Photochemical mechanism of an atypical algal phytochrome ChemBioChem (IF 2.847) Pub Date : 2018-03-15 Nigel Scrutton, Uzma Choudry, Derren Heyes, Samantha Hardman, Michiyo Sakuma, Igor Sazanovich, Joyce Woodhouse, Eugenio De La Mora, Martin Pederson, Michael Wulff, Martin Weik, Giorgio Schiro
Phytochromes are bilin-containing photoreceptors that are typically sensitive to the red / far-red region of the visible spectrum. Recently phytochromes from certain eukaryotic algae have become attractive targets for optogenetic applications because of their unique ability to respond to multiple wavelengths of light. Here, we have used a combination of time-resolved spectroscopy and structural approaches across picosecond to second timescales to map photochemical mechanisms and structural change in this atypical group of phytochromes. The photochemistry of an orange / far-red light sensitive algal phytochrome from Dolihomastix tenuilepis has been investigated using a combination of visible, infra-red and X-ray scattering probes. The entire photocycle, correlated with accompanying structural changes in the cofactor / protein, are reported. Our study identifies a complex photocycle for this atypical phytochrome. It also highlights a need to combine outcomes from a range of biophysical approaches to unravel complex photochemical and macromolecular processes in multi-domain photoreceptor proteins that are the basis of biological light-mediated signaling.
Enzymatic Synthesis of Lignin-Based Concrete Dispersing Agents ChemBioChem (IF 2.847) Pub Date : 2018-03-15 Dagmara Jankowska, Tobias Heck, Mark Schubert, Alpaslan Yerlikaya, Christophe Weymuth, Daniel Rentsch, Irene Schober, Michael Richter
Lignin is the most abundant aromatic biopolymer functioning as an integral compound of woody materials. In its unmodified form it shows limited water solubility and is relatively unreactive. Thus, biotechnological lignin valorisation for high performance applications is highly underexploited. To expand its application, a new synthesis route towards novel concrete dispersing agents was developed. The route relies on a lignin functionalization by enzymatic transformation. Screening of lignin modifying systems resulted in functionalized lignin polymers with improved solubility in aqueous systems. Due to grafting of sulfanilic acid or p-aminobenzoic acid by fungal laccases, lignin became soluble in water at pH≤4 or pH≤7, respectively. Products were analysed and evaluated in miniaturized application tests in cement paste and mortar. Their dispersing properties match performance criteria of commercially available lignosulfonates. The study exemplifies new perspectives for the use of lignin.
Dual Maleimides Tagging for Relative and Absolute Quantitation of Cysteine-Containing Peptides by MALDI-TOF MS ChemBioChem (IF 2.847) Pub Date : 2018-03-15 Jian Liu, Fuzhong Ouyang, Zhihao Zhao, Ruifang Gao, Rui Shi, Enhui Wu, Rui Lv, Guoqiang Xu
A dual maleimides (DuMal) tagging method has been developed for both relative and absolute quantitation of cysteine-containing peptides (CCPs) in combination with MALDI-TOF mass spectrometry. We choose a pair of maleimides with the minimal difference in their chemical structures, including N-Methylmaleimide (NMM) and N-Ethylmaleimide (NEM), which allow for tagging CCPs in the Michael Addition reaction with a high efficiency rapidly (~minutes). We have validated that the DuMal Tagging technique is sensitive and reliable in quantitative analysis of CCPs. Absolute quantitation of CCPs can be achieved with the detection limit as low as 7.3 nM. Relative quantitation of CCPs can be performed in various sample mixtures with consistent results of the ratio (CV < 5%). The DuMal Tagging technique provides a sensitive and accurate approach for the quantitation of biomolecules containing thiol reactive sites, thus promising protein detection, disease diagnosis, and biomarker discovery associated with post-translational modifications of cysteines.
Protein-Polymer Microcapsule for PCR Technology ChemBioChem (IF 2.847) Pub Date : 2018-03-14 Zhijun Chen, Xiaoliang Wang, Yang Liu, Jiao Liu
Protein-polymer microcapsules attracted much attention due to their special feature and potential in biological use. How to make the most of this type of bio-abiotic hybrid material is an intriguing question. Nevertheless, several unsatisfactory technical issues significantly limited the application of these materials. For instance, introducing various biomolecules and cross-linking for the capsules remain challenging and problematic. In this report, recombinant mCherry protein was covalently linked with PNIPAAm to form amphiphilic protein-polymer conjugates, assembled into microcapsules. These microcapsules were thermoresistant and can be used for polymerase chain reaction (PCR) reaction. In this setting, the reactant molecules can be readily-easily introduced into the microcapsules and cross-linking and water-oil phase transition are not obligatory. This protein-polymer microcapsule granted PCR system has potential in various biological applications.
Nanodisc-targeted STD NMR reveals atomistic details of ligand binding to lipid environments ChemBioChem (IF 2.847) Pub Date : 2018-03-14 Anthony Watts
Saturation transfer difference (STD) NMR constitutes one of the most popular ligand-based NMR techniques for the study of protein−ligand interactions. This is due to its robustness and the fact that it is focused on the signals of the ligand, without any need for NMR information of the macromolecular target. This technique is most commonly applied to systems involving different types of ligands (e.g. small organic molecules, carbohydrates, or lipids) and a protein as the target, where the latter is selectively saturated. However, only a few examples have been reported where membrane mimetics are the macromolecular binding partners. Here, we have employed STD-NMR to investigate the interactions of the neurotransmitter dopamine to mimetics of lipid bilayers, such as nanodiscs, by saturation of the latter. In particular, the interactions between dopamine and model lipid nanodiscs formed from charged and zwitterionic lipids have been resolved at the atomic level. The results, in agreement with previous isothermal titration calorimetry (ITC) studies, show that dopamine preferential binds to negatively charged model membranes, but also provides detailed atomistic insights into the mode of interaction of dopamine to membrane mimetics. Our findings provide relevant structural information for the design of lipid-based drug carriers of dopamine, structural analogues, and are of generic applicability to other systems.
Chloro-functionalized photo-crosslinking BODIPY for glutathione sensing and sub-cellular trafficking ChemBioChem (IF 2.847) Pub Date : 2018-03-14 Dhiraj P. Murale, Seong Cheol Hong, Md. Mamunul Haque, Jun-Seok Lee
Glutathione (GSH) is one of major anti-oxidant inside cells that regulates oxido-reduction homeostasis. Recently, there have been extensive efforts to visualize GSH in live cells, but most of the probes available today are simple detection sensor without detail cellular localization. Here, we have developed a new fluorescent probe (pcBD2-Cl), which are cell permeable and selectively react with GSH in-situ. The in-situ GSH labeled probe (pcBD2-GSH) exhibited quenched fluorescence, but subsequent binding to cellular abundant Glutathione S-transferase (GST) recovers the fluorescence intensity that makes possible to imaging GSH-GST complex in live cells. Interactions between probe and GST was confirmed using photo- crosslinking in intact live cell conditions. Interestingly, we found isomers of chloro-functionalized BODIPY compounds behaved very distinctively inside the cells. Following co-staining imaging with MitoTracker and mitochondria fractionation upon LPS mediated ROS induction experiment showed pcBD2-GSH was accumulated in mitochondria. This is the first example of live cell imaging probe that can visualize translocation of glutathione from cytosol to mitochondria.
A RNA-cleaving Catalytic DNA Accelerated by Freezing ChemBioChem (IF 2.847) Pub Date : 2018-03-14 Tianmeng Yu, Wenhu Zhou, Juewen Liu
The EtNa DNAzyme was isolated during the isopropanol precipitation step of an in vitro selection effort. Although inactive with the intended cofactor, its RNA cleavage activity was observed under a few conditions. With Na+, EtNa is highly active in around 50% ethanol, while in water it is highly active with Ca2+. In this work, we show that the EtNa DNAzyme is accelerated by freezing in water in the presence of Na+. The apparent Kd reached 6.2 mM Na+ under the frozen condition, over 20-fold tighter than that in water at room temperature. With 10 mM Na+, the EtNa has a cleavage rate of 0.12 h-1 after freezing at -20 °C. This effect is unique to EtNa, while all the other tested DNAzymes were inhibited by freezing except for the Na+-specific NaA43. Freezing also inhibited the EtNa if Ca2+ was used. We attribute this to the concentration of EtNa and Na+ in the micropockets between ice crystals, but divalent metals may misfold DNA. Overall, we have systematically studied the effect of freezing on the RNA-cleavage activity of DNAzymes. The DNAzyme sequence and the metal ion species are both crucial to determine the effect of freezing.
