ChemBioChem

Design of Photocontrolled RNA-Binding Peptidomimetics.

Publication Date: 2012 Feb 2 PMID: 22302640
Authors: Mart, R. J. - Wysoczanski, P. - Kneissl, S. - Ricci, A. - Brancale, A. - Allemann, R. K.
Journal: Chembiochem

Positively constrained: The first examples of photocontrolled RNA binding peptides are described. The large number of positively charged sides chains in the Rev response element (RRE) of an HIV type I targeting alpha-helix imposes constraints on the choice of azobenzene-derived crosslinker.

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Synthesis of Cyclic Peptides and Cyclic Proteins via Ligation of Peptide Hydrazides.

Publication Date: 2012 Feb 2 PMID: 22302623
Authors: Zheng, J. S. - Tang, S. - Guo, Y. - Chang, H. N. - Liu, L.
Journal: Chembiochem

Intramolecular ligation of peptide hydrazides is reported to occur readily, causing the lactamization of fully unprotected peptides in an epimerization-free manner. This method relies on the routine procedures of Fmoc solid-phase peptide synthesis. It can be used to prepare cyclic peptides and cyclic proteins under simpler, mild conditions at lower costs.

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In-Cell Solid-State NMR as a Tool to Study Proteins in Large Complexes.

Publication Date: 2012 Feb 1 PMID: 22298299
Authors: Reckel, S. - Lopez, J. J. - Lohr, F. - Glaubitz, C. - Dotsch, V.
Journal: Chembiochem

A major limitation of solution NMR is molecular tumbling, which is often too slow for detection. Here we demonstrate that solid-state NMR spectroscopy in combination with flash freezing of cells can be used to detect proteins in the cellular environment and provides information on backbone chemical shifts.

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An Enzyme Catalyzing O-Prenylation of the Glucose Moiety of Fusicoccin A, a Diterpene Glucoside Produced by the Fungus Phomopsis amygdali.

Publication Date: 2012 Jan 27 PMID: 22287087
Authors: Noike, M. - Liu, C. - Ono, Y. - Hamano, Y. - Toyomasu, T. - Sassa, T. - Kato, N. - Dairi, T.
Journal: Chembiochem

Isoprenoids form the largest family of compounds found in nature. Isoprenoids are often attached to other moieties such as aromatic compounds, indoles/tryptophan, and flavonoids. These reactions are catalyzed by three phylogenetically distinct prenyltransferases: soluble aromatic prenyltransferases identified mainly in actinobacteria, soluble indole prenyltransferases mostly in fungi, and membrane-bound prenyltransferases in various organisms. Fusicoccin A (FC A) is a diterpene glycoside produced by the plant-pathogenic fungus Phomopsis amygdali and has a unique O-prenylated glucose moiety. In this study, we identified for the first time, from a genome database of P. amygdali, a gene (papt) encoding a prenyltransferase that reversibly transfers dimethylallyl diphosphate (DMAPP) to the 6'-hydroxy group of the glucose moiety of FC A to yield an O-prenylated sugar. An in vitro assay with a recombinant enzyme was also developed. Detailed analyses with recombinant PAPT showed that the enzyme is likely to be a monomer and requires no divalent cations. The optimum pH and temperature were 8.0 and 50 degrees C, respectively. K(m) values were calculated as 0.49+/-0.037 muM for FC P (a plausible intermediate of FC A biosynthesis) and 8.3+/-0.63 muM for DMAPP, with a k(cat) of 55.3+/-3.3x10(-3) s. The enzyme did not act on representative substrates of the above-mentioned three types of prenyltransferase, but showed a weak transfer activity of geranyl diphosphate to FC P.

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Sequence Determinants Governing the Topology and Biological Activity of a Lasso Peptide, Microcin J25.

Publication Date: 2012 Jan 27 PMID: 22287061
Authors: Ducasse, R. - Yan, K. P. - Goulard, C. - Blond, A. - Li, Y. - Lescop, E. - Guittet, E. - Rebuffat, S. - Zirah, S.
Journal: Chembiochem

Microcin J25 is a potent antibacterial peptide produced by Escherichia coli AY25. It displays a lasso structure, which consists of a knot involving an N-terminal macrolactam ring through which the C-terminal tail is threaded and sterically trapped. In this study, we rationally designed and performed site-specific mutations in order to pinpoint the sequence determinants of the lasso topology. Structures of the resulting variants were analysed by a combination of methods (mass spectrometry, NMR spectroscopy, enzymatic digestion), and correlated to the antibacterial activity. The selected mutations resulted in the production of branched-cyclic or lasso variants. The C-terminal residues below the ring (Tyr20, Gly21) and the size of the macrolactam ring were revealed to be critical for both the lasso scaffold and bioactivity, while shortening the loop region (Tyr9-Ser18) or extending the C-terminal tail below the ring did not alter the lasso structure, but differentially affected the antibacterial activity. These results provide new insights for the bioengineering of antibacterial agents using a lasso peptide as template.

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A FlAsH Reporter for Protein-Dimerization Triggers.

Publication Date: 2012 Jan 25 PMID: 22278994
Authors: Stafforst, T.
Journal: Chembiochem

A FlAsH of potential: The specific binding of the biarsenical probe to the tetracysteine motif has matured as a tool for cell biology studies. Combining two such binders in one probe generates a useful reporter of protein dimerization events. The current state of art and the perspective for future developments are highlighted.

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NMR as an Effective Tool for the Structure Determination of Lasso Peptides.

Publication Date: 2012 Jan 25 PMID: 22278977
Authors: Xie, X. - Marahiel, M. A.
Journal: Chembiochem

Lasso peptides: unique structure and unusual stability: The highly ordered structures of lasso peptides comprising 16-21 amino acids render them unusually stable. The extremely neat 2D NMR spectra obtained in organic solvents makes NMR a powerful tool for determining these lasso structures.

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Redundant Pathways of Sunscreen Biosynthesis in a Cyanobacterium.

Publication Date: 2012 Jan 25 PMID: 22278966
Authors: Spence, E. - Dunlap, W. C. - Shick, J. M. - Long, P. F.
Journal: Chembiochem

Route of the sun block: According to empirical evidence, sun-screening mycosporine-like amino acids (MAAs) in Eukarya originate from the shikimic acid pathway, whereas in cyanobacteria, biosynthesis of the MAA shinorine reportedly occurs through the pentose phosphate pathway. However, gene deletion shows that the cyanobacterium Anabaena variabilis ATCC 29143 does not biosynthesise shinorine exclusively by this route.

