Chemistry & Biology

In This Issue ()
Publication date: 17 December 2015 Source:Chemistry & Biology, Volume 22, Issue 12
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Vitamin C as Cancer Destroyer, Investigating Sulfhydration, and the Variability in CFTR Interactome ()
Publication date: 17 December 2015 Source:Chemistry & Biology, Volume 22, Issue 12 Each month, Chemistry & Biology Select highlights a selection of research reports from the recent literature. These highlights are a snapshot of interesting research done across the field of chemical biology. Our December 2015 selection includes an insight into how vitamin C destroys cancer cells, a new method that makes possible the investigatation of sulfhydration, and the mapping of the CFTR interactome and how it depends on the environmental conditions and differs between wild-type and disease-causing mutant.
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Unsaturated Lipid Assimilation by Mycobacteria Requires Auxiliary cis-trans Enoyl CoA Isomerase ()
Publication date: 17 December 2015 Source:Chemistry & Biology, Volume 22, Issue 12 Author(s): Sonali Srivastava, Sarika Chaudhary, Lipi Thukral, Ce Shi, Rinkoo D. Gupta, Radhika Gupta, K. Priyadarshan, Archana Vats, Asfarul S. Haque, Rajan Sankaranarayanan, Vivek T. Natarajan, Rakesh Sharma, Courtney C. Aldrich, Rajesh S. Gokhale Mycobacterium tuberculosis (Mtb) can survive in hypoxic necrotic tissue by assimilating energy from host-derived fatty acids. While the expanded repertoire of β-oxidation auxiliary enzymes is considered crucial for Mtb adaptability, delineating their functional relevance has been challenging. Here, we show that the Mtb fatty acid degradation (FadAB) complex cannot selectively break down cis fatty acyl substrates. We demonstrate that the stereoselective binding of fatty acyl substrates in the Mtb FadB pocket is due to the steric hindrance from Phe287 residue. By developing a functional screen, we classify the family of Mtb Ech proteins as monofunctional or bifunctional enzymes, three of which complement the FadAB complex to degrade cis fatty acids. Crystal structure determination of two cis-trans enoyl coenzyme A (CoA) isomerases reveals distinct placement of active-site residue in Ech enzymes. Our studies thus reveal versatility of Mtb lipid-remodeling enzymes and identify an essential role of stand-alone cis-trans enoyl CoA isomerases in mycobacterial biology. Graphical abstract image Teaser Mtb can utilize host lipids as one of the energy sources during infection. Srivastava et al. demonstrate that the assimilation of cis fatty acids by Mtb through β-oxidation machinery requires auxiliary 3-cis 2-trans enoyl CoA isomerases.
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Transcriptional Profiling of a Selective CREB Binding Protein Bromodomain Inhibitor Highlights Therapeutic Opportunities ()
Publication date: 17 December 2015 Source:Chemistry & Biology, Volume 22, Issue 12 Author(s): Eugene L. Piatnitski Chekler, Jessica A. Pellegrino, Thomas A. Lanz, R. Aldrin Denny, Andrew C. Flick, Jotham Coe, Jonathan Langille, Arindrajit Basak, Shenping Liu, Ingrid A. Stock, Parag Sahasrabudhe, Paul D. Bonin, Kevin Lee, Mathew T. Pletcher, Lyn H. Jones Bromodomains are involved in transcriptional regulation through the recognition of acetyl lysine modifications on diverse proteins. Selective pharmacological modulators of bromodomains are lacking, although the largely hydrophobic nature of the pocket makes these modules attractive targets for small-molecule inhibitors. This work describes the structure-based design of a highly selective inhibitor of the CREB binding protein (CBP) bromodomain and its use in cell-based transcriptional profiling experiments. The inhibitor downregulated a number of inflammatory genes in macrophages that were not affected by a selective BET bromodomain inhibitor. In addition, the CBP bromodomain inhibitor modulated the mRNA level of the regulator of G-protein signaling 4 (RGS4) gene in neurons, suggesting a potential therapeutic opportunity for CBP inhibitors in the treatment of neurological disorders. Graphical abstract image Teaser Chekler et al. designed, synthesized, and transcriptionally profiled a selective inhibitor of the bromodomain of CREB binding protein. The inhibitor, called PF-CBP1, modulated key inflammatory genes in macrophages and downregulated RGS4 (a gene linked to Parkinson's disease) in neurons.
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Halophilic Protein Adaptation Results from Synergistic Residue-Ion Interactions in the Folded and Unfolded States ()
Publication date: 17 December 2015 Source:Chemistry & Biology, Volume 22, Issue 12 Author(s): Gabriel Ortega, Tammo Diercks, Oscar Millet Halophilic organisms thrive in environments with extreme salt concentrations and have adapted by allowing molar quantities of cosolutes, mainly KCl, to accumulate in their cytoplasm. To cope with this high intracellular salinity, halophilic organisms modified the chemical composition of their proteins to enrich their surface with acidic and short polar side chains, while lysines and bulky hydrophobic residues got depleted. We have emulated the evolutionary process of haloadaptation with natural and designed halophilic polypeptides and applied novel nuclear magnetic resonance (NMR) methodology to study the different mechanisms contributing to protein haloadaptation at a per residue level. Our analysis of an extensive set of NMR observables, determined over several proteins, allowed us to disentangle the synergistic contributions of protein haloadaptation: cation exclusion and electrostatic repulsion between negatively charged residues destabilize the denatured state ensemble while cumulative weak cation-protein interactions stabilize the folded conformations. Graphical abstract image Teaser We show that the synergy of protein-ion effects in the folded and the unfolded states governs halophilic proteins adaptation to hypersaline media.
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Reciprocal Regulation of ERα and ERβ Stability and Activity by Diptoindonesin G ()
Publication date: 17 December 2015 Source:Chemistry & Biology, Volume 22, Issue 12 Author(s): Zibo Zhao, Lu Wang, Taryn James, Youngeun Jung, Ikyon Kim, Renxiang Tan, F. Michael Hoffmann, Wei Xu ERβ is regarded as a “tumor suppressor” in breast cancer due to its anti-proliferative effects. However, unlike ERα, ERβ has not been developed as a therapeutic target in breast cancer due to loss of ERβ in aggressive cancers. In a small-molecule library screen for ERβ stabilizers, we identified Diptoindonesin G (Dip G), which significantly increases ERβ protein stability while decreasing ERα protein levels. Dip G enhances the transcription and anti-proliferative activities of ERβ, while attenuating the transcription and proliferative effects of ERα. Further investigation revealed that instead of targeting ER, Dip G targets the CHIP E3 ubiquitin ligase shared by ERα and ERβ. Thus, Dip G is a dual-functional moiety that reciprocally controls ERα and ERβ protein stability and activities via an indirect mechanism. The ERβ stabilization effects of Dip G may enable the development of ERβ-targeted therapies for human breast cancers. Graphical abstract image Teaser We identified a small molecule Diptoindonesin G that reciprocally stabilizes ERβ and destabilizes ERα in breast cancer cells. Dip G represents a new class of selective estrogen receptor modulator (SERMs) that could restore the balance of ERα and ERβ for the treatment of human breast cancer.