A Chimeric Styrene Monooxygenase with Increased Efficiency in Asymmetric Biocatalytic Epoxidation ChemBioChem (IF 2.847) Pub Date : 2018-03-12 Maria L. Corrado, Tanja Knaus, Francesco G. Mutti
Similar but not the same: First Kinetic and Structural Analyses of a Methanol Dehydrogenase Containing a Europium Ion in the Active Site ChemBioChem (IF 2.847) Pub Date : 2018-03-10 Bérénice Jahn, Arjan Pol, Henning Lumpe, Thomas Barends, Andreas Dietl, Carmen Hogendoorn, Huub Op den Camp, Lena Daumann
Since the discovery of the biological relevance of rare earth elements (REE) for numerous different bacteria, the questions of the advantage of REE in the active site of methanol dehydrogenase (MDH) over calcium(II) and why bacteria prefer light REE have been a subject of debate. Here we report the cultivation and purification of the strictly REE-dependent methanotrophic bacterium Methyl-acidiphilum fumariolicum SolV with europium(III) as well as structural and kinetic analyses of the first Eu-substituted methanol dehydrogenase. Crystal structure determination of the Eu-MDH demonstrated that overall no major structural changes were induced by converting to this REE. Circular Dichroism (CD) measurements were used to determine optimal conditions for kinetic assays and inductively-coupled plasma mass-spectrometry (ICP-MS) showed 70% incorporation of Eu in the enzyme. Our studies explain why bacterial growth of SolV with Eu3+ is significantly slower than with La3+/Ce3+/Pr3+: Eu-MDH possesses a decreased catalytic efficiency and affinity for the substrate. Although rare earth elements have similar properties, the differences in ionic radii and coordination numbers across the series significantly impact MDH efficiency.
Deletion of the C-26 Methyl Substituent from the Bryostatin Analogue Merle 23 has Negligible Impact on Its Biological Profile or Potency ChemBioChem (IF 2.847) Pub Date : 2018-03-08 Xiguang Zhao, Noemi Kedei, Aleksandra M. Michalowski, Nancy E Lewin, Gary E. Keck, Peter M. Blumberg
Important strides are being made in understanding the structural features of bryostatin 1, a candidate therapeutic agent for cancer and dementia, conferring its potency for protein kinase C and the unique spectrum of biological responses which it induces. A critical pharmacophoric element in bryostatin 1 is a secondary hydroxyl, whereas a primary hydroxyl group plays the analogous role in binding of the phorbol esters to protein kinase C. Here, we describe the synthesis of a bryostatin homolog where the hydroxyl group is primary, as in the phorbol esters, and show that its biological activity is almost indistinguishable from that of the corresponding compound with the secondary hydroxyl group.
An Unusual FAAL-ACP Didomain in Ambruticin Biosynthesis ChemBioChem (IF 2.847) Pub Date : 2018-03-08 Franziska Hemmerling, Karen Lebe, Johannes Wunderlich, Frank Hahn
The divinylcyclopropane (DVC) fragment of the ambruticins is proposed to be formed via a unique polyene cyclization mechanism in which the unusual didomain AmbG plays a key role. It is proposed to activate the branched thioester carboxylic acid resulting from the polyene cyclisation and to transfer it to its associated ACP. After oxidative decarboxylation, the intermediate is channeled back into polyketide synthase (PKS) processing. AmbG was previously annotated as an adenylation-thiolation didomain with a very unusual substrate selectivity code, but has not yet been biochemically studied. Based on sequence and homology model analysis, we reannotate AmbG as a fatty acyl:adenylate ligase-acyl carrier protein didomain with unusual substrate specificity. The expected adenylate-forming activity on fatty acids was confirmed by in vitro studies. AmbG also adenylates a number of structurally diverse carboxylic acids, including functionalized fatty acids, unsaturated and aromatic carboxylic acids. HPLC-MS analysis and competition experiments showed that AmbG preferentially acylates its ACP with long-chain hydrophobic acids and tolerates a p-system and a branch near the carboxylic acid. AmbG is the first characterized example of an FAAL-ACP didomain that is centrally located in a PKS and apparently activates a polyketidic intermediate. This is an important step towards deeper biosynthetic studies such as a partial reconstitution of the ambruticin pathway to elucidate DVC formation.
Rigid Peptide Macrocycles via On-Resin Glaser Stapling ChemBioChem (IF 2.847) Pub Date : 2018-03-07 Philip A. Cistrone, Anthony P. Silvestri, Jordi C. J. Hintzen, Philip E. Dawson
Peptide macrocycles are widely utilized in the development of high affinity ligands including stapled α-helices. The linear rigidity of a 1,3-diynyl linkage provides an optimal distance (7 Å) between β- carbons of the i, i+4 amino acid side chains, suggesting its utility in stabilizing α-helical structures. Here we report the development of an on-resin strategy for an intramolecular Glaser reaction between two alkyne-terminated side chains using copper chloride, an essential bpy-diol ligand, and diisopropylethylamine, at room temperature. The efficiency of this ligation is illustrated by the synthesis of (i, i+4), (i, i+5), (i, i+6), and (i, i+7)-stapled BCL-9 α-helical peptides using the unnatural amino acid propargyl serine. Overall, this procedurally-simple method relies on inexpensive and widely available reagents to generate low molecular weight 23-, 26-, 29-, and 32-membered peptide macrocycles.
The ULTIMATE Reagent: A Universal Photo-Cleavable & Clickable Reagent for the Regiospecific and Reversible End Labeling of Any Nucleic Acid ChemBioChem (IF 2.847) Pub Date : 2018-03-07 Piyush K. Jain, Simon H. Friedman
There is a need for methods to chemically incorporate photocleavable labels into synthetic and biologically sourced nucleic acids in a chemically defined and reversible manner. We have previously demonstrated that the light cleaved diazo-DMNPE group (diazo di-methoxy nitro phenyl ethyl) has a remarkable regiospecificity for modifying terminally phosphorylated siRNA. Building on this observation, we have identified conditions by which a new diazo-DMNPE reagent (diazo-DMNPE-azide or DDA) is able to singly modify any nucleic acid (RNA, DNA, single stranded, double stranded, 3' or 5' phosphate). It can then be modified with any clickable reagent to incorporate arbitrary labels such as fluorophores into the nucleic acid. Finally, native nucleic acid can be regenerated directly through photolysis of the reagent. Use of the described approach should allow for the tagging of any nucleic acid, from any source, natural or unnatural, while allowing for the light-induced regeneration of native nucleic acid.
Probing the outstanding local hydrophobicity increase of peptide sequences induced by trifluoromethylated amino acids incorporation ChemBioChem (IF 2.847) Pub Date : 2018-03-07 Charlène Gadais, Emmanuelle Devillers, Vincent Gasparik, Evelyne Chelain, Julien Pytkowicz, Thierry Brigaud
In order to accurately probe the local hydrophobicity increase of peptide sequences by trifluoromethylated amino acids (TfmAAs) incorporation, the chromatographic hydrophobicity indexes (φ0) of three series of tripeptides containing three unnatural trifluoromethylated amino acids have been measured and compared to the indexes of their non-fluorinated analogs. The hydrophobic contribution of each fluorinated amino acids was quantified by varying the position and the protection of (R) and (S)-α-trifluoromethyl alanine (TfmAla), (S)-trifluoromethyl cysteine (TfmCys) and (L)-trifluoromethionine (TFM) in a short peptide sequence. As a general trend, a strong increase of hydrophobicity was precisely measured even exceeding the high hydrophobic contribution of the natural isoleucine amino acid. This study validates the incorporation of trifluoromethylated amino acids into peptide sequences as a rational strategy for the fine tuning of hydrophobic peptide-protein interactions.
Tracking Down a Novel Steroid Hydroxylating Promiscuous Cytochrome P450, CYP154C8 from Streptomyces sp. W2233-SM ChemBioChem (IF 2.847) Pub Date : 2018-03-07 Bikash Dangi, Ki-Hwa Kim, Sang-Ho Kang, Tae-Jin Oh
CYP154C8 from Streptomyces sp. was identified as a novel cytochrome P450 with substrate flexibility to different sets of steroids. The in vitro reaction of these steroids with CYP154C8 revealed interesting product formation patterns with the same group of steroids. Nuclear magnetic resonance study revealed the major product of corticosterone hydroxylated at the C21 position; while progesterone, androstenedione, testosterone, and 11-ketoprogesterone were exclusively hydroxylated at 16α position. However, the 16α-hydroxylated product of progesterone was further hydroxylated to yield dihydroxylated products. 16-hydroxyprogesterone was hydroxylated at two positions yielding 2α,16α-dihydroxyprogesterone and 6β,16α-dihydroxyprogesterone. To our knowledge, this is the first report of generation of such products in enzymatic hydroxylation by CYP450. Considering the importance of modified steroids as pharmaceutical components, CYP154C8 has immense potential to be utilized in bioproduction of the hydroxylated derivative compounds directly employed for pharmaceutical applications.