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Discovery of the Rhizopodin Biosynthetic Gene Cluster in Stigmatella aurantiaca Sg a15 by Genome Mining.

Publication Date: 2012 Jan 25 PMID: 22278953
Authors: Pistorius, D. - Muller, R.
Journal: Chembiochem

The field of bacterial natural product research is currently undergoing a paradigm change concerning the discovery of natural products. Previously most efforts were based on isolation of the most abundant compound in an extract, or on tracking bioactivity. However, traditional activity-guided approaches are limited by the available test panels and frequently lead to the rediscovery of already known compounds. The constantly increasing availability of bacterial genome sequences provides the potential for the discovery of a huge number of new natural compounds by in silico identification of biosynthetic gene clusters. Examination of the information on the biosynthetic machinery can further prevent rediscovery of known compounds, and can help identify so far unknown biosynthetic pathways of known compounds. By in silico screening of the genome of the myxobacterium Stigmatella aurantiaca Sg a15, a trans-AT polyketide synthase/non-ribosomal peptide synthetase (PKS/NRPS) gene cluster was identified that could not be correlated to any secondary metabolite known to be produced by this strain. Targeted gene inactivation and analysis of extracts from the resulting mutants by high performance liquid chromatography coupled to high resolution mass spectrometry (HPLC-HRMS), in combination with the use of statistical tools resulted in the identification of a compound that was absent in the mutants extracts. By matching with our in-house database of myxobacterial secondary metabolites, this compound was identified as rhizopodin. A detailed analysis of the rhizopodin biosynthetic machinery is presented in this manuscript.

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A Single Active Site Mutation Inverts Stereoselectivity of 16-Hydroxylation of Testosterone Catalyzed by Engineered Cytochrome P450 BM3.

Publication Date: 2012 Jan 24 PMID: 22275147
Authors: Venkataraman, H. - de Beer, S. B. - van Bergen, L. A. - van Essen, N. - Geerke, D. P. - Vermeulen, N. P. - Commandeur, J. N.
Journal: Chembiochem

Inversion of stereoselectivity: screening of a minimal mutant library revealed a cytochrome P450 BM3 variant M01 A82W S72I capable of producing 16 alpha-OH-testosterone. Remarkably, a single active site mutation S72I in M01 A82W inverted the stereoselectivity of hydroxylation from 16 beta to 16 alpha. Introduction of S72I mutation in another 16 beta-OH-selective variant M11 V87I, also resulted in similar inversion of stereoselectivity.

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Identification of Hydrophobic Tags for the Degradation of Stabilized Proteins.

Publication Date: 2012 Jan 23 PMID: 22271667
Authors: Tae, H. S. - Sundberg, T. B. - Neklesa, T. K. - Noblin, D. J. - Gustafson, J. L. - Roth, A. G. - Raina, K. - Crews, C. M.
Journal: Chembiochem

New HyTs are a knockout: We previously reported that labeling HaloTag proteins with low molecular weight hydrophobic tags (HyTs) leads to targeted degradation of HaloTag fusion proteins. In this report, we employed a chemical approach to extend this hydrophobic tagging methodology to highly stabilized proteins by synthesizing and evaluating a library of HyTs, which led to the identification of HyT36.

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An Adaptable Luminescence Resonance Energy Transfer Assay for Measuring and Screening Protein-Protein Interactions and their Inhibition.

Publication Date: 2012 Jan 23 PMID: 22271654
Authors: Yapici, E. - Reddy, D. R. - Miller, L. W.
Journal: Chembiochem

Protein-protein interactions (PPIs) are central to biological processes and represent an important class of therapeutic targets. Here we show that the interaction between FK506-binding protein 12 fused to green fluorescent protein (GFP-FKBP) and the rapamycin-binding domain of mTor fused to Escherichia coli dihydrofolate reductase (FRB-eDHFR) can be sensitively detected (signal-to-background ratio (S/B)>100) and accurately quantified within an impure cell lysate matrix using a luminescence resonance energy transfer (LRET) assay. Ascomycin-mediated inhibition of GFP-FKBP-rapamycin-FRB-eDHFR complex formation was also detected at high S/B ratio (>80) and Z'-factor (0.89). The method leverages the selective, stable binding of trimethoprim (TMP)-terbium complex conjugates to eDHFR, and time-resolved, background-free detection of the long-lifetime (~ms) terbium-to-GFP LRET signal that indicates target binding. TMP-eDHFR labeling can be adapted to develop high-throughput screening assays and complementary, quantitative counter-screens for a wide variety of PPI targets with a broad range of affinities that may not be amenable to purification.

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Stereoselective Hydride Transfer by Aryl-Alcohol Oxidase, a Member of the GMC Superfamily.

Publication Date: 2012 Jan 23 PMID: 22271643
Authors: Hernandez-Ortega, A. - Ferreira, P. - Merino, P. - Medina, M. - Guallar, V. - Martinez, A. T.
Journal: Chembiochem

Primary alcohol oxidation by aryl-alcohol oxidase (AAO), a flavoenzyme providing H(2) O(2) to ligninolytic peroxidases, is produced by concerted proton and hydride transfers, as shown by substrate and solvent kinetic isotope effects (KIEs). Interestingly, when the reaction was investigated with synthesized (R)- and (S)-alpha-deuterated p-methoxybenzyl alcohol, a primary KIE ( approximately 6) was observed only for the R enantiomer, revealing that the hydride transfer is highly stereoselective. Docking of p-methoxybenzyl alcohol at the buried crystal active site, together with QM/MM calculations, showed that this stereoselectivity is due to the position of the hydride- and proton-receiving atoms (flavin N5 and His502 Nepsilon, respectively) relative to the alcohol Calpha-substituents, and to the concerted nature of transfer (the pro-S orientation corresponding to a 6 kcal mol(-1) penalty with respect to the pro-R orientation). The role of His502 is supported by the lower activity (by three orders of magnitude) of the H502A variant. The above stereoselectivity was also observed, although activities were much lower, in AAO reactions with secondary aryl alcohols (over 98 % excess of the R enantiomer after treatment of racemic 1-(p-methoxyphenyl)ethanol, as shown by chiral HPLC) and especially with use of the F501A variant. This variant has an enlarged active site that allow better accommodation of the alpha-substituents, resulting in higher stereoselectivity (S/R ratios) than is seen with AAO. High enantioselectivity in a member of the GMC oxidoreductase superfamily is reported for the first time, and shows the potential for engineering of AAO for deracemization purposes.