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Unraveling the B. pseudomallei Heptokinase WcbL: From Structure to Drug Discovery ()
Publication date: 17 December 2015 Source:Chemistry & Biology, Volume 22, Issue 12 Author(s): Mirella Vivoli, Michail N. Isupov, Rebecca Nicholas, Andrew Hill, Andrew E. Scott, Paul Kosma, Joann L. Prior, Nicholas J. Harmer Gram-negative bacteria utilize heptoses as part of their repertoire of extracellular polysaccharide virulence determinants. Disruption of heptose biosynthesis offers an attractive target for novel antimicrobials. A critical step in the synthesis of heptoses is their 1-O phosphorylation, mediated by kinases such as HldE or WcbL. Here, we present the structure of WcbL from Burkholderia pseudomallei. We report that WcbL operates through a sequential ordered Bi-Bi mechanism, loading the heptose first and then ATP. We show that dimeric WcbL binds ATP anti-cooperatively in the absence of heptose, and cooperatively in its presence. Modeling of WcbL suggests that heptose binding causes an elegant switch in the hydrogen-bonding network, facilitating the binding of a second ATP molecule. Finally, we screened a library of drug-like fragments, identifying hits that potently inhibit WcbL. Our results provide a novel mechanism for control of substrate binding and emphasize WcbL as an attractive anti-microbial target for Gram-negative bacteria. Graphical abstract image Teaser Vivoli et al. present the structure and mechanism of the bacterial sugar kinase WcbL. WcbL demonstrates unusual kinetics: its cooperativity for ATP switches on binding of the sugar substrate. Screening of a fragment library identified a potent inhibitor of WcbL.
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Biosynthesis of Neocarazostatin A Reveals the Sequential Carbazole Prenylation and Hydroxylation in the Tailoring Steps ()
Publication date: 17 December 2015 Source:Chemistry & Biology, Volume 22, Issue 12 Author(s): Sheng Huang, Somayah Sameer Elsayed, Meinan Lv, Jioji Tabudravu, Mostafa E. Rateb, Roland Gyampoh, Kwaku Kyeremeh, Rainer Ebel, Marcel Jaspars, Zixin Deng, Yi Yu, Hai Deng Neocarazostatin A (NZS) is a bacterial alkaloid with promising bioactivities against free radicals, featuring a tricyclic carbazole nucleus with a prenyl moiety at C-6 of the carbazole ring. Here, we report the discovery and characterization of the biosynthetic pathway of NZS through genome mining and gene inactivation. The in vitro assays characterized two enzymes: NzsA is a P450 hydroxylase and NzsG is a new phytoene-synthase-like prenyltransferase (PTase). This is the first reported native PTase that specifically acts on the carbazole nucleus. Finally, our in vitro reconstituted experiment demonstrated a coupled reaction catalyzed by NzsG and NzsA tailoring the NZS biosynthesis. Graphical abstract image Teaser Huang et al. identified the gene cluster directing the biosynthesis of neocarazostatin A, characterized two new enzymes responsible for the late stage of the biosynthesis, and reconstituted in vitro the biotransformation from the biosynthetic intermediate to neocarazostatin A.
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Human ISPD Is a Cytidyltransferase Required for Dystroglycan O-Mannosylation ()
Publication date: 17 December 2015 Source:Chemistry & Biology, Volume 22, Issue 12 Author(s): Moniek Riemersma, D. Sean Froese, Walinka van Tol, Udo F. Engelke, Jolanta Kopec, Monique van Scherpenzeel, Angel Ashikov, Tobias Krojer, Frank von Delft, Marco Tessari, Anna Buczkowska, Ewa Swiezewska, Lucas T. Jae, Thijn R. Brummelkamp, Hiroshi Manya, Tamao Endo, Hans van Bokhoven, Wyatt W. Yue, Dirk J. Lefeber A unique, unsolved O-mannosyl glycan on α-dystroglycan is essential for its interaction with protein ligands in the extracellular matrix. Defective O-mannosylation leads to a group of muscular dystrophies, called dystroglycanopathies. Mutations in isoprenoid synthase domain containing (ISPD) represent the second most common cause of these disorders, however, its molecular function remains uncharacterized. The human ISPD (hISPD) crystal structure showed a canonical N-terminal cytidyltransferase domain linked to a C-terminal domain that is absent in cytidyltransferase homologs. Functional studies demonstrated cytosolic localization of hISPD, and cytidyltransferase activity toward pentose phosphates, including ribulose 5-phosphate, ribose 5-phosphate, and ribitol 5-phosphate. Identity of the CDP sugars was confirmed by liquid chromatography quadrupole time-of-flight mass spectrometry and two-dimensional nuclear magnetic resonance spectroscopy. Our combined results indicate that hISPD is a cytidyltransferase, suggesting the presence of a novel human nucleotide sugar essential for functional α-dystroglycan O-mannosylation in muscle and brain. Thereby, ISPD deficiency can be added to the growing list of tertiary dystroglycanopathies. Graphical abstract image Teaser Guided by X-ray crystallography and biochemical studies in ISPD knockout and overexpressing cell models, we implicated human ISPD in the synthesis of a novel human nucleotide sugar required for dystroglycan O-mannosylation in muscle and brain.
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Synthetic Peptides as cGMP-Independent Activators of cGMP-Dependent Protein Kinase Iα ()
Publication date: 17 December 2015 Source:Chemistry & Biology, Volume 22, Issue 12 Author(s): Thomas M. Moon, Nathan R. Tykocki, Jessica L. Sheehe, Brent W. Osborne, Werner Tegge, Joseph E. Brayden, Wolfgang R. Dostmann PKG is a multifaceted signaling molecule and potential pharmaceutical target due to its role in smooth muscle function. A helix identified in the structure of the regulatory domain of PKG Iα suggests a novel architecture of the holoenzyme. In this study, a set of synthetic peptides (S-tides), derived from this helix, was found to bind to and activate PKG Iα in a cyclic guanosine monophosphate (cGMP)-independent manner. The most potent S-tide derivative (S1.5) increased the open probability of the potassium channel KCa1.1 to levels equivalent to saturating cGMP. Introduction of S1.5 to smooth muscle cells in isolated, endothelium-denuded cerebral arteries through a modified reversible permeabilization procedure inhibited myogenic constriction. In contrast, in endothelium-intact vessels S1.5 had no effect on myogenic tone. This suggests that PKG Iα activation by S1.5 in vascular smooth muscle would be sufficient to inhibit augmented arterial contractility that frequently occurs following endothelial damage associated with cardiovascular disease. Graphical abstract image Teaser The control of vascular smooth muscle relaxation and blood flow are tightly linked to the activity of cGMP-dependent protein kinase (PKG). Moon et al. demonstrate the development and assay of a class of novel vasodilators that are selective cGMP-independent PKG Iα activators.