Improving the activity and stability of human galactokinase for therapeutic and biotechnological applications ChemBioChem (IF 2.847) Pub Date : 2018-03-05 Margaret McAuley, Noel Mesa-Torres, Aisling McFall, Sarah Morris, Meilan Huang, Angel Pey, David J Timson
Galactokinase catalyses the site- and stereospecific phosphorylation of α-D-galactose. As such it has attracted interest as a biocatalyst for the introduction of phosphate groups into monosaccharides. However, attempts to broaden the substrate range of human galactokinase have generally resulted in substantially reduced activity. The enzyme also has biotechnological potential in enzyme replacement therapy (ERT) for type II galactosemia. The return-to-consensus approach can be used to identify residues which can be altered to increase protein stability and enzyme activity. This approach identified six residues of potential interest in human galactokinase. Some of the single consensus variants increased the catalytic turnover of the enzyme (M60V, D268E, A334S and G373S), but none resulted in improved stability. When all six changes were introduced into the protein (M60V/M180V/D268E/A334S/R366Q/G373S) thermal stability was increased. Molecular dynamics simulations suggested that these changes altered the protein's conformation at key sites. The number of salt bridges and hydrogen bonds was also increased. Combining the six consensus variations with Y379W (a variant with greater substrate promiscuity) increased the stability of this variant and its turnover towards some substrates. Thus, the six consensus variants can be used to stabilise catalytically interesting variants of human galactokinase and may also be useful if the protein were to be used in ERT.
Heat Shock Proteins Revisited: Using a Mutasynthetically Generated Reblastatin Library to Compare the Inhibition of Human and Leishmania Hsp90s ChemBioChem (IF 2.847) Pub Date : 2018-03-05 Sona Mohammadi-Ostad-Kalayeh, Frank Stahl, Thomas Scheper, Klaus Kock, Christian Herrmann, Fernanda Aparecida Heleno Batista, Júlio César Borges, Florenz Sasse, Simone Eichner, Jekaterina Ongouta, Carsten Zeilinger, Andreas Kirschning
The N-terminal domain of pullulanase from Anoxybacillus sp. WB42 modulates enzyme specificity and thermostability ChemBioChem (IF 2.847) Pub Date : 2018-03-01 Jianfeng Wang, Zhongmei Liu, Zhemin Zhou
Anoxybacillus sp. WB42 pullulanase (PulWB42) is a novel thermophilic amylopullulanase which is assigned to the glycoside hydrolase family 13 subfamily 14 (GH13_14) type I pullulanases in the carbohydrate-active enzymes database. Its N-terminal domain (Met1-Phe101) was identified as the carbohydrate-binding module 68 (CBM68) via homology modeling. The N-domain-deleted PulWB42 exhibited an equivalent Km for pullulan and significant decreases in pullulytic activity, amylose selectivity, and thermostability as compared to PulWB42 having high alpha-amylase-to-pullulanase activity ratio. Furthermore, the replacement of Ala90 or Arg93 significantly changed the substrate specificity and catalytic efficiency of PulWB42, while Q87A, L173D, and H5A/R6A/T7A showed improvements in thermostability and changes in catalytic kinetics. Therefore, the N-domain of PulWB42 is not essential for catalysis, but it does modulate enzyme catalysis, especially with respect to substrate specificity. The modulation was achieved mainly by the Leu86-Arg93 segment adjacent to the CBM48 domain and the catalytic domain A in the modelled structure of PulWB42.
Alkoxy Tetrazine Substitution at a Boron Center: A Strategy for Synthesizing Highly Fluorogenic Hydrophilic Probes ChemBioChem (IF 2.847) Pub Date : 2018-02-28 Min Wu, Xiaoai Wu, Yayue Wang, Lei Gu, Jiao You, Haoxing Wu, Ping Feng
New 4-Amino-1,2,3-Triazole Inhibitors of Indoleamine 2,3-Dioxygenase Form a Long-Lived Complex with the Enzyme and Display Exquisite Cellular Potency ChemBioChem (IF 2.847) Pub Date : 2018-02-28 Julie Anne Christine Alexandre, Michael Kenneth Swan, Mike John Latchem, Dean Boyall, John Robert Pollard, Stuart Wynn Hughes, James Westcott
Photomodulating Gene Expression by Using Caged siRNAs with Single-Aptamer Modification ChemBioChem (IF 2.847) Pub Date : 2018-02-28 Liangliang Zhang, Changmai Chen, Xinli Fan, Xinjing Tang
Mannose-Decorated Multicomponent Supramolecular Polymers Trigger Effective Uptake into Antigen-Presenting Cells ChemBioChem (IF 2.847) Pub Date : 2018-02-27 David Straßburger, Natascha Stergiou, Moritz Urschbach, Hajime Yurugi, Daniel Spitzer, Dieter Schollmeyer, Edgar Schmitt, Pol Besenius
We describe a modular route to prepare functional self-assembling dendritic peptide amphiphiles decorated with mannosides (Man) to effectively target antigen-presenting cells such as macrophages. The monomeric building blocks were equipped with tetraethylene glycols (TEG) or labelled with a Cy3 fluorescent probe. Experiments on the uptake of the multifunctional supramolecular particles into murine macrophages (Mϕs) were monitored by confocal microscopy and fluorescence-activated cell sorting (FACS). Mannose-decorated supramolecular polymers trigger a significantly higher cellular uptake and distribution, compared to TEG carrying bare polymers. No cytotoxicity or negative impact on cytokine production of the treated Mϕs was observed, emphasizing their biocompatibility. The modular nature of the multicomponent supramolecular polymer co-assembly protocol is a promising platform to develop fully synthetic multifunctional vaccines, for example in cancer immunotherapy.
Structural Characterization of i-Motif Structure in the Human Acetyl-CoA Carboxylase 1 Gene Promoters and their Role in the Regulation of Gene Expression ChemBioChem (IF 2.847) Pub Date : 2018-02-27 K Muniyappa, Mangesh Hanumant Kaulage, Santanu Bhattacharya
The G/C-rich sequences within the promoters (PI and PII) of human acetyl-CoA carboxylase 1 (ACC1) gene play a vital role in determining hormone- or diet-inducible expression of ACC1. The PI and PII promoters contain consecutive runs of 3 and 3-5 G/C base pairs respectively. Typically, stretches of C-rich sequences that coexist with stretches of guanines have the capacity to form another four-stranded secondary structure known as an i-motif. However, studies on i-motif structure are limited and its functional significance is unclear. In the current study, using a combination of different techniques, we demonstrate that C-rich single-stranded DNA derived from ACC1 promoters I and II form intramolecular i-motif structures and affect normal DNA metabolic processes. Additionally, the C-rich strands of promoter I and II in duplex DNA adopt i-motif conformation in crowded solution environments at neutral pH. Notably, i-motif forming sequences of PI and PII promoters suppressed luciferase gene transcription in HeLa cells. Further, substitution of a nucleotide sequence that has no potential to form i-motif structure increases luciferase gene expression. These results support the idea that C-rich sequences within ACC1 PI and PII promoters can form intramolecular i-motif structures, cause suppression of transcription thus revealing functional significance of C-rich sequences in the regulation of ACC1 gene expression.
Efficient synthesis of light-triggered circular antisense oligonucleotides targeting cellular protein expression ChemBioChem (IF 2.847) Pub Date : 2018-02-25 Linlin Yang, Hyun-Bum Kim, Jai-Yoon Sul, Sean B. Yeldell, James H. Eberwine, Ivan J. Dmochowski
Light-activated ("caged") antisense oligonucleotides are powerful molecules for regulating gene expression at sub-micron spatial resolution, through the focal modulation of endogenous cellular processes. Cyclized caged oligos are particularly promising structures based on their inherent stability and similarity to naturally occurring circular DNA and RNA molecules. Here we introduce an efficient route for cyclizing an antisense oligodeoxynucleotide incorporating a photocleavable linker. Oligo cyclization was achieved for several sequences in nearly quantitative yield through intramolecular copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). Caging stability and light activation were characterized by FRET efficiency, denaturing gel assay and melting temperature measurement. Finally, a cyclized caged oligo was designed to target gfap, which gave 10-fold reduction in GFAP protein upon photoactivation in astrocytes.