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Photocaged DNA Provides New Levels of Transcription Control.

Publication Date: 2012 Jan 23 PMID: 22271631
Authors: Ceo, L. M. - Koh, J. T.
Journal: Chembiochem

Spot lit: Photocaged nucleic acids have been used to regulate gene expression through the action of light. Whereas most methods target mRNAs, DNA decoys have recently been used to target DNA transcription by targeting specific DNA-transcription-factor interactions. This has allowed researchers to "turn-off" transcription through the action of light on caged nucleic acids for the first time.

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Base Pairing at the Abasic Site in DNA Duplexes and Its Application in Adenosine Aptasensors.

Publication Date: 2012 Jan 23 PMID: 22271570
Authors: Pang, Y. - Xu, Z. - Sato, Y. - Nishizawa, S. - Teramae, N.
Journal: Chembiochem

The binding of nucleosides to abasic site (AP site)-containing DNA duplexes (AP-DNAs) carrying complementary nucleosides opposite the AP site was investigated by thermal denaturation and isothermal titration calorimetric (ITC) experiments. Purine nucleosides show high affinities (K(d) =14.1 muM for adenosine and 41.8 muM for guanosine) for binding to the AP-DNAs, and the interactions are driven primarily by the enthalpy change, similarly to the case of DNA intercalators. In contrast, pyrimidine nucleosides do not show noticeable binding to the AP-DNAs, thus suggesting that stacking interaction at the AP site plays a key role in the binding of purine nucleosides to the AP-DNAs, as revealed by ITC measurements. Next, to apply an AP-DNA as an aptasensor for adenosine, a competitive assay between adenosine and AP-site-binding fluorescent ligand was performed. The assay employs a fluorescent ligand, riboflavin, that binds to the AP site in a DNA duplex, thereby causing fluorescence quenching. By adding adenosine to the riboflavin/AP-DNA complex, the binding of adenosine to the AP site causes release of riboflavin from the AP site, thereby resulting in restoration of riboflavin fluorescence. AP-DNAs can serve as a new class of aptasensors-a limit of detection of 0.7 muM was obtained for adenosine. In contrast to conventional aptasensors for adenosine, the present method shows high selectivity for adenosine over the other nucleotides (AMP, ADP and ATP). The method does not require covalent labelling of fluorophores, and thus it is cost-effective; finally, the method was successfully demonstrated to be applicable for the detection of adenosine in horse serum.

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The Myxobacterial Compounds Spirangien A and Spirangien M522 are Potent Inhibitors of IL-8 Expression.

Publication Date: 2012 Jan 23 PMID: 22271561
Authors: Reboll, M. R. - Ritter, B. - Sasse, F. - Niggemann, J. - Frank, R. - Nourbakhsh, M.
Journal: Chembiochem

Elevated expression of interleukin-8 (IL-8) has been implicated in inflammatory diseases, in tumor growth, and in angiogenesis. The aim of this study was to identify natural or synthetic compounds that suppress IL-8 production in response to interleukin-1 (IL-1), the natural inflammatory stimulus of the IL-8 gene. We therefore developed an IL-1-inducible cell-based screening assay by stable integration of an IL-8 reporter gene into HeLa S3 cells. The screening of heterogeneous compound libraries revealed several compounds that displayed an inhibitory effect on the reporter gene expression. Following hit validation, we focused on the most efficient compound, spirangien A, and its chemical derivate spirangien M522. Detailed analysis shows that both compounds are potent inhibitors of the endogenous IL-8 gene transcription. Furthermore, both compounds decelerate the phosphorylation and degradation of IkappaBalpha, the key regulator of the IL-1-stimulated NF-kappaB signaling pathway. Our study has identified the two spirangiens A and M522 as potent inhibitors of IL-1/NF-kappaB-mediated IL-8 gene expression.

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Effects of Single Genetic Damage in Carbohydrate-Recognizing Proteins in Mouse Serum N-Glycan Profile Revealed by Simple Glycotyping Analysis.

Publication Date: 2012 Jan 24 PMID: 22271523
Authors: Amano, M. - Hashimoto, R. - Nishimura, S. I.
Journal: Chembiochem

Gene knock-out of C-type lectin receptors expressed in dendritic cells induced significant alteration of serum N-glycans compared with that of gender-matched controls. Glycotyping analysis suggested that putative-core fucosylation is strongly influenced by differences in the dominant mechanisms after carbohydrate recognition by pattern-recognition receptors, endocytosis of ligands, or induction of cytokines/chemokines. However, the loss of galectin-9, a ligand for T-helper type 1-specific cell-surface molecule, did not affect most N-glycan profiles. Interestingly, lack of the Chst3 gene (chondroitin 6-sulfotransferase) appeared to influence markedly the expression of most N-glycans, especially highly modified glycoforms bearing multiple Neu5Gc, Fuc, and LacNAc units. In contrast, genetic mutations in B4galnt1 and B4galnt2 (GalNAc transferase, responsible for the synthesis of many gangliosides) induced no discernable alteration. These results indicate that the biosynthesis of N-glycans of serum glycoproteins can be affected not only by direct genetic mutations in the glycosyltransferases but also by changes in metabolite availability in sugar nucleotide synthesis and Golgi N-glycosylation pathways caused concertedly in whole cells, tissues, and organs by milder deficiencies in immune cell-surface lectins. Many common chronic conditions, such as autoimmunity, metabolic syndrome, and aging/dementia result.

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Chemical Biology Approaches Reveal Conserved Features of a C-Terminal Processing PDZ Protease.