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Differential Regulation of Specific Sphingolipids in Colon Cancer Cells during Staurosporine-Induced Apoptosis ()
Publication date: 17 December 2015 Source:Chemistry & Biology, Volume 22, Issue 12 Author(s): Virginia del Solar, Darleny Y. Lizardo, Nasi Li, Jerod J. Hurst, Christopher J. Brais, G. Ekin Atilla-Gokcumen Apoptosis is accompanied by distinct morphological changes at the plasma and organelle membrane level. Involvement of certain lipids in apoptosis has been established; however, we have limited understanding of the specific lipid structures that participate in this process. We used untargeted comparative lipidomics to study the changes in lipid composition during staurosporine-induced apoptosis in HCT-116. Our results revealed that ceramides, dihydroceramides, and sphingomyelins, with defined acyl chains, constitute the majority of changes in the lipidome. Expression levels and activities of enzymes responsible for the biosynthesis of lipids that change suggest that de novo synthesis causes these specific changes. Further analysis of the lipidome during apoptosis in other cancer and non-cancer cell lines suggested that accumulation of ceramides and dihydroceramides is specific to cancer cells. Taken together, our data propose that these molecules are regulated at the lipid-specific level during apoptosis and that this regulation differs between cancer and non-cancer cells. Graphical abstract image Teaser del Solar et al. employ comparative lipidomics to analyze the changes in lipid composition during staurosporine-induced apoptosis in cancer and non-cancer cells, and show that specific ceramides and dihydroceramides accumulate in cancer cells but not in non-cancer cells.
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DIVERSE System: De Novo Creation of Peptide Tags for Non-enzymatic Covalent Labeling by In Vitro Evolution for Protein Imaging Inside Living Cells ()
Publication date: 17 December 2015 Source:Chemistry & Biology, Volume 22, Issue 12 Author(s): Takashi Kawakami, Koji Ogawa, Naoki Goshima, Tohru Natsume Polypeptide-tag/small-molecule pairs for specific cellular protein labeling are useful for visualizing cellular proteins and controlling their activity. Here, we report the development of an in vitro evolution-based (poly)peptide tag identification system named the DIVERSE (Directed In Vitro Evolution of Reactive peptide tags via Sequential Enrichment) system. In this system, an extremely diverse (1014) library of peptide tags, displayed by covalent attachment to their encoding cDNAs, is continuously prepared from the DNA library in a one-pot approach. Using this system, we demonstrated de novo creation of non-enzymatically covalent-labeling peptide tags for a synthetic small-molecule target from a random peptide library. Protein labeling with these tags was applicable to N- and C-terminal fusions, multiple different proteins and fluorophores, and intracellular labeling. The DIVERSE system can be used not only for the de novo creation of polypeptide tags but also sequence optimization of existing polypeptide tags from extremely diverse libraries. Graphical abstract image Teaser Kawakami et al. developed an in vitro evolution-based (poly)peptide tag identification system named the DIVERSE (Directed In Vitro Evolution of Reactive peptide tags via Sequential Enrichment) system. Using the DIVERSE system, the authors demonstrated de novo creation of peptide tags for non-enzymatic, covalent protein labeling.
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In This Issue ()
Publication date: 19 November 2015 Source:Chemistry & Biology, Volume 22, Issue 11
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Dissecting How Mtb Makes Its Wall, Buffering Endosomal pH, and Discovery of Ribocil ()
Publication date: 19 November 2015 Source:Chemistry & Biology, Volume 22, Issue 11 Each month, Chemistry & Biology Select highlights a selection of research reports from the recent literature. These highlights are a snapshot of interesting research done across the field of chemical biology. Our November 2015 selection includes an insight into non-overlapping biosynthetic pathways that lead to formation of Mycobacterium tuberculosis peptidoglycan, a new method to not only measure but also buffer the endosomal pH using nanoparticles, and a demonstration that non-coding RNAs can be a target for antibiotic discovery.
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Geometrically Precise Building Blocks: the Self-Assembly of β-Peptides ()
Publication date: 19 November 2015 Source:Chemistry & Biology, Volume 22, Issue 11 Author(s): Romila D. Gopalan, Mark P. Del Borgo, Adam I. Mechler, Patrick Perlmutter, Marie-Isabel Aguilar Peptides comprised entirely of β-amino acids, or β-peptides, have attracted substantial interest over the past 25 years due to their unique structural and chemical characteristics. β-Peptides form well-defined secondary structures that exhibit different geometries compared with their α-peptide counterparts, giving rise to their foldamer classification. β-Peptide foldamers can be functionalized easily and are metabolically stable and, together with the predictable side-chain topography, have led to the design of a growing number of bioactive β-peptides with a range of biological targets. The strategic engineering of chemical and topographic properties has also led to the design of β-peptide mimics of higher-order oligomers. More recently, the ability of these peptides to self-assemble into complex structures of controlled geometries has been exploited in materials applications. The focus of this mini-review is on how the unique structural features of β-peptide assemblies have been exploited in the design of self-assembled proteomimetic bundles and nanomaterials. Teaser Peptides comprised entirely of β-amino acids form unique structures that self-assemble to form ion channels, proteomimetic bundles, and DNA mimics. This structural template also allows the tailored design of new nanomaterials with unique physical properties for application in nanotechnology and biomedicine.
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Substrate Flexibility of a Mutated Acyltransferase Domain and Implications for Polyketide Biosynthesis ()
Publication date: 19 November 2015 Source:Chemistry & Biology, Volume 22, Issue 11 Author(s): Kenny Bravo-Rodriguez, Stephan Klopries, Kyra R.M. Koopmans, Uschi Sundermann, Samir Yahiaoui, Julia Arens, Susanna Kushnir, Frank Schulz, Elsa Sanchez-Garcia Polyketides are natural products frequently used for the treatment of various diseases, but their structural complexity hinders efficient derivatization. In this context, we recently introduced enzyme-directed mutasynthesis to incorporate non-native extender units into the biosynthesis of erythromycin. Modeling and mutagenesis studies led to the discovery of a variant of an acyltransferase domain in the erythromycin polyketide synthase capable of accepting a propargylated substrate. Here, we extend molecular rationalization of enzyme-substrate interactions through modeling, to investigate the incorporation of substrates with different degrees of saturation of the malonic acid side chain. This allowed the engineered biosynthesis of new erythromycin derivatives and the introduction of additional mutations into the AT domain for a further shift of the enzyme's substrate scope. Our approach yields non-native polyketide structures with functional groups that will simplify future derivatization approaches, and provides a blueprint for the engineering of AT domains to achieve efficient polyketide synthase diversification. Graphical abstract image Teaser Molecular modeling of polyketide synthase domains can yield insights into the structure and function of these giant and complex enzymes. Based on modeling, mutations are devised that shift the substrate scope of an acyltransferase domain of a polyketide synthase toward the incorporation of artificial building blocks into the biosynthesis of the important antibiotic erythromycin to generate new derivatives.
