selective agonists moderately reduced the exocytotic responses in β- and α-cells, respectively, whereas SSTR4- and SSTR5-specific agonists were ineffective. SST also reduced voltage-gated P/Q-type Ca²⁺ currents in β-cells, but normalization of Ca²⁺ influx to control levels by prolonged depolarizations only partially restored exocytosis. We conclude that SST inhibits secretion from both human β- and α-cells by activating GIRK and suppressing electrical activity, reducing P/Q-type Ca²⁺ currents, and directly inhibiting exocytosis. These effects are mediated predominantly by SSTR2 in both cell types.
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| 22946511 |
Receptor activity-modifying protein-dependent impairment of calcitonin receptor splice variant Delta(1-47)hCT((a)) function. |
10.1111/j.1476-5381.2012.02197.x |
Br. J. Pharmacol. |
Receptor activity-modifying protein-dependent impairment of calcitonin receptor splice variant Delta(1-47)hCT((a)) function.
Abstract
- Background and purpose: Alternative splicing expands proteome diversity to GPCRs. Distinct receptor variants have been identified for a secretin family GPCR, the calcitonin receptor (CTR). The possible functional contributions of these receptor variants are further altered by their potential interactions with receptor activity-modifying proteins (RAMPs). One variant of the human CTR lacks the first 47 residues at its N terminus [Δ(1-47)hCT((a)) ]. However, very little is known about the pharmacology of this variant or its ability to interact with RAMPs to form amylin receptors. Experimental approach: Δ(1-47)hCT((a)) was characterized both with and without RAMPs in Cos7 and/or HEK293S cells. The receptor expression (ELISA assays) and function (cAMP and pERK1/2 assays) for up to six agonists and two antagonists were determined. Key
Results: Despite lacking 47 residues at the N terminus, Δ(1-47)hCT((a)) was still able to express at the cell surface, but displayed a generalized reduction in peptide potency. Δ(1-47)hCT((a)) retained its ability to interact with RAMP1 and formed a functional amylin receptor; this also appeared to be the case with RAMP3. On the other hand, its interaction with RAMP2 and resultant amylin receptor was reduced to a greater extent. Conclusions and implications: Δ(1-47)hCT((a)) acts as a functional receptor at the cell surface. It exhibits altered receptor function, depending on whether it associates with a RAMP and which RAMP it interacts with. Therefore, the presence of this variant in tissues will potentially contribute to altered peptide binding and signalling, depending on the RAMP distribution in tissues.
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| 22998630 |
Identification and characterization of the echinocandin B biosynthetic gene cluster from Emericella rugulosa NRRL 11440 |
10.1021/ja307220z. |
J Am Chem Soc |
Identification and characterization of the echinocandin B biosynthetic gene cluster from Emericella rugulosa NRRL 11440
Abstract
- Echinocandins are a family of fungal lipidated cyclic hexapeptide natural products. Due to their effectiveness as antifungal agents, three semisynthetic derivatives have been developed and approved for treatment of human invasive candidiasis. All six of the amino acid residues are hydroxylated, including 4R,5R-dihydroxy-L-ornithine, 4R-hydroxyl-L-proline, 3S,4S-dihydroxy-L-homotyrosine, and 3S-hydroxyl-4S-methyl-L-proline. We report here the biosynthetic gene cluster of echinocandin B 1 from Emericella rugulosa NRRL 11440 containing genes encoding for a six-module nonribosomal peptide synthetase EcdA, an acyl-AMP ligase EcdI, and oxygenases EcdG, EcdH, and EcdK. We showed EcdI activates linoleate as linoleyl-AMP and installs it on the first thiolation domain of EcdA. We have also established through ATP-PP(i) exchange assay that EcdA loads L-ornithine in the first module. A separate hty gene cluster encodes four enzymes for de novo generation of L-homotyrosine from acetyl-CoA and 4-hydroxyphenyl-pyruvate is found from the sequenced genome. Deletions in the ecdA, and htyA genes validate their essential roles in echinocandin B production. Five predicted iron-centered oxygenase genes, ecdG, ecdH, ecdK, htyE, and htyF, in the two separate ecd and hty clusters are likely to be the tailoring oxygenases for maturation of the nascent NRPS lipohexapeptidolactam product.
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| 23012369 |
Design, synthesis, structural and functional characterization of novel melanocortin agonists based on the cyclotide kalata B1 |
10.1074/jbc.M112.395442. |
J Biol Chem |
Design, synthesis, structural and functional characterization of novel melanocortin agonists based on the cyclotide kalata B1
Abstract
- Cyclotides are useful scaffolds to stabilize bioactive peptides.
Four melanocortin analogues of kalata B1 were synthesized. One is a selective MC4R agonist.
The analogues retain the native kalata B1 scaffold and introduce a designed pharmacological activity, validating cyclotides as protein engineering scaffolds.
A novel type of melanocortin agonist has been developed, with potential as a drug lead for treating obesity. Obesity is an increasingly important global health problem that lacks current treatment options. The melanocortin receptor 4 (MC4R) is a target for obesity therapies because its activation triggers appetite suppression and increases energy expenditure. Cyclotides have been suggested as scaffolds for the insertion and stabilization of pharmaceutically active peptides. In this study, we explored the development of appetite-reducing peptides by synthesizing MC4R agonists based on the insertion of the His-Phe-Arg-Trp sequence into the cyclotide kalata B1. The ability of the analogues to fold similarly to kalata B1 but display MC4R activity were investigated. Four peptides were synthesized using t-butoxycarbonyl peptide chemistry with a C-terminal thioester to facilitate backbone cyclization. The structures of the peptides were found to be similar to kalata B1, evaluated by Hα NMR chemical shifts. KB1(GHFRWG;23-28) had a K(i) of 29 nm at the MC4R and was 107 or 314 times more selective over this receptor than MC1R or MC5R, respectively, and had no detectable binding to MC3R. The peptide had higher affinity for the MC4R than the endogenous agonist, α-melanocyte stimulation hormone, but it was less potent at the MC4R, with an EC(50) of 580 nm for activation of the MC4R. In conclusion, we synthesized melanocortin analogues of kalata B1 that preserve the structural scaffold and display receptor binding and functional activity. KB1(GHFRWG;23-28) is potent and selective for the MC4R. This compound validates the use of cyclotides as scaffolds and has the potential to be a new lead for the treatment of obesity.
|
| 23013396 |
Simultaneous quantification of davalintide, a novel amylin-mimetic peptide, and its active metabolite in beagle and rat plasma by online SPE and LC-MS/MS |
10.4155/bio.12.184. |
Bioanalysis |
Simultaneous quantification of davalintide, a novel amylin-mimetic peptide, and its active metabolite in beagle and rat plasma by online SPE and LC-MS/MS
Abstract
- Davalintide, an investigational therapeutic peptide for the treatment of obesity, is rapidly metabolized by enzymatic cleavage of its N-terminal lysine residue to produce an active des-Lys metabolite in vivo. While a sensitive ELISA assay is available, it is unable to distinguish davalintide from its metabolite. Consequently, we developed an online SPE-LC-MS/MS method for simultaneous quantification of the drug and its active metabolite in beagle and rat plasma samples and compared the resulting pharmacokinetic profiles with those determined by ELISA.
The total concentration of active drug measured by ELISA correlated well with the total concentration of davalintide and its metabolite using online SPE-LC-MS/MS.
The technique is a viable alternative to immunochemistry-based methods for peptide quantitation in terms of sensitivity, reproducibility and specificity, and importantly, does not require developing antibody-based reagents.
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