Publication Date: 2012 Jan 20 PMID: 22267294
Authors: Weski, J. - Meltzer, M. - Spaan, L. - Monig, T. - Oeljeklaus, J. - Hauske, P. - Vouilleme, L. - Volkmer, R. - Boisguerin, P. - Boyd, D. - Huber, R. - Kaiser, M. - Ehrmann, M.
Journal: Chembiochem

Several proteases like the high temperature requirement A (HtrA) protein family containing internal or C-terminal PDZ domains play key roles in protein quality control in the cell envelope of Gram-negative bacteria. While several HtrA proteases have been extensively characterized, many features of C-terminal processing proteases such as tail-specific protease (Tsp) are still unknown. To fully understand these cellular control systems, individual domains need to be targeted by specific peptides acting as activators or inhibitors. Here, we describe the identification and design of potent inhibitors and activators of Tsp. Suitable synthetic substrates of Tsp were identified and served as a basis for the generation of boronic acid-based peptide inhibitors. In addition, a proteomic screen of E. coli cell envelope proteins using a synthetic peptide library was performed to identify peptides capable of amplifying Tsp's proteolytic activity. The implications of these findings for the regulation of PDZ proteases and for future mechanistic studies are discussed.

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Glycosylation Assists Binding of HIV Protein gp120 to Human CD4 Receptor.

Publication Date: 2012 Jan 20 PMID: 22266649
Authors: Wilhelm, D. - Behnken, H. N. - Meyer, B.
Journal: Chembiochem

The role of glycosylation of proteins on its binding affinity is not well understood. Even a monosaccharide (magenta) placed at a glycosylation site can significantly enhance binding of peptides to their receptor. If glycosylated, an HIV protein binds stronger and faster to its primary receptors on human cells.

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"Clicking" on the Lights To Reveal Bacterial Social Networking.

Publication Date: 2012 Jan 19 PMID: 22262670
Authors: Clevenger, K. D. - Fast, W.
Journal: Chembiochem

"No man is an island."([1]) With apologies to John Donne, the same could be said for a bacterium. The discovery of bacterial quorum sensing and its relevance to microbial ecology and pathogenesis have fueled the increasing scrutiny of the molecular mechanisms responsible for the apparent group behavior of microbes.([2]) A number of chemically diverse small molecules act as diffusible signaling molecules that regulate gene expression in a population-dependent manner. Some of these signals, such as the N-acyl-L-homoserine lactones, are produced and sensed by others in the same or closely related species, and other chemical classes of signals are used more broadly for interspecies and even interkingdom communication.([3]) As a field, the study of these microbial social networks has been termed "sociomicrobiology."([4]).

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Imaging the Sialome during Zebrafish Development with Copper-Free Click Chemistry.

Publication Date: 2012 Jan 20 PMID: 22262667
Authors: Dehnert, K. W. - Baskin, J. M. - Laughlin, S. T. - Beahm, B. J. - Naidu, N. N. - Amacher, S. L. - Bertozzi, C. R.
Journal: Chembiochem

The sialome is comprised of sialylated glycoproteins and glycolipids that play essential roles in cell-cell communication. Using azide-modified molecular precursors of sialic acids and copper-free click chemistry, we visualized the spatiotemporal dynamics of the sialome in live zebrafish embryos.

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5-Thiomannosides Block the Biosynthesis of Dolichol-Linked Oligosaccharides and Mimic Class I Congenital Disorders of Glycosylation.

Publication Date: 2012 Jan 19 PMID: 22262650
Authors: Zandberg, W. F. - Gao, N. - Kumarasamy, J. - Lehrman, M. A. - Seidah, N. G. - Pinto, B. M.
Journal: Chembiochem

In a cell-based assay for novel inhibitors, we have discovered that two glycosides of 5-thiomannose, each containing an interglycosidic nitrogen atom, prevented the correct zymogen processing of the prohormone proopiomelanocortinin (POMC) and the transcription factor sterol-regulatory element-binding protein-2 (SREBP-2) in mouse pituitary cells and Chinese hamster ovary (CHO) cells, respectively. In the case of SREBP-2, these effects were correlated with the altered N-linked glycosylation of subtilisin/kexin-like isozyme-1 (SKI-1), the protease responsible for SREBP-2 processing under sterol-limiting conditions. Further examination of the effects of these compounds in CHO cells showed that they cause extensive protein hypoglycosylation in a manner similar to type I congenital disorders of glycosylation (CDGs) since the remaining N-glycans in treated cells were complete (normal) structures. The under-glycosylation of glycoproteins in 5-thiomannoside-treated cells is now shown to be caused by the compromised biosynthesis of the dolichol-linked oligosaccharide (DLO) N-glycosylation donor, although the nucleotide sugars required for the synthesis of DLOs were neither reduced under these conditions, nor were their effects reversed upon the addition of exogenous mannose. Analysis of DLO intermediates by fluorophore-assisted carbohydrate electrophoresis demonstrated that 5-thiomannose-containing glycosides block DLO biosynthesis most likely at a stage prior to the GlcNAc(2) Man(3) intermediate, on the cytosolic face of the endoplasmic reticulum.

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Cell Interaction Study of Amyloid by Using Luminescent Conjugated Polythiophene: Implication that Amyloid Cytotoxicity Is Correlated with Prolonged Cellular Binding.

Publication Date: 2012 Jan 19 PMID: 22262644
Authors: Zako, T. - Sakono, M. - Kobayashi, T. - Sorgjerd, K. - Nilsson, K. P. - Hammarstrom, P. - Lindgren, M. - Maeda, M.
Journal: Chembiochem

Needles and noodles: Studying amyloid toxicity is important for understanding protein misfolding diseases. Using a luminescent conjugated polythiophene, we found that cell binding of nontoxic filamentous amyloids of insulin and beta2-microglobulin was less efficient than that of toxic fibrillar amyloids; this suggests a correlation between amyloid toxicity and cell binding.

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Monocillin II Inhibits Human Breast Cancer Growth Partially by Inhibiting MAPK Pathways and CDK2 Thr160 Phosphorylation.

Publication Date: 2012 Jan 17 PMID: 22253097
Authors: Wei, H. - Xu, L. - Yu, M. - Zhang, L. - Wang, H. - Wei, X. - Ruan, Y.
Journal: Chembiochem

Twenty-two beta-resorcylic acid lactones (RALs) were evaluated for cytotoxicity against human breast cancer cells to find their structure-activity relationship (SAR). Monocillin II, a trans-enone RAL without epoxy and conjugated dienone, was found to have higher activity in inhibiting tumor cell growth in both in vitro experiment and in vivo nude xenografted mice model than its analogue radicicol, an anticancer lead compound. We demonstrated for the first time that monocillin II could arrest breast cancer cell cycle in G1 phase, which might partially be the result of its inhibition effect on the phosphorylation of the Thr160 residue of cyclin dependent kinase 2 (CDK2), a key enzyme in cell-cycle regulation. Moreover, monocillin II exhibited inhibition of heat shock protein 90 (Hsp90) and depleted its target proteins, Raf-1 and A-Raf, which are involved in Ras/Raf/MEK/ERK mitogen-activated protein kinase (MAPK) pathway. Remarkably, we found that monocillin II could inhibit activation of MAPKs including ERK, JNK and p38, which might be involved in the inactivation of CDK2. These results suggest that monocillin II has potential therapeutic benefits in breast cancer prevention and intervention.