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Bioorthogonal Labeling of Ghrelin Receptor to Facilitate Studies of Ligand-Dependent Conformational Dynamics ()
Publication date: 19 November 2015 Source:Chemistry & Biology, Volume 22, Issue 11 Author(s): Minyoung Park, Bjørn B. Sivertsen, Sylvia Els-Heindl, Thomas Huber, Birgitte Holst, Annette G. Beck-Sickinger, Thue W. Schwartz, Thomas P. Sakmar Ghrelin receptor (GhrR) is a promising drug target because of its central role in energy homeostasis. GhrR, known for high constitutive activity, is thought to display multi-state conformations during activation and signaling. We used genetically encoded unnatural amino acids and bioorthogonal labeling reactions to engineer multiple fluorescent donor-acceptor pairs to probe ligand-directed structural changes in GhrR. We demonstrate how conformational dynamics of a G-protein-coupled receptor can be measured in reconstituted systems. Graphical abstract image Teaser Understanding ligand-induced conformations and consequences in ghrelin receptor signaling might assist in drug design. Park et al. use bioorthogonally labeled ghrelin receptors and show that RET-based approaches can reveal distinctive ligand-induced RET signals that are sensitive to inter- and intramolecular conformational changes.
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An L-RNA Aptamer that Binds and Inhibits RNase ()
Publication date: 19 November 2015 Source:Chemistry & Biology, Volume 22, Issue 11 Author(s): Charles Olea, Joachim Weidmann, Philip E. Dawson, Gerald F. Joyce L-RNA aptamers were developed that bind to barnase RNase and thereby inhibit the function of the enzyme. These aptamers were obtained by first carrying out in vitro selection of D-RNAs that bind to the full-length synthetic D-enantiomer of barnase, then reversing the mirror and preparing L-RNAs of identical sequence that similarly bind to natural L-barnase. The resulting L-aptamers bind L-barnase with an affinity of ∼100 nM and function as competitive inhibitors of enzyme cleavage of D-RNA substrates. L-RNA aptamers are resistant to degradation by ribonucleases, thus enabling them to function in biological samples, most notably for applications in molecular diagnostics and therapeutics. In addition to the irony of using RNA to inhibit RNase, L-RNA aptamers such as those described here could be used to measure the concentration or inhibit the function of RNase in the laboratory or in biological systems. Graphical abstract image Teaser Olea et al. select mirror-image RNA molecules, composed of the non-natural L-isomer, for their ability to bind and inhibit RNase, an enzyme that rapidly degrades natural RNA. This was accomplished by selecting natural RNAs that bind the full-length, non-natural isomer of RNase then reversing the mirror to enable L-RNA to protect its natural counterpart.
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Combinatorial Screening Identifies Novel Promiscuous Matrix Metalloproteinase Activities that Lead to Inhibition of the Therapeutic Target IL-13 ()
Publication date: 19 November 2015 Source:Chemistry & Biology, Volume 22, Issue 11 Author(s): Carole Urbach, Nathaniel C. Gordon, Ian Strickland, David Lowne, Cathy Joberty-Candotti, Richard May, Athula Herath, DirkJan Hijnen, Judith L. Thijs, Carla A. Bruijnzeel-Koomen, Ralph R. Minter, Florian Hollfelder, Lutz Jermutus The practical realization of disease modulation by catalytic degradation of a therapeutic target protein suffers from the difficulty to identify candidate proteases, or to engineer their specificity. We identified 23 measurable, specific, and new protease activities using combinatorial screening of 27 human proteases against 24 therapeutic protein targets. We investigate the cleavage of monocyte chemoattractant protein 1, interleukin-6 (IL-6), and IL-13 by matrix metalloproteinases (MMPs) and serine proteases, and demonstrate that cleavage of IL-13 leads to potent inhibition of its biological activity in vitro. MMP-8 degraded human IL-13 most efficiently in vitro and ex vivo in human IL-13 transgenic mouse bronchoalveolar lavage. Hence, MMP-8 is a therapeutic protease lead against IL-13 for inflammatory conditions whereby reported genetic and genomics data suggest an involvement of MMP-8. This work describes the first exploitation of human enzyme promiscuity for therapeutic applications, and reveals both starting points for protease-based therapies and potential new regulatory networks in inflammatory disease. Teaser Proteases with therapeutically relevant activities are generally unknown and cannot be tailor-made. Urbach et al. explore the substrate promiscuity of natural proteases to successfully identify starting scaffolds for novel biological therapies.
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Probing the Substrate Specificity and Protein-Protein Interactions of the E. coli Fatty Acid Dehydratase, FabA ()
Publication date: 19 November 2015 Source:Chemistry & Biology, Volume 22, Issue 11 Author(s): Kara Finzel, Chi Nguyen, David R. Jackson, Aarushi Gupta, Shiou-Chuan Tsai, Michael D. Burkart Microbial fatty acid biosynthetic enzymes are important targets for areas as diverse as antibiotic development to biofuel production. Elucidating the molecular basis of chain length control during fatty acid biosynthesis is crucial for the understanding of regulatory processes of this fundamental metabolic pathway. In Escherichia coli, the acyl carrier protein (AcpP) plays a central role by sequestering and shuttling the growing acyl chain between fatty acid biosynthetic enzymes. FabA, a β-hydroxyacyl-AcpP dehydratase, is an important enzyme in controlling fatty acid chain length and saturation levels. FabA-AcpP interactions are transient in nature and thus difficult to visualize. In this study, four mechanistic crosslinking probes mimicking varying acyl chain lengths were synthesized to systematically probe for modified chain length specificity of 14 FabA mutants. These studies provide evidence for the AcpP-interacting “positive patch,” FabA mutations that alter substrate specificity, and the roles that the FabA “gating residues” play in chain length control. Graphical abstract image Teaser Finzel et al. utilized synthetic probes and FabA mutations to detect modified fatty acid dehydratase (FabA)-substrate and FabA-acyl carrier protein (AcpP) interactions. Altering FabA led to the first gain-of-function activity for shorter chain length fatty acid substrates.
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Development of a Clickable Probe for Profiling of Protein Glutathionylation in the Central Cellular Metabolism of E. coli and Drosophila ()
Publication date: 19 November 2015 Source:Chemistry & Biology, Volume 22, Issue 11 Author(s): Shan Feng, Yuling Chen, Fan Yang, Lei Zhang, Yiyi Gong, Gulishana Adilijiang, Yan Gao, Haiteng Deng Protein glutathionylation is an important post-translational modification that regulates many cellular processes, including energy metabolism, signal transduction, and protein homeostasis. Global profiling of glutathionylated proteins (denoted as glutathionylome) is crucial for understanding redox-regulated signal transduction. Here, we developed a novel method based on click reaction and proteomics to enrich and identify the glutathionylated peptides in Escherichia coli and Drosophila lysates, in which 937 and 1,930 potential glutathionylated peptides were identified, respectively. Bioinformatics analysis showed that the cysteine residue next to negatively charged amino acid residues has a higher frequency of glutathionylation. Importantly, we found that most proteins associated with metabolic pathways were glutathionylated and that the glutathionylation sites of metabolic enzymes were highly conserved among different species. Our results indicate that the glutathione analog is a useful tool to characterize protein glutathionylation, and glutathionylation of metabolic enzymes, which play important roles in regulating cellular metabolism, is conserved. Graphical abstract image Teaser Protein glutathionylation plays a crucial role in various cellular processes. Feng et al. describe a novel method based on click reaction to profile glutathionylated proteins and sites in E. coli and Drosophila. The selectivity and conservatism of glutathionylation are characterized.