Spatial Control of Inducible Cre-Mediated Recombination Using Cyanine Photocages ChemBioChem (IF 2.847) Pub Date : 2018-02-22 Alexander Gorka, Tsuyoshi Yamamoto, Jianjian Zhu, Martin Schnermann
Optical control over protein expression could provide a means to interrogate a range of biological processes. One approach has employed caged ligands of the estrogen receptor (ER) in combination with broadly used ligand-dependent Cre recombinase proteins. Existing approaches used UV or blue wavelengths, which hinders their application in tissue settings. Additionally, issues of payload diffusion can impede fine spatial control over the recombination process. Here we detail the chemical optimization a near-infrared (NIR) light-activated variant of the ER antagonist cyclofen. These studies result in modification of both the caging group and payload with lipophilic ¬n-butyl esters. The appendage of esters to the cyanine cage improves cellular uptake and retention. The installation of a 4-piperidyl ester enables high spatial resolution of the light-initiated Cre-mediated recombination event. These studies describe chemical modifications with potentially general utility for improving spatial control of intracellular caging strategies. Additionally, these efforts will enable future applications that use these molecules in complex physiological settings.
Tracking the oxygen status in the cell nucleus by using a Hoechst−tagged phosphorescent ruthenium complex ChemBioChem (IF 2.847) Pub Date : 2018-02-21 Kazuhito Tanabe, Daiki Hara, Yui Umehara, Aoi Son, Wataru Asahi, Sotaro Misu, Ryohsuke Kurihara, Teruyuki Kondo
Molecular oxygen in living cells is distributed and consumed inhomogeneously, depending on the activity of each organelle. Therefore, tractable methods that can be used to monitor the oxygen status in each organelle are needed to understand cellular function. Here, we report the design of a novel oxygen-sensing probe for use in the cell nucleus. We prepared Ru−Hoechsts, which consist of a phosphorescent ruthenium complex linked to a Hoechst 33258 moiety, and characterized their properties as oxygen sensors. The Hoechst unit shows high DNA−binding properties in the nucleus, and the ruthenium complex shows oxygen−dependent phosphorescence. Thus, Ru−Hoechsts accumulated in the cell nucleus and shows oxygen−dependent signals that could be monitored. Among the Ru−Hoechsts prepared in this study, Ru−Hoechst b, in which the ruthenium complex and Hoechst unit were linked by a hexyl chain, showed the most suitable properties for monitoring the oxygen status. Ru−Hoechsts are novel probes with high potential for visualizing oxygen fluctuations in the nucleus.
MSN-on-a-Chip: Cell-Based Screenings Made Possible on a Small Molecule Microarray of Native Natural Products ChemBioChem (IF 2.847) Pub Date : 2018-02-21 Shao Q. Yao, Bo Peng, Changmin Yu, Shubo Du, Si Si Liew, Xin Mao, Peiyan Yuan, Zhenkun Na
Standard small molecule microarray (SMM) is not well-suited for cell-based screening assays. Of the few attempts made thus far to render SMM cell-compatible, all were met with major limitations. We report herein the first mesoporous silica nanoparticle (MSN)-on-a-chip platform capable of conducting high-throughput cell-based screening on SMM. By making use of a glass surface on which hundreds of mesoporous silica nanoparticles, each encapsulated with a different native natural product, were immobilized in spatially defined manner, followed by on-chip mammalian cell growth and on-demand compound release, high-content screening was successfully carried out with readily available phenotypic detection methods. By combining this novel MSN-on-a-chip system with siRNA technology for the first time, we discovered (+)-Usniacin possesses synergistic inhibitory properties similar to Olaparib (an FDA-approved drug) in BRCA1-knockdown cancer cells.
Targeted delivery of activatable fluorescent probe for detection of furin activity in living cells ChemBioChem (IF 2.847) Pub Date : 2018-02-21 Jianguo Lin, Xueyu Zhao, Gaochao Lv, Ying Peng, Qingzhu Liu, Xi Li, Shanshan Wang, Ke Li, Ling Qiu
Furin, a protein convertase, play a crucial role in the progression of tumor. In this work, a new fluorescent probe 1 consisting of a peptide Arg-val-Arg-Arg (RVRR) and aminoluciferin fluorophore was designed and prepared for responsive and activatable detecting furin activity in vitro and in living cells. We demonstrated that probe 1 could be response toward furin with an "off-on" florescence signal to generate ~3.58 folds enhancement of the fluorescence intensity in vitro. Fluorescence imaging in MDA-MB-468 and LoVo cells showed that the probe 1 could be cleaved by the overexpressed furin with fluorescence turn-on in the MDA-MB-468 cells, which is capable of discrimination between furin-overexpressing and furin-deficient cell lines. The research indicated that probe 1 had great potential on detecting furin activity in living cells.
Near-infrared light-controlled gene expression and protein targeting in neurons and non-neuronal cells. ChemBioChem (IF 2.847) Pub Date : 2018-02-21 Vladislav Verkhusha, Taras Redchuk, Maksim Karasev, Evgeniya Omelina
Near-infrared (NIR) light-inducible binding of bacterial phytochrome BphP1 to its engineered partner QPAS1 is used for optical protein regulation in mammalian cells. However, there are no data on the application of the BphP1-QPAS1 pair in cells derived from various mammalian tissues. Here, we tested functionality of two BphP1-QPAS1-based optogenetic tools, such as an NIR and blue light-sensing system for control of protein localization (iRIS) and an NIR light-sensing system for transcription activation (TA), in several cell types including cortical neurons. We found that the performance of these optogenetic tools often rely on physiological properties of a specific cell type, such as nuclear transport, which may limit applicability of blue light-sensitive component of iRIS. In contrast, the NIR-light-sensing part of iRIS performed well in all tested cell types. The TA system showed the best performance in HeLa, U-2 OS and HEK-293 cells. Small size of the QPAS1 component allows designing AAV viral particles, which were applied to deliver the TA system to neurons.
Fluorine-Mediated Editing of a G-Quadruplex Folding Pathway ChemBioChem (IF 2.847) Pub Date : 2018-02-20 Jonathan Dickerhoff, Klaus Weisz
A (3+1)-hybrid-type G-quadruplex was substituted within its central tetrad by a single 2'-fluoro-modified guanosine. Driven by the anti-favoring nucleoside analog, a novel quadruplex fold with an inversion of a single G-tract and the conversion of a propeller into a lateral loop emerges. In addition, scalar couplings across hydrogen bonds demonstrate the formation of intra- and interresidual F***H8-C8 pseudohydrogen bonds within the modified quadruplexes. Alternative folding can be rationalized by the impact of fluorine on intermediate species based on a kinetic partitioning mechanism. Apparently, chemical or other environmental perturbations are able to redirect folding of a quadruplex, possibly modulating its regulatory role in physiological processes.
Amide Neighbouring Group Effects in Peptides: Phenylalanine as Relay Amino Acid in Long-Distance Electron Transfer ChemBioChem (IF 2.847) Pub Date : 2018-02-19 Uta Wille, Joses Nathanael, Luke Gamon, Meike Cordes, Paul Rablen, Thomas Bally, Katharina Fromm, Bernd Giese
Nature uses proteins as medium for long-distance electron transfer (ET) to carry out redox reactions in distant compartments. This ET occurs either by a single-step superexchange or through a multi-step charge hopping process, which uses side chains of amino acids as stepping stones. In this study we demonstrate that Phe can act as a relay amino acid for long-distance electron hole transfer through peptides. The considerably increased susceptibility of the aromatic ring to oxidation is caused by the lone pairs of neighbouring amide carbonyl groups, which stabilize the Phe radical cation. This neighbouring amide group effect helps to better understand the mechanism of extracellular electron transfer through conductive protein filaments (pili) of anaerobic bacteria during mineral respiration.
Substrate binding drives active site closing of human blood group B galactosyltransferase as revealed by hot-spot labeling and NMR experiments ChemBioChem (IF 2.847) Pub Date : 2018-02-19 Thomas Peters, Sophie Weissbach, Friedemann Flügge
Crystallography has shown that human blood group A (GTA) and B (GTB) glycosyltransferases undergo transitions between "open", "semi-closed", and "closed" conformations upon substrate binding. However, the time scales of corresponding conformational reorientations are unknown. Crystal structures show that Trp and Met residues are located at "conformational hot spots" of the enzymes. Therefore, we have utilized 15N-side chain labeling of Trp residues, and 13C-methyl labeling of Met residues to study substrate induced conformational transitions of GTB. Chemical shift perturbations (CSPs) of Met and Trp residues in direct contact with substrate ligands reflect binding kinetics, whereas CSPs of Met and Trp residues at remote sites reflect conformational changes of the enzyme upon substrate binding. Acceptor binding is fast on the chemical shift time scale with rather small CSPs in the range of less than ca. 20 Hz. Donor binding matches the intermediate exchange regime yielding an estimate for exchange rate constants around 200 - 300 Hz. Donor or acceptor binding to GTB saturated with acceptor or donor substrate, respectively, is slow (<10 Hz) as are coupled protein motions, reflecting mutual allosteric control of donor and acceptor binding. Remote CSPs suggest that substrate binding drives the enzyme into the closed state required for catalysis. These findings should contribute to a better understanding of the mechanism of glycosyltransfer of GTA and GTB.