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The Importance of Peripheral Sequences in Determining the Metal Selectivity of an in Vitro-Selected Co(2+) -Dependent DNAzyme.

Publication Date: 2012 Jan 17 PMID: 22250000
Authors: Nelson, K. E. - Ihms, H. E. - Mazumdar, D. - Bruesehoff, P. J. - Lu, Y.
Journal: Chembiochem

DNAzymes are catalytically active DNA molecules that use metal cofactors for their enzymatic functions. While a growing number of DNAzymes with diverse functions and metal selectivities have been reported, the relationships between metal ion selectivity, conserved sequences and structures responsible for selectivity remain to be elucidated. To address this issue, we report biochemical assays of a family of previously reported in vitro selected DNAzymes. This family includes the clone 11 DNAzyme, which was isolated by positive and negative selection, and the clone 18 DNAzyme, which was isolated by positive selection alone. The clone 11 DNAzyme has a higher selectivity for Co(2+) over Pb(2+) compared with clone 18. The reasons for this difference are explored here through phylogenetic comparison, mutational analysis and stepwise truncation. A novel DNAzyme truncation method incorporated a nick in the middle of the DNAzyme to allow for truncation close to the nicked site while preserving peripheral sequences at both ends of the DNAzyme. The results demonstrate that peripheral sequences within the substrate binding arms, most notably the stem loop, loop II, are sufficient to restore its selectivity for Co(2+) over Pb(2+) to levels observed in clone 11. A comparison of these sequences' secondary structures and Co(2+) selectivities suggested that metastable structures affect metal ion selectivity. The Co(2+) selectivity of the clone 11 DNAzyme showed that the metal ion binding and selectivities of small, in vitro selected DNAzymes may be more complex than previously appreciated, and that clone 11 may be more similar to larger ribozymes than to other small DNAzymes in its structural complexity and behavior. These factors should be taken into account when metal-ion selectivity is required in rationally designed DNAzymes and DNAzyme-based biosensors.

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Preview: ChemBioChem 3/2012.

Publication Date: 2012 Jan 23 PMID: 22246812
Authors:
Journal: Chembiochem

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Abstracts.

Publication Date: 2012 Jan 23 PMID: 22246811
Authors:
Journal: Chembiochem

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Natural and Engineered Plasmin Inhibitors: Applications and Design Strategies.

Publication Date: 2012 Jan 11 PMID: 22238174
Authors: Swedberg, J. E. - Harris, J. M.
Journal: Chembiochem

The serine protease plasmin is ubiquitously expressed throughout the human body in the form of the zymogen plasminogen. Conversion to active plasmin occurs through enzymatic cleavage by plasminogen activators. The plasminogen activator/plasmin system has a well-established function in the removal of intravascular fibrin deposition through fibrinolysis and the inhibition of plasmin activity; this has found widespread clinical use in reducing perioperative bleeding. Increasing evidence also suggests diverse, although currently less defined, roles for plasmin in a number of physiological and pathological processes relating to extracellular matrix degradation, cell migration and tissue remodelling. In particular, dysregulation of plasmin has been linked to cancer invasion/metastasis and various chronic inflammatory conditions; this has prompted efforts to develop inhibitors of this protease. Although a number of plasmin inhibitors exist, they commonly suffer from poor potency and/or specificity of inhibition that either results in reduced efficacy or prevents clinical use. Consequently, there is a need for further development of high-affinity plasmin inhibitors that maintain selectivity over other serine proteases. This review summarises clearly defined and potential applications for plasmin inhibition. The properties of naturally occurring and engineered plasmin inhibitors are discussed in the context of current knowledge regarding plasmin structure, specificity and function. This includes design strategies to obtain the potency and specificity of inhibition in addition to controlled temporal and spatial distribution tailored for the intended use.

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Tuning the Activity of Mitochondria-Penetrating Peptides for Delivery or Disruption.

Publication Date: 2012 Jan 11 PMID: 22238158
Authors: Horton, K. L. - Pereira, M. P. - Stewart, K. M. - Fonseca, S. B. - Kelley, S. O.
Journal: Chembiochem

Mitochondrially targeted agents have the capacity to be both vehicles for the delivery of bioactive agents and mitochondrial disrupters and show promise for the treatment of various diseases. Engineering these agents to specifically accumulate or disrupt the mitochondrion is challenging, as there is a fine line between characteristics of the molecules that accomplish each task. Here, we assess the physicochemical properties governing mitochondrial matrix accumulation or membrane disruption caused by mitochondria-penetrating peptides. Increases in peptide length and hydrophobicity were uncovered as the dominant factors in deriving membrane disruptive activity. Shorter, less hydrophobic peptides did not disrupt the mitochondrial membrane, but rather accumulated in the mitochondrial matrix without interfering with cellular activity. These shorter peptides, however, can trigger cytochrome c release through activation of the permeability transition pore complex (PTPC), but only at very high concentrations. This study illustrates that the activity of a mitochondria-localizing agent can be controlled through alterations in peptide hydrophobicity and dosing concentrations.

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Colorful Calcium Sensors.

Publication Date: 2012 Jan 10 PMID: 22234942
Authors: Lindenburg, L. - Merkx, M.
Journal: Chembiochem

(R)evolution of protein-based calcium sensors: Expanding the toolbox of genetically encoded calcium sensors with new colors and traits is important for understanding calcium signaling and its relation to other intracellular pathways. Campbell and co-workers have used a new directed-evolution strategy to develop a rich palette of new sensors, including the first red-shifted, genetically encoded calcium sensor.

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Simultaneous Purification and Site-Specific Modification of Pyrroline-Carboxy-Lysine Proteins.