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Visualization of Compartmentalized Kinase Activity Dynamics Using Adaptable BimKARs ()
Publication date: 19 November 2015 Source:Chemistry & Biology, Volume 22, Issue 11 Author(s): Charlene Depry, Sohum Mehta, Ruojing Li, Jin Zhang The ability to monitor kinase activity dynamics in live cells greatly aids the study of how signaling events are spatiotemporally regulated. Here, we report on the adaptability of bimolecular kinase activity reporters (bimKARs) as molecular tools to enhance the real-time visualization of kinase activity. We demonstrate that the bimKAR design is truly versatile and can be used to monitor a variety of kinases, including JNK, ERK, and AMPK. Furthermore, bimKARs can have significantly enhanced dynamic ranges over their unimolecular counterparts, allowing the elucidation of previously undetectable kinase activity dynamics. Using these newly designed bimKARs, we investigate the regulation of AMPK by protein kinase A (PKA) in the plasma membrane, and demonstrate that PKA can both negatively and positively regulate AMPK activity in the same cell. Graphical abstract image Teaser Depry et al. demonstrate the versatility of FRET-based bimolecular kinase activity reporters (bimKARs) as tools for improving the visualization of signaling dynamics in living cells, and reveal that PKA acts simultaneously as both a positive and negative regulator of AMPK signaling at the plasma membrane.
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Biochemical Studies of Mycobacterial Fatty Acid Methyltransferase: A Catalyst for the Enzymatic Production of Biodiesel ()
Publication date: 19 November 2015 Source:Chemistry & Biology, Volume 22, Issue 11 Author(s): Nektaria Petronikolou, Satish K. Nair Transesterification of fatty acids yields the essential component of biodiesel, but current processes are cost-prohibitive and generate waste. Recent efforts make use of biocatalysts that are effective in diverting products from primary metabolism to yield fatty acid methyl esters in bacteria. These biotransformations require the fatty acid O-methyltransferase (FAMT) from Mycobacterium marinum (MmFAMT). Although this activity was first reported in the literature in 1970, the FAMTs have yet to be biochemically characterized. Here, we describe several crystal structures of MmFAMT, which highlight an unexpected structural conservation with methyltransferases that are involved in plant natural product metabolism. The determinants for ligand recognition are analyzed by kinetic analysis of structure-based active-site variants. These studies reveal how an architectural fold employed in plant natural product biosynthesis is used in bacterial fatty acid O-methylation. Graphical abstract image Teaser Mycobacterial fatty acid methyltransferases are employed as biocatalysts for the production of biodiesel. Petronikolou and Nair describe structural and biochemical characterization of a mycobacterial fatty acid methyltransferase, reveal an unexpected homology to enzymes involved in plant primary metabolism, and provide insights into substrate preference.
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Small-Molecule Disruption of RAD52 Rings as a Mechanism for Precision Medicine in BRCA-Deficient Cancers ()
Publication date: 19 November 2015 Source:Chemistry & Biology, Volume 22, Issue 11 Author(s): Gurushankar Chandramouly, Shane McDevitt, Katherine Sullivan, Tatiana Kent, Antonio Luz, J. Fraser Glickman, Mark Andrake, Tomasz Skorski, Richard T. Pomerantz Suppression of RAD52 causes synthetic lethality in BRCA-deficient cells. Yet pharmacological inhibition of RAD52, which binds single-strand DNA (ssDNA) and lacks enzymatic activity, has not been demonstrated. Here, we identify the small molecule 6-hydroxy-DL-dopa (6-OH-dopa) as a major allosteric inhibitor of the RAD52 ssDNA binding domain. For example, we find that multiple small molecules bind to and completely transform RAD52 undecamer rings into dimers, which abolishes the ssDNA binding channel observed in crystal structures. 6-OH-Dopa also disrupts RAD52 heptamer and undecamer ring superstructures, and suppresses RAD52 recruitment and recombination activity in cells with negligible effects on other double-strand break repair pathways. Importantly, we show that 6-OH-dopa selectively inhibits the proliferation of BRCA-deficient cancer cells, including those obtained from leukemia patients. Taken together, these data demonstrate small-molecule disruption of RAD52 rings as a promising mechanism for precision medicine in BRCA-deficient cancers. Graphical abstract image Teaser Chandramouly et al. show that the small molecule 6-hydroxy-DL-dopa prevents RAD52 from binding to single-strand DNA by disrupting oligomeric ring structures of the protein, and selectively kills BRCA-deficient cell lines and leukemia patient cells by allosterically inactivating RAD52.
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Engineering Duplex RNAs for Challenging Targets: Recognition of GGGGCC/CCCCGG Repeats at the ALS/FTD C9orf72 Locus ()
Publication date: 19 November 2015 Source:Chemistry & Biology, Volume 22, Issue 11 Author(s): Jiaxin Hu, Jing Liu, Liande Li, Keith T. Gagnon, David R. Corey A GGGGCC expansion within an intronic region of the C9orf72 gene forms RNA foci that are associated with one-third of familial amyotrophic lateral sclerosis and one-quarter of frontotemporal dementia. The C9orf72 locus also expresses an antisense transcript with a CCCCGG expansion that forms foci and may contribute to disease. Synthetic agents that bind these hexanucleotide repeats and block foci would be leads for therapeutic discovery. We have engineered duplex RNAs to enable them to recognize difficult C/G targets. Recognition inhibits foci formed by both GGGGCC and CCCCGG RNA. Our findings show that a single duplex RNA can be used to recognize both disease-related C9orf72 transcripts. More broadly, we extend RNAi to previously inaccessible C/G sequences and provide another example of target recognition in human cells by nuclear RNAi. Graphical abstract image Teaser A GGGGCC hexanucleotide expansion within the C9orf72 gene can cause familial amyotrophic lateral sclerosis and frontotemporal dementia. Hu et al. have engineered duplex RNAs to enable them to recognize difficult C/G targets and inhibit potential disease-causing foci formed by both GGGGCC and CCCCGG RNA.