Clickable polyamine derivatives as chemical probes for the polyamine transport system ChemBioChem (IF 2.847) Pub Date : 2018-02-16 Roeland Vanhoutte, Jan Pascal Kahler, Shaun Martin, Sarah van Veen, Steven Verhelst
Polyamines are essential for cell growth and differentiation, but their trafficking by the polyamine transport system is not fully understood. In this paper, we describe the synthesis of several azido-derivatized polyamines for easy conjugation by click chemistry. Attachment of a BODIPY-dye gave fluorescent polyamine probes, which were tested in cell culture. The linear probe series showed superior cellular uptake compared with the probes where the dye was attached to a branch on one of the central amines. Interestingly, the linear probes accumulated rapidly in cancer cells (MCF-7), but not in non-tumorigenic cells (MCF-10A). The fluorescent polyamine probes are therefore applicable to the study of polyamine trafficking, whereas the azido-polyamines may be further utilized to transport cargo into cancer cells by exploiting the polyamine transport system.
MicroRNA Detection by DNA-Mediated Liposome Fusion ChemBioChem (IF 2.847) Pub Date : 2018-02-16 Coline Jumeaux, Olov Wahlsten, Stephan Block, Eunjung Kim, Rona Chandrawati, Philip D. Howes, Fredrik Höök, Molly M. Stevens
Synthetic Cancer-Targeting Innate Immune Stimulators Give Insights into Avidity Effects ChemBioChem (IF 2.847) Pub Date : 2018-02-16 Anne C. Conibear, André J. G. Pötgens, Karine Thewes, Claudia Altdorf, Clarissa Hilzendeger, Christian F. W. Becker
Development and Validation of 2D Difference Intensity Analysis for Chemical Library Screening by Protein-Detected NMR Spectroscopy ChemBioChem (IF 2.847) Pub Date : 2018-02-16 John M. Egner, Davin R. Jensen, Michael D. Olp, Nolan W. Kennedy, Brian F. Volkman, Francis C. Peterson, Brian C. Smith, R. Blake Hill
Light-dependent cytoplasmic recruitment enhances the dynamic range of a nuclear import photoswitch ChemBioChem (IF 2.847) Pub Date : 2018-02-14 Hayretin Yumerefendi, Hui Wang, Daniel J Dickinson, Andrew Lerner, Per Malkus, Bob Goldstein, Klaus Hahn, Brian Kuhlman
Cellular signal transduction is often regulated at multiple steps in order to achieve more complex logic or precise control of a pathway. For instance, some signaling mechanisms couple allosteric activation with localization to achieve high signal to noise. Here, we create a system for light activated nuclear import that incorporates two levels of control. It consists of a nuclear import photoswitch, Light Activated Nuclear Shuttle (LANS), and a protein engineered to preferentially interact with LANS in the dark, Zdk2. First, Zdk2 is tethered to a location in the cytoplasm, which sequesters LANS in the dark. Second, LANS incorporates a nuclear localization signal (NLS) that is sterically blocked from binding to the nuclear import machinery in the dark. When activated with light, LANS both dissociates from its tethered location and exposes its NLS, which leads to nuclear accumulation. We demonstrate that this coupled system improves the dynamic range of LANS in mammalian cells, yeast, and C. elegans and provides tighter control of transcription factors that have been fused to LANS.
Lysine-241 has a role in coupling 2OG turnover with substrate oxidation during KDM4-catalysed histone demethylation ChemBioChem (IF 2.847) Pub Date : 2018-02-14 Rebecca L Hancock, Martine I Abboud, Tristan J Smart, Emily Flashman, Akane Kawamura, Christopher J Schofield, Richard Hopkinson
The JmjC histone demethylases (KDMs) play important roles in modulating histone methylation states and have the potential to be regulated by oxygen availability. Lys-241 of the KDM4 subfamily is proposed to be important in oxygen binding by KDM4A. We report evidence that, although K241 is unlikely to be directly involved in oxygen binding, it has an important role in coupling 2-oxoglutarate cosubstrate oxidation with lysine demethylase activity. The results suggest that compounds promoting uncoupling of substrate oxidation are of interest as JmjC-KDM inhibitors.
Optogenetic Control by Pulsed Illumination ChemBioChem (IF 2.847) Pub Date : 2018-02-14 Julia Hennemann, Roman S Iwasaki, Tamara N Grund, Ralph P Diensthuber, Florian Richter, Andreas Möglich
Sensory photoreceptors evoke numerous adaptive responses in Nature and serve as light-gated actuators in optogenetics to enable the spatiotemporally precise, reversible and noninvasive control of cellular events. The output of optogenetic circuits can often be dialed in by varying illumination quality, quantity and duration. Here, we devise a programmable matrix of light-emitting diodes to efficiently probe the response of optogenetic systems to intermittently applied light of varying intensity and pulse frequency. Circuits for light-regulated gene expression markedly differed in their responses to pulsed illumination of a single color which sufficed for sequentially triggering them. In addition to quantity and quality, the pulse frequency of intermittent light hence provides a further input variable for output control in optogenetics and photobiology. Pulsed illumination schemes allow the reduction of overall light dose and facilitate the multiplexing of several light-dependent actuators and reporters.
Alanine and lysine scans of the LL-37 derived peptide fragment KR-12 reveal key residues for antimicrobial activity ChemBioChem (IF 2.847) Pub Date : 2018-02-11 Sunithi Gunasekera, Taj Muhammad, Adam A. Strömstedt, K. Johan Rosengren, Ulf Göransson
The human host defence peptide LL-37 is a broad-spectrum antibiotic with immunomodulatory functions. Residues 18-29 in LL-37 have previously been identified as a minimal peptide (KR-12) that retains antibacterial activity with decreased cytotoxicity. In this study, we generated analogues of KR-12 by Ala and Lys scans to identify key elements for activity. These were tested against a panel of human pathogens and for membrane permeabilization on liposomes. Replacements of hydrophobic and cationic residues with Ala were detrimental for antibiotic potency. Substitutions by Lys increased activity as long as the increase in cationic density did not disrupt the amphiphilic disposition of the helical structure. Importantly, substitutions showed differential effects against different organisms. Replacement of Gln5 with Lys and Asp9 with Ala or Lys improved broad-spectrum activity the most, each resulting in up to eight-fold increase in potency against Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. The improved analogues displayed no significant toxicity against human cells, depicting KR-12 as a tuneable template for antibiotic development.
Dual Labeling of the CBP/p300 KIX domain for 19F NMR leads to identification of a new small molecule binding site ChemBioChem (IF 2.847) Pub Date : 2018-02-11 Clifford Thomas Gee, Keith E. Arntson, Edward J. Koleski, Rachel Lynn Staebell, William Charles Krause Pomerantz
Protein-Observed Fluorine NMR Spectroscopy (PrOF NMR) is an emerging technique for screening and characterizing small molecule-protein interactions. The choice of which amino acid to label for PrOF NMR can be critical for analysis. Here we report the first use of a protein containing two different fluoroaromatic amino acids for NMR studies. Using the KIX domain of the CBP/p300 as a model system, we examine ligand binding of several small molecules elaborated from our previous fragment screen and identify a new ligand binding site distinct from those used by native transcription factors. This site was further supported by computational modeling (FTMap and Schrödinger) and 1H-15N HSQC/HMQC NMR spectroscopy. Metabolic labelling with multiple fluorinated amino acids provides useful probes for further studying ligand binding and has led to new insight for allosterically regulating transcription-factor protein interactions with small molecules.
Synthesis and Biological Evaluation of Fluorescent Bryostatin Analogues ChemBioChem (IF 2.847) Pub Date : 2018-02-09 Thomas J. Cummins, Noemi Kedei, Agnes Czikora, Nancy E. Lewin, Sharon Kirk, Mark E. Petersen, Kevin M. McGowan, Jin-Qiu Chen, Xiaoling Luo, Randall C. Johnson, Sarangan Ravichandran, Peter M. Blumberg, Gary E. Keck
To investigate the cellular distribution of tumor-promoting vs nontumor-promoting bryostatin analogues, we synthesized fluorescently labeled variants of two bryostatin derivatives that have previously shown either phorbol ester-like or bryostatin-like biological activity on U937 cells. These new fluorescent analogues both displayed high affinity for PKC binding and retained the basic properties of the parent, unlabeled compounds in U937 assays. The fluorescent compounds showed similar patterns of intracellular distribution in cells, however, arguing against an existing hypothesis that varying patterns of intracellular distribution contributed to the difference in biological activity. In further characterization, the fluorescent compounds revealed a slow rate of cellular uptake; correspondingly, they showed reduced activity for cellular responses that are only transient upon treatment with phorbol ester or bryostatin 1.