Publication Date: 2012 Jan 5 PMID: 22223621
Authors: Chiu, H. P. - Grunewald, J. - Hao, X. - Brock, A. - Okach, L. - Uno, T. - Geierstanger, B. H.
Journal: Chembiochem

Sticky residue: Pyrroline-carboxy-lysine (Pcl) can be readily incorporated into proteins expressed in E. coli and mammalian cells by using the pyrrolysyl tRNA/tRNA synthetase pair. Pcl can be used as a single amino acid purification tag and can be site-specifically modified with functional probes during the elution process.

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A New Concept for Glycosyltransferase Inhibitors: Nonionic Mimics of the Nucleotide Donor of the Human Blood Group B Galactosyltransferase.

Publication Date: 2012 Jan 5 PMID: 22223604
Authors: Schaefer, K. - Albers, J. - Sindhuwinata, N. - Peters, T. - Meyer, B.
Journal: Chembiochem

Glycosyltransferases play an important role in the formation of oligosaccharides and glycoconjugates. To find suitable and selective inhibitors for this class of enzymes is still challenging. Here, we describe a novel concept that allows the design of inhibitors based on the structure of the donor substrate binding pocket. As a first step we describe the design, synthesis and analysis of inhibitors of the human blood group B galactosyltransferase (GTB). This enzyme served as a model system to study the concept, which can be used for easy access of glycosyltransferase inhibitors in general. In silico docking of bicyclic heteroaromatic ligands to GTB and experimental verification of binding affinities by saturation transfer difference NMR (STD NMR) spectroscopy gave 9-N-pentityl uric acid derivatives as non-ionic mimics of UDP. Two derivatives were synthesized and showed inhibitory activity for GTB as determined by competitive STD NMR experiments and by a radiolabeled enzyme assay.

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A New Chemical Handle for Protein AMPylation at the Host-Pathogen Interface.

Publication Date: 2012 Jan 23 PMID: 22213418
Authors: Broncel, M. - Serwa, R. A. - Tate, E. W.
Journal: Chembiochem

Tagging protein AMPylation: A new chemical reporter for AMPylation, recently identified as a key post-translational modification during bacterial infection, is a robust tool for detecting and identifying AMPylated proteins in vitro.

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A general chemical ligation approach towards isopeptide-linked ubiquitin and ubiquitin-like assay reagents.

Publication Date: 2012 Jan 23 PMID: 22213387
Authors: Geurink, P. P. - El Oualid, F. - Jonker, A. - Hameed, D. S. - Ovaa, H.
Journal: Chembiochem

Thiolysine-mediated chemical ligation has generated fluorescence polarisation assay reagents based on isopeptide-linked ubiquitin-like protein conjugates. These have been used to monitor the activity of ubiquitin(-like) proteases. Thus, it is now possible to generate assay reagents that contain substrate-derived elements around the isopeptide linkage, with no practical limitation.

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Live-Cell dSTORM of Cellular DNA Based on Direct DNA Labeling.

Publication Date: 2012 Jan 23 PMID: 22213360
Authors: Benke, A. - Manley, S.
Journal: Chembiochem

We have implemented the super-resolution method of direct stochastic optical reconstruction microscopy (dSTORM) to image nuclear and mitochondrial DNA in living cells. We also demonstrate time-lapse imaging, all using a dye that associates directly with cellular DNA: the commercially available dye Picogreen.

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The thorny way to the mechanism of ribosomal Peptide-bond formation.

Publication Date: 2012 Jan 23 PMID: 22213275
Authors: Pech, M. - Nierhaus, K. H.
Journal: Chembiochem

Per aspera ad astra: Kinetic isotope effects have indicated a new and comprehensive picture of the central ribosomal enzymatic activity, peptide-bond formation. To this end, isotopes were incorporated in the positions highlighted in red.

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The Natural Product Betulinic Acid Inhibits C/EBP Family Transcription Factors.

Publication Date: 2012 Jan 23 PMID: 22213238
Authors: Hollis, A. - Sperl, B. - Graber, M. - Berg, T.
Journal: Chembiochem

Nature does it: Small-molecule inhibitors of transcription factors are highly sought after, but only a limited number are known to date. Here, we report the pentacyclic triterpenoid betulinic acid as a pan-specific inhibitor of the C/EBP transcription factor family, proteins that play prominent roles in adipogenesis and tumorigenesis.

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Terpenoids are Widespread in Actinomycetes: A Correlation of Secondary Metabolism and Genome Data.

Publication Date: 2012 Jan 23 PMID: 22213220
Authors: Citron, C. A. - Gleitzmann, J. - Laurenzano, G. - Pukall, R. - Dickschat, J. S.
Journal: Chembiochem

The genomes of all bacteria with publicly available sequenced genomes have been screened for the presence of sesquiterpene cyclase homologues, resulting in the identification of 55 putative geosmin synthases, 23 homologues of 2-methylisoborneol synthases, and 98 other sesquiterpene cyclase homologues. Most of these enzymes by far were found in actinomycetes. The terpenoid volatiles from 35 strains, including 31 actinomycetes and four strains from other taxa, were collected by using a closed-loop stripping apparatus and identified by GC-MS. All of these bacteria apart from one strain encode sesquiterpene cyclase homologues in their genomes. The identified volatile terpenoids were grouped according to structural similarities and their biosynthetic relationship, and the results of these analyses were correlated to the available genome information, resulting in valuable new insights into bacterial terpene biosynthesis.

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Functionalization of oxidases with peroxidase activity creates oxiperoxidases: a new breed of hybrid enzyme capable of cascade chemistry.

Publication Date: 2012 Jan 23 PMID: 22213198
Authors: Winter, R. T. - van den Berg, T. E. - Colpa, D. I. - van Bloois, E. - Fraaije, M. W.
Journal: Chembiochem

The covalent flavoprotein alditol oxidase (AldO) from Streptomyces coelicolor A3(2) was endowed with an extra catalytic functionality by fusing it to a microperoxidase. Purification of the construct resulted in the isolation of a synthetic bifunctional enzyme that was both fully covalently flavinylated and heminylated: an oxiperoxidase. Characterization revealed that both oxidase and peroxidase functionalities were active, with the construct functioning as a single-component xylitol biosensor. In an attempt to reduce the size of the oxidase-peroxidase fusion, we replaced portions of the native AldO sequence with the bacterial cytochrome c CXXCH heme-binding motif. By mutating only three residues of the AldO protein we were able to create a functional oxidase-peroxidase hybrid.

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Arginine Mimetics Using alpha-Guanidino Acids: Introduction of Functional Groups and Stereochemistry Adjacent to Recognition Guanidiniums in Peptides.