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Combining Suppression of Stemness with Lineage-Specific Induction Leads to Conversion of Pluripotent Cells into Functional Neurons ()
Publication date: 19 November 2015 Source:Chemistry & Biology, Volume 22, Issue 11 Author(s): Debasish Halder, Gyeong-Eon Chang, Debojyoti De, Eunji Cheong, Kyeong Kyu Kim, Injae Shin Sox2 is a key player in the maintenance of pluripotency and stemness, and thus inhibition of its function would abrogate the stemness of pluripotent cells and induce differentiation into several types of cells. Herein we describe a strategy that relies on a combination of Sox2 inhibition with lineage-specific induction to promote efficient and selective differentiation of pluripotent P19 cells into neurons. When P19 cells transduced with Skp protein, an inhibitor of Sox2, are incubated with a neurogenesis inducer, the cells are selectively converted into neurons that generate depolarization-induced sodium currents and action potentials. This finding indicates that the differentiated neurons are electrophysiologically active. Signaling pathway studies lead us to conclude that a combination of Skp with the neurogenesis inducer enhances neurogenesis in P19 cells by activating Wnt and Notch pathways. The present differentiation protocol could be valuable to selectively generate functionally active neurons from pluripotent cells. Graphical abstract image Teaser Halder et al. demonstrated that a combination of Sox2 inhibition with lineage-specific induction led to efficient and selective differentiation of pluripotent P19 cells into neurons. Differentiated neuronal cells exhibited voltage-dependent inward and outward sodium currents and depolarization-induced action potentials.
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Host-Microbe Protein Interactions during Bacterial Infection ()
Publication date: 19 November 2015 Source:Chemistry & Biology, Volume 22, Issue 11 Author(s): Devin K. Schweppe, Christopher Harding, Juan D. Chavez, Xia Wu, Elizabeth Ramage, Pradeep K. Singh, Colin Manoil, James E. Bruce Interspecies protein-protein interactions are essential mediators of infection. While bacterial proteins required for host cell invasion and infection can be identified through bacterial mutant library screens, information about host target proteins and interspecies complex structures has been more difficult to acquire. Using an unbiased chemical crosslinking/mass spectrometry approach, we identified interspecies protein-protein interactions in human lung epithelial cells infected with Acinetobacter baumannii. These efforts resulted in identification of 3,076 crosslinked peptide pairs and 46 interspecies protein-protein interactions. Most notably, the key A. baumannii virulence factor, OmpA, was identified as crosslinked to host proteins involved in desmosomes, specialized structures that mediate host cell-to-cell adhesion. Co-immunoprecipitation and transposon mutant experiments were used to verify these interactions and demonstrate relevance for host cell invasion and acute murine lung infection. These results shed new light on A. baumannii-host protein interactions and their structural features, and the presented approach is generally applicable to other systems. Graphical abstract image Teaser Pathogenic bacteria exploit host resources through difficult-to-determine interactions between bacterial and host proteins. Schweppe et al. identified interspecies protein interactions during Acinetobacter baumannii infection of lung epithelia by protein crosslinking and mass spectrometry. Host protein targets for bacterial virulence factors were revealed, and crosslinked sites provide structural information for interspecies interactions during infection.
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Optogenetic Inhibitor of the Transcription Factor CREB ()
Publication date: 19 November 2015 Source:Chemistry & Biology, Volume 22, Issue 11 Author(s): Ahmed M. Ali, Jakeb M. Reis, Yan Xia, Asim J. Rashid, Valentina Mercaldo, Brandon J. Walters, Katherine E. Brechun, Vitali Borisenko, Sheena A. Josselyn, John Karanicolas, G. Andrew Woolley Current approaches for optogenetic control of transcription do not mimic the activity of endogenous transcription factors, which act at numerous sites in the genome in a complex interplay with other factors. Optogenetic control of dominant negative versions of endogenous transcription factors provides a mechanism for mimicking the natural regulation of gene expression. Here we describe opto-DN-CREB, a blue-light-controlled inhibitor of the transcription factor CREB created by fusing the dominant negative inhibitor A-CREB to photoactive yellow protein (PYP). A light-driven conformational change in PYP prevents coiled-coil formation between A-CREB and CREB, thereby activating CREB. Optogenetic control of CREB function was characterized in vitro, in HEK293T cells, and in neurons where blue light enabled control of expression of the CREB targets NR4A2 and c-Fos. Dominant negative inhibitors exist for numerous transcription factors; linking these to optogenetic domains offers a general approach for spatiotemporal control of native transcriptional events. Graphical abstract image Teaser Ali et al. use protein engineering to create opto-DN-CREB, a blue-light-controlled specific inhibitor of the transcription factor CREB.
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Molecular Basis of Spectral Diversity in Near-Infrared Phytochrome-Based Fluorescent Proteins ()
Publication date: 19 November 2015 Source:Chemistry & Biology, Volume 22, Issue 11 Author(s): Daria M. Shcherbakova, Mikhail Baloban, Sergei Pletnev, Vladimir N. Malashkevich, Hui Xiao, Zbigniew Dauter, Vladislav V. Verkhusha Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes (BphPs) are the probes of choice for deep-tissue imaging. Detection of several processes requires spectrally distinct NIR FPs. We developed an NIR FP, BphP1-FP, which has the most blue-shifted spectra and the highest fluorescence quantum yield among BphP-derived FPs. We found that these properties result from the binding of the biliverdin chromophore to a cysteine residue in the GAF domain, unlike natural BphPs and other BphP-based FPs. To elucidate the molecular basis of the spectral shift, we applied biochemical, structural and mass spectrometry analyses and revealed the formation of unique chromophore species. Mutagenesis of NIR FPs of different origins indicated that the mechanism of the spectral shift is general and can be used to design multicolor NIR FPs from other BphPs. We applied pairs of spectrally distinct point cysteine mutants to multicolor cell labeling and demonstrated that they perform well in model deep-tissue imaging. Graphical abstract image Teaser We show that the biliverdin chromophore can bind to cysteine in the GAF domain of bacterial phytochromes and derived near-infrared fluorescent proteins, resulting in their blue shift and high quantum yield. This opens up the way to develop spectrally distinct near-infrared fluorescent proteins for multicolor imaging.
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Profiling of Free Fatty Acids Using Stable Isotope Tagging Uncovers a Role for Saturated Fatty Acids in Neuroexocytosis ()
Publication date: 19 November 2015 Source:Chemistry & Biology, Volume 22, Issue 11 Author(s): Vinod K. Narayana, Vanesa M. Tomatis, Tong Wang, David Kvaskoff, Frederic A. Meunier The phospholipase-catalyzed release of free fatty acids (FFAs) from phospholipids is implicated in many critical biological processes such as neurotransmission, inflammation, and cancer. However, determining the individual change in FFAs generated during these processes has remained challenging due to the limitations of current methods, and has hampered our understanding of these key mediators. Here, we developed an “iTRAQ”-like method for profiling FFAs by stable isotope tagging (FFAST), based on the differential labeling of the carboxyl group and designed to resolve analytical variance, through a multiplexed assay in cells and subcellular fractions. With nanomolar sensitivity, this method revealed a spectrum of saturated FFAs elicited during stimulation of exocytosis that was identical in neurons and neurosecretory cells. Purified secretory vesicles also generated these FFAs when challenged with cytosol. Our multiplex method will be invaluable to assess the range of FFAs generated in other physiological and pathological settings. Graphical abstract image Teaser Narayana et al. developed a free fatty acid stable isotope tagging (FFAST) method that enables multiplexed quantification of endogenous free fatty acids with nanomolar sensitivity. Here, they uncover an unexpected variety of change in free fatty acids generated during neuroexocytosis in neurons and chromaffin cells.