Discovery of a short chain dehydrogenase from Catharanthus roseus that produces a novel monoterpene indole alkaloid ChemBioChem (IF 2.847) Pub Date : 2018-02-09 Anna Stavrinides, Evangelos Tatsis, Thu-Thuy Dang, Lorenzo Caputi, Clare Stevenson, David Lawson, Bernd Schneider, Sarah O'Connor
Monoterpene indole alkaloids, a large class of plant natural products, derive from the biosynthetic intermediate strictosidine aglycone. Strictosidine aglycone, which can exist as a variety of isomers, can be reduced to form numerous different structures. We have discovered a short chain alcohol dehydrogenase (SDR) from monoterpene indole alkaloid producing plants (Catharanthus roseus and Rauvolfia serpentina) that reduce strictosidine aglycone and produce an alkaloid that does not correspond to any previously reported compound. Here we report the structural characterization of this product, which we have named vitrosamine, as well as the crystal structure of the SDR. This discovery highlights the structural versatility of the strictosidine aglycone biosynthetic intermediate and expands the range of enzymatic reactions that SDRs can catalyze. This discovery further highlights how a sequence-based gene mining discovery approach in plants can reveal cryptic chemistry that would not be uncovered by classical natural product chemistry approaches.
A miniaturized E. coli green light sensor with high dynamic range ChemBioChem (IF 2.847) Pub Date : 2018-02-08 Nicholas Ting Xun Ong, Jeffrey J Tabor
Genetically-engineered photoreceptors enable unrivaled control over gene expression. Previously, we ported the Synechocystis PCC 6803 CcaSR two-component system, which is activated by green light and de-activated by red, into E. coli, resulting in a sensor with 6-fold dynamic range. Later, we optimized pathway protein expression levels and the output promoter sequence to decrease transcriptional leakiness and increase the dynamic range to approximately 120-fold. These CcaSR v1.0 and 2.0 systems have been used for precise quantitative, temporal, and spatial control of gene expression for a variety of applications. Recently, others have deleted two PAS domains of unknown function from the CcaS sensor histidine kinase in a CcaSR v1.0-like system. Here, we apply these deletions to CcaSR v2.0, resulting in a v3.0 light sensor with 4-fold lower leaky output and nearly 600-fold dynamic range. We demonstrate that the PAS domain deletions have no deleterious effect on CcaSR green light sensitivity or response dynamics. CcaSR v3.0 is the best performing engineered bacterial green light sensor available, and should have broad applications in fundamental and synthetic biology studies.
Npom-protected NONOate enables light-triggered NO/cGMP signalling in primary vascular smooth muscle cells ChemBioChem (IF 2.847) Pub Date : 2018-02-08 Anna Sofia Stroppel, Michael Paolillo, Thomas Ziegler, Robert Feil, Thorsten Stafforst
Diazeniumdiolates (NONOates) are a class of nitric oxide (NO)-releasing substances widely used in studies of NO/cGMP signalling. Since spatiotemporal control is highly desirable for such purposes, we synthesised a novel Npom-caged pyrrolidine NONOate. A kinetic analysis together with a Griess Assay show the photo-dependent release of NO with high quantum yield (UV light). In primary vascular smooth muscle cells (VSMCs), our compound was able to reliably induce a fast increase in cGMP as measured with a genetically encoded FRET-based cGMP sensor and was further validated by the phosphorylation of the downstream target vasodilator-stimulated phospho¬protein (VASP). Due to the facile synthesis, good decaging kinetics, and its capability to activate cGMP signalling in a fast and efficient manner, Npom-protected NONOates allow for improved spatiotemporal control of NO/cGMP signalling.
Design of a short thermally stable α-helix embedded in a macrocycle ChemBioChem (IF 2.847) Pub Date : 2018-02-07 Haifan Wu, Arusha Acharyya, Yibing Wu, Lijun Liu, Hyunil Jo, Feng Gai, William DeGrado
Although helices play key roles in peptide-protein and protein-protein interactions, the helical conformation is generally unstable for short peptides (10 - 15 residues) in aqueous solution in the absence of their binding partners. Thus, stabilizing the helical conformation of peptides can lead to increases in binding potency, specificity, and stability towards proteolytic degradation. Helices have been successfully stabilized by introducing sidechain-to-sidechain cross-links within the central portion of the helix. However, this approach leaves the helical ends free, leading to fraying and exposure of the non-hydrogen bonded amide groups to solvent. Here, we develop a "capped-strapped" peptide strategy to stabilize helices by embedding the entire length of the helix within a macrocycle, which also includes a semi-rigid organic template as well as end-capping interactions. We have designed a 10-residue capped-strapped helical peptide that behaves like a mini-protein, with a cooperative thermal unfolding transition and Tm ~ 70 °C, unprecedented for helical peptides of this length. The NMR structure determination confirmed the design, and X-ray crystallography revealed a novel quaternary structure with implications for foldamer design.
Screening for selective protein inhibitors using IANUS peptide array ChemBioChem (IF 2.847) Pub Date : 2018-02-07 Frank Erdmann, Erik Prell, Günther Jahreis, Gunter Fischer, Miroslav Malesevic
The IANUS (Induced orgANization of strUcture by matrix-assisted togethernesS) peptide array was build up on a hypothesis that two peptides from two interacting protein epitopes are also able to interact when immobilized on a solid support. This peptide-peptide interactions can be visualized by FRET effects caused by close proximity of fluorescent labeled peptide chains. The biggest advantage of this low-resolution method is that no soluble protein probe or selective antibodies are needed. We applied the IANUS peptide array to screen for peptides from a phospatase Calcineurin (CaN) that binds to regulatory domain of the nuclear factor of activated T cells (NFAT). Peptides derived from the IANUS-library hits were tested for their ability to suppress i) the NFAT dephosphorylation by CaN, ii) the NFkB dephosphorylation by CaN, and iii) the CaN phosphatase activity. The best hit, peptide FPSLITIFSAPNYLDVYN, appeared to be a protease-resistant and cell-permeable NFAT inhibitor.
Structural Diversity and Anticancer Activity of Marine-Derived Elastase Inhibitors: Key Features and Mechanisms Mediating the Antimetastatic Effects in Invasive Breast Cancer ChemBioChem (IF 2.847) Pub Date : 2018-02-05 Fatma H Al-Awadhi, Valerie J Paul, Hendrik Luesch
Three new 3-amino-6-hydroxy-2-piperidone (Ahp)-containing cyclic depsipeptides named loggerpeptins A‒C (1‒3) along with molassamide (4) were discovered from a marine cyanobacterium, extending the structural diversity of this prevalent scaffold of cyanobacterial serine protease inhibitors. Molassamide (4), containing the 2-amino-butenoic (Abu) unit in the cyclic core, was the most potent and selective analogue against human neutrophil elastase (HNE). Given the growing evidence supporting the role of HNE in breast cancer progression and metastasis, we assessed the cellular effects of compounds 3 and 4 in the context of targeting invasive breast cancer. Both compounds inhibited the cleavage of the elastase substrate CD40 in biochemical assays; however, only 4 exhibited significant cellular activity. Since CD40 and other receptor proteolytic processing culminates in NFKB activation, we assessed the effects on the expression of target genes, including ICAM-1. ICAM-1 is also a direct target of elastase, and in our studies compound 4 attenuated both elastase-induced ICAM-1 gene expression and ICAM-1 proteolytic processing by elastase, revealing a potential dual effect on migration through modulation of gene expression and proteolytic processing. Molassamide (4) specifically inhibited the elastase-mediated migration of highly invasive triple negative breast cancer cells.
Loganic Acid Methyltransferase: Insights into the specificity of methylation on an iridoid glycoside ChemBioChem (IF 2.847) Pub Date : 2018-02-05 Nektaria Petronikolou, Allison Hollatz, Mary A. Schuler, Satish K. Nair
: Loganin is an iridoid glycoside of interest as both an intermediate in the biosynthesis of indole alkaloids in plants and as a bioactive compound itself. The loganic acid methyltransferase catalyzes the methylation of a monoterpenoid glycoside precursor to produce loganin, and demonstrates stereospecificity for the (6S,7R) substrate. Here, we biochemically characterize this biocatalyst and elucidate the basis for its strict substrate specificity. These studies may help facilitate the design of new classes of monoterpenoid indole alkaloids of pharmaceutical interest.