Publication Date: 2012 Jan 23 PMID: 22213184
Authors: Balakrishnan, S. - Scheuermann, M. J. - Zondlo, N. J.
Journal: Chembiochem

Arginine residues are broadly employed for specific biomolecular recognition, including in protein-protein, protein-DNA, and protein-RNA interactions. Arginine recognition commonly exploits the potential for bidentate electrostatic and hydrogen-bonding interactions. However, in arginine residues, the guanidinium functional group is located at the terminus of a flexible hydrocarbon side chain, which lacks the functionality to contribute to specific arginine-mediated recognition and may entropically disfavor binding. In order to enhance the potential for specificity and affinity in arginine-mediated molecular recognition, we have developed an approach to the synthesis of peptides that incorporates an alpha-guanidino acid as a novel arginine mimetic. alpha-Guanidino acids, derived from alpha-amino acids, with guanidinylation of the amino group, were incorporated stereospecifically into peptides on solid phase via coupling of an Fmoc amino acid to diaminopropionic acid (Dap), Fmoc deprotection, guanidinylation of the amine on solid phase, and deprotection, generating a peptide containing an alpha-functionalized arginine mimetic. This approach was examined by incorporating arginine mimetics into ligands for the Src, Grb, and Crk SH3 domains at the site of the key recognition arginine. Protein binding was examined for peptides containing guanidino acids derived from Gly, L-Val, L-Phe, L-Trp, D-Val, D-Phe, and D-Trp. We demonstrate that paralogue specificity and target site affinity may be modulated with the use of alpha-guanidino acid-derived arginine mimetics, generating peptides that exhibit enhanced Src specificity by selection against Grb and peptides that reverse the specificity of the native peptide ligand, with enhancements in Src target specificity of up to 15-fold (1.6 kcal mol(-1) ).

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Multifunctions of MelB, a Fungal Tyrosinase from Aspergillus oryzae.

Publication Date: 2012 Jan 23 PMID: 22213164
Authors: Fujieda, N. - Murata, M. - Yabuta, S. - Ikeda, T. - Shimokawa, C. - Nakamura, Y. - Hata, Y. - Itoh, S.
Journal: Chembiochem

The pro form of melB tyrosinase from the melB gene of Aspergillus oryzae was over-produced from E. coli and formed a homodimer that exhibited the spectral features of met-tyrosinase. In the presence of NH(2) OH (reductant), the proenzyme bound dioxygen to give a stable (mu-eta(2) :eta(2) -peroxo)dicopper(II) species (oxy form), thus indicating that the pro form tyrosinase can function as an oxygen carrier or storage protein like hemocyanin. The pro form tyrosinase itself showed no catalytic activity toward external substrates, but proteolytic digestion with trypsin activated it to induce tyrosinase activity. Mass spectroscopy analyses, mutagenesis experiments, and colorimetry assays have demonstrated that the tryptic digestion induced cleavage of the C-terminal domain (Glu458-Ala616), although the dimeric structure of the enzyme was retained. The structural changes induced by proteolytic digestion might open the entrance to the enzyme active site for substrate incorporation.

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Improvement of RNAi Activity and Strand Selectivity of RISC Formation by Modified siRNA Involving Intercalators near 5' Termini.

Publication Date: 2012 Jan 23 PMID: 22213122
Authors: Ito, H. - Urushihara, M. - Liang, X. - Asanuma, H.
Journal: Chembiochem

RISC avoidance: By introducing an intercalator such as azobenzene into the 5'-end of the sense strand at a D-threoninol linker, RNAi activity was greatly improved. This enhancement of RNAi activity was attributed to selective loading of the antisense strand into RISC and the suppression of RISC assembly with the modified sense strand.

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Incorporation of manganese complexes into xylanase: new artificial metalloenzymes for enantioselective epoxidation.

Publication Date: 2012 Jan 23 PMID: 22190469
Authors: Allard, M. - Dupont, C. - Munoz Robles, V. - Doucet, N. - Lledos, A. - Marechal, J. D. - Urvoas, A. - Mahy, J. P. - Ricoux, R.
Journal: Chembiochem

Here we report the best artificial metalloenzyme to date for the selective oxidation of aromatic alkenes; it was obtained by noncovalent insertion of Mn(III) -meso-tetrakis(p-carboxyphenyl)porphyrin [Mn(TpCPP), 1-Mn] into a host protein, xylanase 10A from Streptomyces lividans (Xln10A). Two metallic complexes-N,N'-ethylene bis(2-hydroxybenzylimine)-5,5'-dicarboxylic acid Mn(III) [(Mn-salen), 2-Mn] and 1-Mn-were associated with Xln10A, and the two hybrid biocatalysts were characterised by UV-visible spectroscopy, circular dichroism and molecular modelling. Only the artificial metalloenzyme based on 1-Mn and Xln10A was studied for its catalytic properties in the oxidation of various substituted styrene derivatives by KHSO(5) : after optimisation, the 1-Mn-Xln10A artificial metalloenzyme was able to catalyse the oxidation of para-methoxystyrene by KHSO(5) with a 16 % yield and the best enantioselectivity (80 % in favour of the R isomer) ever reported for an artificial metalloenzyme.

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Direct Observation of Metabolic Differences in Living Escherichia Coli Strains K-12 and BL21.

Publication Date: 2012 Jan 23 PMID: 22190455
Authors: Meier, S. - Jensen, P. R. - Duus, J. O.
Journal: Chembiochem

Direct observation of metabolism on a timescale of seconds shows altered usage, due to genomic differences, of one specific reaction in living E. coli K-12 and BL21. The resulting phenotypic data vary strongly when directly detecting biochemical reactions at work.

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Towards Quantitative Computer-Aided Studies of Enzymatic Enantioselectivity: The Case of Candida antarctica Lipase A.

Publication Date: 2012 Jan 23 PMID: 22190449
Authors: Frushicheva, M. P. - Warshel, A.
Journal: Chembiochem

The prospect for consistent computer-aided refinement of stereoselective enzymes is explored by simulating the hydrolysis of enantiomers of an alpha-substituted ester by wild-type and mutant Candida antarctica lipase A, using several strategies. In particular, we focused on the use of the empirical valence bond (EVB) method in a quantitative screening for enantioselectivity, and evaluate both k(cat) and k(cat) /K(M) of the R and S stereoisomers. We found that an extensive sampling is essential for obtaining converging results. This requirement points towards possible problems with approaches that use a limited conformational sampling. However, performing the proper sampling appears to give encouraging results and to offer a powerful tool for the computer-aided design of enantioselective enzymes. We also explore faster strategies for identifying mutations that will help in augmenting directed-evolution experiments, but these approaches require further refinement.