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Quantitative Lipoproteomics in Clostridium difficile Reveals a Role for Lipoproteins in Sporulation ()
Publication date: 19 November 2015 Source:Chemistry & Biology, Volume 22, Issue 11 Author(s): Thomas M. Charlton, Andrea Kovacs-Simon, Stephen L. Michell, Neil F. Fairweather, Edward W. Tate Bacterial lipoproteins are surface exposed, anchored to the membrane by S-diacylglyceryl modification of the N-terminal cysteine thiol. They play important roles in many essential cellular processes and in bacterial pathogenesis. For example, Clostridium difficile is a Gram-positive anaerobe that causes severe gastrointestinal disease; however, its lipoproteome remains poorly characterized. Here we describe the application of metabolic tagging with alkyne-tagged lipid analogs, in combination with quantitative proteomics, to profile protein lipidation across diverse C. difficile strains and on inactivation of specific components of the lipoprotein biogenesis pathway. These studies provide the first comprehensive map of the C. difficile lipoproteome, demonstrate the existence of two active lipoprotein signal peptidases, and provide insights into lipoprotein function, implicating the lipoproteome in transmission of this pathogen. Graphical abstract image Teaser Bacterial lipoproteins are S-diacylglyceryl modified, surface anchored proteins, which play important roles at the host-pathogen interface. We use metabolic tagging, combined with inactivation of lipoprotein biosynthesis, to profile the Clostridium difficile lipoproteome, revealing a role for lipoproteins in transmission of this pathogen.
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In This Issue ()
Publication date: 22 October 2015 Source:Chemistry & Biology, Volume 22, Issue 10
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Nuclear Pore Complex: From Structural View to Chemical Tools ()
Publication date: 22 October 2015 Source:Chemistry & Biology, Volume 22, Issue 10 Author(s): Richard W. Wong Nuclear pore complexes (NPCs) are the macromolecular turnstiles between the cytoplasm and the nucleus that control the trafficking of proteins, RNAs and viruses. The giant NPC structures are extremely complex. Here, I highlight several recent findings on NPC architectures, and briefly discuss how chemical biologists might use this information to design synthetic devices and improve strategies for nuclear drug delivery.
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Targeting Mycobacterial Enzymes with Natural Products ()
Publication date: 22 October 2015 Source:Chemistry & Biology, Volume 22, Issue 10 Author(s): Elwira Sieniawska Tuberculosis (TB) is a recurring threat to contemporary civilization. It affects not only those within developing countries, but has also appeared again in places where it was once considered eradicated. TB co-infection in patients infected by HIV is, at the time of writing, the most common cause of death. In the field of searching for new antimycobacterial drug leads, compounds of natural origin still remain a promising source. The review is intended to gather information about natural products (metabolites of plants, fungi, bacteria, and marine sponges) that show activity against mycobacterial enzymes. Here, natural metabolites are presented as being inhibitors/activators of the mycobacterial enzymes involved in mycobacterial growth in vitro (ClpC1, ClpP, MurE ligase, mycothiol S-conjugate amidase, β-ketoacyl-ACP synthase, InhA) and in vivo, as regards the host cell (PtpB). Each enzyme is briefly described so as to generate an understanding of its role in mycobacterial growth and engender a perception of the mechanism of action of the studied natural compounds. Furthermore, after the introduction of the enzyme, its inhibitors are listed and exactly characterized. Teaser Mycobacterial enzymes are targets of important antibiotics and new drug leads. Sieniawska reviews the natural products originating from plants, fungi, bacteria, and marine sponges as sources of inhibitors/activators of mycobacterial enzymes involved in mycobacterial growth in vitro and in vivo.
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XBP1s Links the Unfolded Protein Response to the Molecular Architecture of Mature N-Glycans ()
Publication date: 22 October 2015 Source:Chemistry & Biology, Volume 22, Issue 10 Author(s): Mahender B. Dewal, Andrew S. DiChiara, Aristotelis Antonopoulos, Rebecca J. Taylor, Chyleigh J. Harmon, Stuart M. Haslam, Anne Dell, Matthew D. Shoulders The molecular architecture of the mature N-glycome is dynamic, with consequences for both normal and pathologic processes. Elucidating cellular mechanisms that modulate the N-linked glycome is, therefore, crucial. The unfolded protein response (UPR) is classically responsible for maintaining proteostasis in the secretory pathway by defining levels of chaperones and quality control proteins. Here, we employ chemical biology methods for UPR regulation to show that stress-independent activation of the UPR’s XBP1s transcription factor also induces a panel of N-glycan maturation-related enzymes. The downstream consequence is a distinctive shift toward specific hybrid and complex N-glycans on N-glycoproteins produced from XBP1s-activated cells, which we characterize by mass spectrometry. Pulse-chase studies attribute this shift specifically to altered N-glycan processing, rather than to changes in degradation or secretion rates. Our findings implicate XBP1s in a new role for N-glycoprotein biosynthesis, unveiling an important link between intracellular stress responses and the molecular architecture of extracellular N-glycoproteins. Graphical abstract image Teaser The molecular architecture of the N-glycome is regulated by poorly defined mechanisms. Dewal et al. now demonstrate that the unfolded protein response plays a critical role in N-glycan maturation, unveiling a functional link between intracellular proteostasis and extracellular N-glycoprotein structures.
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Characterization of the Biosynthetic Gene Cluster for Benzoxazole Antibiotics A33853 Reveals Unusual Assembly Logic ()
Publication date: 22 October 2015 Source:Chemistry & Biology, Volume 22, Issue 10 Author(s): Meinan Lv, Junfeng Zhao, Zixin Deng, Yi Yu A33853, which shows excellent bioactivity against Leishmania, is a benzoxazole-family compound formed from two moieties of 3-hydroxyanthranilic acid and one 3-hydroxypicolinic acid. In this study, we have identified the gene cluster responsible for the biosynthesis of A33853 in Streptomyces sp. NRRL12068 through genome mining and heterologous expression. Bioinformatics analysis and functional characterization of the orfs contained in the gene cluster revealed that the biosynthesis of A33853 is directed by a group of unusual enzymes. In particular, BomK, annotated as a ketosynthase, was found to catalyze the amide bond formation between 3-hydroxypicolinic and 3-hydroxyanthranilic acid during the assembly of A33853. BomJ, a putative ATP-dependent coenzyme A ligase, and BomN, a putative amidohydrolase, were further proposed to be involved in the benzoxazole formation in A33853 according to gene deletion experiments. Finally, we have successfully utilized mutasynthesis to generate two analogs of A33853, which were reported previously to possess excellent anti-leishmanial activity. Graphical abstract image Teaser Lv et al. unveil the pathway that directs the biosynthesis of the promising anti-leishmanial drug lead A33853, characterize a group of unusual enzymes responsible for the skeleton assembly of A33853, and generate two analogs of A33853 via mutasynthesis.