Construction of T-motif-based DNA nanostructures using enzymatic reactions ChemBioChem (IF 2.847) Pub Date : 2018-02-04 Ryo Kageyama, Ibuki Kawamata, Kaori Tanabe, Yuki Suzuki, Shin-ichiro Nomura, Satoshi Murata
The most common way to fabricate DNA nanostructures is to mix individually synthesized DNA oligomers in one pot. However, if DNA nanostructures could be produced using enzymatic reactions, they could be applied in various environments, including in vivo. Here, we report an enzymatic method that we developed to construct a DNA nanostructure from a simple motif called a T-motif. A long, repeated structure was replicated from a circular template by rolling circle amplification (RCA) and then cleaved into T-motif segments by restriction enzymes. These motifs have been successfully assembled into a ladder-like nanostructure without purification or controlled annealing. We believe that our approach is widely applicable to constructing a variety of DNA nanostructures using enzymatic reactions.
The unfold story of general anesthesia ChemBioChem (IF 2.847) Pub Date : 2018-02-04 Ferenc Zsila
Inhalational anesthetics are routinely employed in the clinical practice to accomplish general anesthesia. Concerns have recently been emerged regarding deleterious impact of these volatile agents on cognitive performance, immune functions, tumor recurrence and metastasis. These agents have shown to modify the gene expression pattern as well as cell signaling in tumor cells but the underlying molecular mechanisms remain a matter of conjecture. Regulatory/signaling proteins either of cytosolic or membrane origin abundantly contain intrinsically disordered sequences, the conformational pliability of which is pivotal in their biological functions. It is well known that chloroform (an anesthetic itself), trifluoroethanol, hexafluoroisopropanol and related haloalcohols markedly affect the structure of disordered proteins and protein regions inducing folding, misfolding or even aggregation. Taking into consideration physicochemical similarities and protein interaction modes of these volatile solvents and inhaled anesthetics, it is postulated that administration of these drugs can also modify the secondary structure of disordered protein segments. Accordingly, pharmacological effects of anesthetics may, at least in part, be mediated via conformational perturbations of intrinsic disorder-based regulatory protein networks of cells.
Lucifer-Based Environment-Sensitive Fluorescent PNA Probe for Imaging Poly(A) RNAs ChemBioChem (IF 2.847) Pub Date : 2018-02-03 Seergazhi G Srivatsan, Pramod M Sabale, Uddhav B Ambi
Fluorescence-based oligonucleotide (ON) hybridization probes greatly aid the detection and profiling of RNA sequences in the cells. However, certain limitations like target accessibility and hybridization efficiency in cellular environment hamper their broad application as RNAs can form complex and stable structures. In this context, we have developed a robust hybridization probe suitable for imaging RNA in cells by combining the properties of (i) a new microenvironment-sensitive fluorescent nucleobase analog, derived by attaching the Lucifer chromophore (1,8-naphthalimide) at the 5-position of uracil, and (ii) peptide nucleic acid (PNA), which is capable of forming stable hybrids with RNA. The fluorescence of Lucifer chromophore-labeled PNA base analog, when incorporated into PNA oligomers and hybridized to complementary and mismatched ONs, is highly responsive to its neighboring base environment. Notably, the PNA base reports the presence of an adenine repeat in an RNA ON with reasonable enhancement in fluorescence. This feature of the emissive analog enabled the construction of a poly(T) PNA probe for the efficient visualization of polyadenylated [poly(A)] RNAs in cells, an important motif, which plays vital roles in the lifecycle of many types of RNA. Our results demonstrate that such responsive fluorescent nucleobase analogs, when judiciously placed in PNA oligomers, could generate useful hybridization probes to detect and also image nucleic acid sequences in cell.
Site-specific labelling of native mammalian proteins for single-molecule FRET measurements ChemBioChem (IF 2.847) Pub Date : 2018-02-02 Alexander Gust, Leonhard Jakob, Daniela Zeitler, Astrid Bruckmann, Kevin Kramm, Sarah Willkomm, Philip Tinnefeld, Gunter Meister, Dina Grohmann
Human cells are complex entities in which molecular recognition and selection are critical for cellular processes often driven by structural changes and dynamic interactions. Biomolecules appear in different chemical states and modifications like phosphorylation affect their function. Hence, using proteins in their chemically native state in biochemical and biophysical assays is essential. Single-molecule FRET measurements allow the exploration of the structure, function and dynamics of biomolecules but cannot be fully exploited for the human proteome as a method for the site-specific coupling of organic dyes into native, non-recombinant mammalian proteins is lacking. We address this issue showing the site-specific engineering of fluorescent dyes into human proteins based on bioorthogonal reactions. We show the applicability of the method to study functional and posttranslationally modified proteins on the single-molecule level, among them the hitherto inaccessible human Argonaute 2.
Single-molecule kinetics of an enzyme in the presence of multiple substrates ChemBioChem (IF 2.847) Pub Date : 2018-02-02 Divya Singh, srabanti chaudhury
In this work, we study the catalytic activity of a single enzyme in the presence of multiple substrates. We broadly discuss three different mechanisms of bisubstrate binding, namely ordered sequential, random sequential and ping-pong non-sequential pathway. Using the chemical master equation approach we obtain exact expressions for the waiting time distributions the mean turnover time and the randomness parameter as a function of the substrate concentration, such that both concentrations are fixed but one of them is changed quasistatically. The randomness parameter is not equal to unity at intermediate to high substrate concentration, indicating the presence of multiple rate limiting steps in the reaction pathway in all the three modes of bisubstrate binding. This arises due to transitions between the free enzyme and the enzyme-substrate complexes that occur on a comparable time scales. Such turnover statistics of the single enzyme can also distinguish between the different types of bisubstrate binding mechanisms.
Mechanism-based Inhibitor of DNA Cytosine-5 Methyltransferase (DNMT) via a SNAr Reaction with an Oligodeoxyribonucleotide Containing 2-Amino-4-Halopyridine-C-Nucleoside (dXP) ChemBioChem (IF 2.847) Pub Date : 2018-02-02 Kousuke Sato, Yuma Kunitomo, Yukiko Kasai, Shohei Utsumi, Isao Suetake, Shoji Tajima, Satoshi Ichikawa, Akira Matsuda
In chromatin, 5-methylcytosine (mC), which represents the 5th nucleobase in genomic DNA, plays a role as an inducer of epigenetic changes. Tumor cells exhibit aberrant DNA methylation patterns, and inhibition of human DNA cytosine-5 methyltransferase (DNMT), which is responsible for generating mC in CpG sequences, is an effective strategy to treat various cancers. Here, we describe the design, synthesis, and evaluation of the properties of 2-amino-4-halopyridine-C-nucleosides (dXP) and oligodeoxyribonucleotides (ODNs) containing dXP as a novel mechanism-based inhibitor of DNMTs. The designed ODN containing XPpG forms a complex with DNMTs via covalent bonding through a nucleophilic aromatic substitution (SNAr) reaction, and its cell proliferation activity was also investigated. This study suggests that dXP in a CpG sequence of DNA could serve as a potential nucleic acid drug leads in cancer chemotherapy and a useful chemical probe for studies of epigenetics. Our molecular design using a SNAr reaction would be useful for DNMTs and other protein-DNA interactions.
Exploring PTDH–P450BM3 Variants for the Synthesis of Drug Metabolites ChemBioChem (IF 2.847) Pub Date : 2018-01-17 Nina Beyer, Justyna K. Kulig, Marco W. Fraaije, Martin A. Hayes, Dick B. Janssen
Enzymes as Enhancers for the Biodegradation of Synthetic Polymers in Wastewater ChemBioChem (IF 2.847) Pub Date : 2018-01-16 Karolina Haernvall, Sabine Zitzenbacher, Antonino Biundo, Motonori Yamamoto, Michael Bernhard Schick, Doris Ribitsch, Georg M. Guebitz
Characterisation of a Bacterial Galactokinase with High Activity and Broad Substrate Tolerance for Chemoenzymatic Synthesis of 6-Aminogalactose-1-Phosphate and Analogues ChemBioChem (IF 2.847) Pub Date : 2018-01-04 Kun Huang, Fabio Parmeggiani, Edward Pallister, Chuen-Jiuan Huang, Fang-Fang Liu, Qian Li, William R. Birmingham, Peter Both, Baptiste Thomas, Li Liu, Josef Voglmeir, Sabine L. Flitsch
Pseudomonas fluorescens Strain R124 Encodes Three Different MIO Enzymes ChemBioChem (IF 2.847) Pub Date : 2018-01-04 Pál Csuka, Vivien Juhász, Szabolcs Kohári, Alina Filip, Andrea Varga, Péter Sátorhelyi, László Csaba Bencze, Hazel Barton, Csaba Paizs, László Poppe
Effect of Site-Specific Peptide-Tag Labeling on the Biocatalytic Properties of Thermoalkalophilic Lipase from Geobacillus thermocatenulatus ChemBioChem (IF 2.847) Pub Date : 2018-01-04 Oscar Romero, Blanca de las Rivas, David Lopez-Tejedor, Jose M. Palomo
Some contents have been Reproduced by permission of The Royal Society of Chemistry.