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Phosphorylation as a tool to modulate aggregation propensity and to predict fibril architecture.

Publication Date: 2012 Jan 23 PMID: 22174034
Authors: Valette, N. M. - Radford, S. E. - Harris, S. A. - Warriner, S. L.
Journal: Chembiochem

Despite the importance of post-translational modifications in controlling the solubility and conformational properties of proteins and peptides, precisely how the aggregation propensity of different peptide sequences is modulated by chemical modification remains unclear. Here we have investigated the effect of phosphorylation on the aggregation propensity of a 13-residue synthetic peptide incorporating one or more phosphate groups at seven different sites at various pH values. Fibril formation was shown to be inhibited when a single phosphate group was introduced at all seven locations in the peptide sequence at pH 7.5, when the phosphate group is fully charged. By contrast, when the same peptides were analysed at pH 1.1, when the phosphate is fully protonated, fibrils from all seven peptide sequences form rapidly. At intermediate pH values (pH 3.6) when the phosphate group is mono-anionic, the aggregation propensity of the peptides was found to be highly dependent on the position of the phosphate group in the peptide sequence. Using this information, combined with molecular dynamics (MD) simulations of the peptide sequence, we provide evidence consistent with the peptide forming amyloid fibrils with a class 7 architecture. The results highlight the potential utility of phosphorylation as a method of reversibly controlling the aggregation kinetics of peptide sequences both during and after synthesis. Moreover, by exploiting the ability of the phosphate group to adopt different charge states as a function of pH, and combining experimental insights with atomistic information calculated from MD simulations as pH is varied, we show how the resulting information can be used to predict fibril structures consistent with both datasets, and use these to rationalise their sensitivity of fibrillation kinetics both to the location of the phosphate group and its charge state.

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Synthesis and evaluation of a fluorescent non-peptidic cholecystokinin-b/gastrin receptor specific antagonist for cancer cell imaging.

Publication Date: 2012 Jan 23 PMID: 22162268
Authors: Kumari, S. - Chowdhury, J. - Mishra, A. K. - Chandna, S. - Saluja, D. - Chopra, M.
Journal: Chembiochem

Fluorescent labeling has enabled a better understanding of the relationships between receptor location, function, and life cycle. Each of these perspectives contributes new insights into drug action, particularly for G protein-coupled receptors (GPCRs). The aim of this study was to develop a fluorescein derivative, FLUO-QUIN-a novel antagonist of the cholecystokinin-B/gastrin receptor. A radioligand-binding experiment revealed an IC(50) of 4.79 nm, and the antagonist inhibited gastric acid secretion in an isolated lumen-perfused mouse stomach assay (up to 51 % at 100 nm). The fluorescence properties altered upon binding to the receptor, and the fluorophore was quenched to a greater extent when free than in the bound form. FLUO-QUIN specifically bound to human pancreatic carcinoma cells, MiaPaca-2, which are known to express the receptor, as evidenced by rapid clustering followed by time-dependent receptor internalization. This proves the stability of FLUO-QUIN and its ability to penetrate vesicular membranes and reach various cell targets. Hence it might be used as an agent for the detection of CCK-B-receptor-positive tumors by fluorescence imaging.

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Cloning and Heterologous Expression of Three Type II PKS Gene Clusters from Streptomyces bottropensis.

Publication Date: 2012 Jan 23 PMID: 22162248
Authors: Yan, X. - Probst, K. - Linnenbrink, A. - Arnold, M. - Paululat, T. - Zeeck, A. - Bechthold, A.
Journal: Chembiochem

Mensacarcin is a potent cytotoxic agent isolated from Streptomyces bottropensis. It possesses a high content of oxygen atoms and two epoxide groups, and shows cytostatic and cytotoxic activity comparable to that of doxorubicin, a widely used drug for antitumor therapy. Another natural compound, rishirilide A, was also isolated from the fermentation broth of S. bottropensis. Screening a cosmid library of S. bottropensis with minimal PKS-gene-specific primers revealed the presence of three different type II polyketide synthase (PKS) gene clusters in this strain: the msn cluster (mensacarcin biosynthesis), the rsl cluster (rishirilide biosynthesis), and the mec cluster (putative spore pigment biosynthesis). Interestingly, luciferase-like oxygenases, which are very rare in Streptomyces species, are enriched in both the msn cluster and the rsl cluster. Three cosmids, cos2 (containing the major part of the msn cluster), cos3 (harboring the mec cluster), and cos4 (spanning probably the whole rsl cluster) were introduced into the general heterologous host Streptomyces albus by intergeneric conjugation. Expression of cos2 and cos4 in S. albus led to the production of didesmethylmensacarcin (DDMM, a precursor of mensacarcin) and the production of rishirilide A and B (a precursor of rishirilide A), respectively. However, no product was detected from the expression of cos3. In addition, based on the results of isotope-feeding experiments in S. bottropensis, a putative biosynthesis pathway for mensacarcin is proposed.

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Interaction of a tat substrate and a tat signal Peptide with thylakoid lipids at the air-water interface.

Publication Date: 2012 Jan 23 PMID: 22114060
Authors: Kerth, A. - Brehmer, T. - Meister, A. - Hanner, P. - Jakob, M. - Klosgen, R. B. - Blume, A.
Journal: Chembiochem

The Tat machinery enables folded proteins to be translocated across biological membranes. In vitro studies have shown that Tat substrates can interact with membranes prior to translocation. In this study we investigated the initial states of this interaction with thylakoid lipid monolayers at the air-water interface by using monolayer techniques combined with infrared reflection-absorption spectroscopy (IRRAS). We used enhanced green fluorescent protein (EGFP) as a model substrate and the signal peptide SP16 from the 16 kDa protein of the spinach oxygen-evolving complex (OEC16). We found that the signal peptide is essential for the interaction of the model substrate with lipid monolayers. IRRA spectroscopy showed an increased amount of alpha-helical secondary structure elements for the chimeric model substrate i16/EGFP (SP16 fused to EGFP) compared with EGFP; this can be attributed to the signal peptide.

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