- Acc. Chem. Res.
- ACS Appl. Mater. Interfaces
- ACS Biomater. Sci. Eng.
- ACS Catal.
- ACS Cent. Sci.
- ACS Chem. Biol.
- ACS Chem. Neurosci.
- ACS Comb. Sci.
- ACS Earth Space Chem.
- ACS Energy Lett.
- ACS Infect. Dis.
- ACS Macro Lett.
- ACS Med. Chem. Lett.
- ACS Nano
- ACS Omega
- ACS Photonics
- ACS Sens.
- ACS Sustainable Chem. Eng.
- ACS Synth. Biol.
- Acta Biomater.
- Acta Crystallogr. A Found. Adv.
- Acta Mater.
- Adv. Colloid Interface Sci.
- Adv. Electron. Mater.
- Adv. Energy Mater.
- Adv. Funct. Mater.
- Adv. Healthcare Mater.
- Adv. Mater.
- Adv. Mater. Interfaces
- Adv. Opt. Mater.
- Adv. Sci.
- Adv. Synth. Catal.
- AlChE J.
- Anal. Bioanal. Chem.
- Anal. Chem.
- Anal. Chim. Acta
- Anal. Methods
- Angew. Chem. Int. Ed.
- Annu. Rev. Anal. Chem.
- Annu. Rev. Biochem.
- Annu. Rev. Environ. Resour.
- Annu. Rev. Food Sci. Technol.
- Annu. Rev. Mater. Res.
- Annu. Rev. Phys. Chem.
- Appl. Catal. A Gen.
- Appl. Catal. B Environ.
- Appl. Clay. Sci.
- Appl. Energy
- Aquat. Toxicol.
- Arab. J. Chem.
- Asian J. Org. Chem.
- Atmos. Environ.
- Carbohydr. Polym.
- Catal. Commun.
- Catal. Rev. Sci. Eng.
- Catal. Sci. Technol.
- Catal. Today
- Cell Chem. Bio.
- Cem. Concr. Res.
- Ceram. Int.
- Chem. Asian J.
- Chem. Bio. Drug Des.
- Chem. Biol. Interact.
- Chem. Commun.
- Chem. Educ. Res. Pract.
- Chem. Eng. J.
- Chem. Eng. Sci.
- Chem. Eur. J.
- Chem. Mater.
- Chem. Phys.
- Chem. Phys. Lett.
- Chem. Phys. Lipids
- Chem. Rev.
- Chem. Sci.
- Chem. Soc. Rev.
- Chin. J. Chem.
- Combust. Flame
- Compos. Part A Appl. Sci. Manuf.
- Compos. Sci. Technol.
- Compr. Rev. Food Sci. Food Saf.
- Comput. Chem. Eng.
- Constr. Build. Mater.
- Coordin. Chem. Rev.
- Corros. Sci.
- Crit. Rev. Food Sci. Nutr.
- Crit. Rev. Solid State Mater. Sci.
- Cryst. Growth Des.
- Curr. Opin. Chem. Eng.
- Curr. Opin. Colloid Interface Sci.
- Curr. Opin. Environ. Sustain
- Curr. Opin. Solid State Mater. Sci.
- Ecotox. Environ. Safe.
- Electrochem. Commun.
- Electrochim. Acta
- Energy Environ. Sci.
- Energy Fuels
- Energy Storage Mater.
- Environ. Impact Assess. Rev.
- Environ. Int.
- Environ. Model. Softw.
- Environ. Pollut.
- Environ. Res.
- Environ. Sci. Policy
- Environ. Sci. Technol.
- Environ. Sci. Technol. Lett.
- Environ. Sci.: Nano
- Environ. Sci.: Processes Impacts
- Environ. Sci.: Water Res. Technol.
- Eur. J. Inorg. Chem.
- Eur. J. Med. Chem.
- Eur. J. Org. Chem.
- Eur. Polym. J.
- J. Acad. Nutr. Diet.
- J. Agric. Food Chem.
- J. Alloys Compd.
- J. Am. Ceram. Soc.
- J. Am. Chem. Soc.
- J. Am. Soc. Mass Spectrom.
- J. Anal. Appl. Pyrol.
- J. Anal. At. Spectrom.
- J. Antibiot.
- J. Catal.
- J. Chem. Educ.
- J. Chem. Eng. Data
- J. Chem. Inf. Model.
- J. Chem. Phys.
- J. Chem. Theory Comput.
- J. Chromatogr. A
- J. Chromatogr. B
- J. Clean. Prod.
- J. CO2 UTIL.
- J. Colloid Interface Sci.
- J. Comput. Chem.
- J. Cryst. Growth
- J. Dairy Sci.
- J. Electroanal. Chem.
- J. Electrochem. Soc.
- J. Environ. Manage.
- J. Eur. Ceram. Soc.
- J. Fluorine Chem.
- J. Food Drug Anal.
- J. Food Eng.
- J. Food Sci.
- J. Funct. Foods
- J. Hazard. Mater.
- J. Heterocycl. Chem.
- J. Hydrol.
- J. Ind. Eng. Chem.
- J. Inorg. Biochem.
- J. Magn. Magn. Mater.
- J. Mater. Chem. A
- J. Mater. Chem. B
- J. Mater. Chem. C
- J. Mater. Process. Tech.
- J. Mech. Behav. Biomed. Mater.
- J. Med. Chem.
- J. Membr. Sci.
- J. Mol. Catal. A Chem.
- J. Mol. Liq.
- J. Nat. Gas Sci. Eng.
- J. Nat. Prod.
- J. Nucl. Mater.
- J. Org. Chem.
- J. Photochem. Photobiol. C Photochem. Rev.
- J. Phys. Chem. A
- J. Phys. Chem. B
- J. Phys. Chem. C
- J. Phys. Chem. Lett.
- J. Polym. Sci. A Polym. Chem.
- J. Porphyr. Phthalocyanines
- J. Power Sources
- J. Solid State Chem.
- J. Taiwan Inst. Chem. E.
- Macromol. Rapid Commun.
- Mass Spectrom. Rev.
- Mater. Chem. Front.
- Mater. Des.
- Mater. Horiz.
- Mater. Lett.
- Mater. Sci. Eng. A
- Mater. Sci. Eng. R Rep.
- Mater. Today
- Meat Sci.
- Med. Chem. Commun.
- Microchem. J.
- Microchim. Acta
- Micropor. Mesopor. Mater.
- Mol. Biosyst.
- Mol. Cancer Ther.
- Mol. Catal.
- Mol. Nutr. Food Res.
- Mol. Pharmaceutics
- Mol. Syst. Des. Eng.
- Nano Energy
- Nano Lett.
- Nano Res.
- Nano Today
- Nano-Micro Lett.
- Nanomed. Nanotech. Biol. Med.
- Nanoscale Horiz.
- Nat. Catal.
- Nat. Chem.
- Nat. Chem. Biol.
- Nat. Commun.
- Nat. Energy
- Nat. Mater.
- Nat. Med.
- Nat. Methods
- Nat. Nanotech.
- Nat. Photon.
- Nat. Prod. Rep.
- Nat. Protoc.
- Nat. Rev. Chem.
- Nat. Rev. Drug. Disc.
- Nat. Rev. Mater.
- Natl. Sci. Rev.
- Neurochem. Int.
- New J. Chem.
- NPG Asia Mater.
- npj 2D Mater. Appl.
- npj Comput. Mater.
- npj Flex. Electron.
- npj Mater. Degrad.
- npj Sci. Food
- Pharmacol. Rev.
- Pharmacol. Therapeut.
- Photochem. Photobiol. Sci.
- Phys. Chem. Chem. Phys.
- Phys. Life Rev.
- PLOS ONE
- Polym. Chem.
- Polym. Degrad. Stabil.
- Polym. J.
- Polym. Rev.
- Powder Technol.
- Proc. Combust. Inst.
- Prog. Cryst. Growth Ch. Mater.
- Prog. Energy Combust. Sci.
- Prog. Mater. Sci.
- Prog. Photovoltaics
- Prog. Polym. Sci.
- Prog. Solid State Chem.
- Sci. Adv.
- Sci. Bull.
- Sci. Rep.
- Sci. Total Environ.
- Sci. Transl. Med.
- Scr. Mater.
- Sens Actuators B Chem.
- Sep. Purif. Technol.
- Small Methods
- Soft Matter
- Sol. Energy
- Sol. Energy Mater. Sol. Cells
- Solar PRL
- Spectrochim. Acta. A Mol. Biomol. Spectrosc.
- Surf. Sci. Rep.
- Sustainable Energy Fuels