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Three Redundant Synthetases Secure Redox-Active Pigment Production in the Basidiomycete Paxillus involutus ()
Publication date: 22 October 2015 Source:Chemistry & Biology, Volume 22, Issue 10 Author(s): Jana Braesel, Sebastian Götze, Firoz Shah, Daniel Heine, James Tauber, Christian Hertweck, Anders Tunlid, Pierre Stallforth, Dirk Hoffmeister The symbiotic fungus Paxillus involutus serves a critical role in maintaining forest ecosystems, which are carbon sinks of global importance. P. involutus produces involutin and other 2,5-diarylcyclopentenone pigments that presumably assist in the oxidative degradation of lignocellulose via Fenton chemistry. Their precise biosynthetic pathways, however, remain obscure. Using a combination of biochemical, genetic, and transcriptomic analyses, in addition to stable-isotope labeling with synthetic precursors, we show that atromentin is the key intermediate. Atromentin is made by tridomain synthetases of high similarity: InvA1, InvA2, and InvA5. An inactive atromentin synthetase, InvA3, gained activity after a domain swap that replaced its native thioesterase domain with that of InvA5. The found degree of multiplex biosynthetic capacity is unprecedented with fungi, and highlights the great importance of the metabolite for the producer. Graphical abstract image Teaser Diarylcyclopentenones, produced by the symbiotic fungus Paxillus involutus, are redox-active metabolites involved in carbon cycling as they serve Fenton-based decomposition of lignocellulose in forest ecosystems. Braesel et al. show that the fungus uses three enzymes in parallel to secure the key step in diarylcyclopentenone biosynthesis.
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MEK Inhibitors Reverse cAMP-Mediated Anxiety in Zebrafish ()
Publication date: 22 October 2015 Source:Chemistry & Biology, Volume 22, Issue 10 Author(s): Pia R. Lundegaard, Corina Anastasaki, Nicola J. Grant, Rowland R. Sillito, Judith Zich, Zhiqiang Zeng, Karthika Paranthaman, Anders Peter Larsen, J. Douglas Armstrong, David J. Porteous, E. Elizabeth Patton Altered phosphodiesterase (PDE)-cyclic AMP (cAMP) activity is frequently associated with anxiety disorders, but current therapies act by reducing neuronal excitability rather than targeting PDE-cAMP-mediated signaling pathways. Here, we report the novel repositioning of anti-cancer MEK inhibitors as anxiolytics in a zebrafish model of anxiety-like behaviors. PDE inhibitors or activators of adenylate cyclase cause behaviors consistent with anxiety in larvae and adult zebrafish. Small-molecule screening identifies MEK inhibitors as potent suppressors of cAMP anxiety behaviors in both larvae and adult zebrafish, while causing no anxiolytic behavioral effects on their own. The mechanism underlying cAMP-induced anxiety is via crosstalk to activation of the RAS-MAPK signaling pathway. We propose that targeting crosstalk signaling pathways can be an effective strategy for mental health disorders, and advance the repositioning of MEK inhibitors as behavior stabilizers in the context of increased cAMP. Graphical abstract image Teaser Lundegaard et al. identify therapeutic potential for anti-cancer MEK inhibitors to treat anxiety-like cAMP-mediated behaviors in zebrafish. Targeting cAMP-MAPK crosstalk pathways broadens the range of therapeutic targets for mental health disorders. This work illustrates the importance of whole-animal phenotypic screening in anxiety drug discovery and repurposing.
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Development of Potent and Selective Tissue Transglutaminase Inhibitors: Their Effect on TG2 Function and Application in Pathological Conditions ()
Publication date: 22 October 2015 Source:Chemistry & Biology, Volume 22, Issue 10 Author(s): Eduard Badarau, Zhuo Wang, Dan L. Rathbone, Andrea Costanzi, Thomas Thibault, Colin E. Murdoch, Said El Alaoui, Milda Bartkeviciute, Martin Griffin Potent-selective peptidomimetic inhibitors of tissue transglutaminase (TG2) were developed through a combination of protein-ligand docking and molecular dynamic techniques. Derivatives of these inhibitors were made with the aim of specific TG2 targeting to the intra- and extracellular space. A cell-permeable fluorescently labeled derivative enabled detection of in situ cellular TG2 activity in human umbilical cord endothelial cells and TG2-transduced NIH3T3 cells, which could be enhanced by treatment of cells with ionomycin. Reaction of TG2 with this fluorescent inhibitor in NIH3T3 cells resulted in loss of binding of TG2 to cell surface syndecan-4 and inhibition of translocation of the enzyme into the extracellular matrix, with a parallel reduction in fibronectin deposition. In human umbilical cord endothelial cells, this same fluorescent inhibitor also demonstrated a reduction in fibronectin deposition, cell motility, and cord formation in Matrigel. Use of the same inhibitor in a mouse model of hypertensive nephrosclerosis showed over a 40% reduction in collagen deposition. Graphical abstract image Teaser Badarau et al. design and develop high-potency TG2-specific irreversible inhibitors that show reactivity with the intracellular active form of TG2, leading to inhibition of its translocation into the extracellular matrix. The compounds are effective in inhibiting in vitro angiogenesis and hypertensive nephrosclerosis in animal models.
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Elucidation of DnaE as the Antibacterial Target of the Natural Product, Nargenicin ()
Publication date: 22 October 2015 Source:Chemistry & Biology, Volume 22, Issue 10 Author(s): Ronald E. Painter, Gregory C. Adam, Marta Arocho, Edward DiNunzio, Robert G.K. Donald, Karen Dorso, Olga Genilloud, Charles Gill, Michael Goetz, Nichelle N. Hairston, Nicholas Murgolo, Bakela Nare, David B. Olsen, Maryann Powles, Fred Racine, Jing Su, Francisca Vicente, Douglas Wisniewski, Li Xiao, Milton Hammond, Katherine Young Resistance to existing classes of antibiotics drives the need for discovery of novel compounds with unique mechanisms of action. Nargenicin A1, a natural product with limited antibacterial spectrum, was rediscovered in a whole-cell antisense assay. Macromolecular labeling in both Staphylococcus aureus and an Escherichia coli tolC efflux mutant revealed selective inhibition of DNA replication not due to gyrase or topoisomerase IV inhibition. S. aureus nargenicin-resistant mutants were selected at a frequency of ∼1 × 10−9, and whole-genome resequencing found a single base-pair change in the dnaE gene, a homolog of the E. coli holoenzyme α subunit. A DnaE single-enzyme assay was exquisitely sensitive to inhibition by nargenicin, and other in vitro characterization studies corroborated DnaE as the target. Medicinal chemistry efforts may expand the spectrum of this novel mechanism antibiotic. Teaser There is an urgent need for new antibiotics due to continuing emergence of resistance. Painter et al. identified the antibacterial mechanism of nargenicin: inhibition of the replicative bacterial polymerase DnaE. This is the first known example of such an inhibitor.
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Fluorinated Sterols Are Suicide Inhibitors of Ergosterol Biosynthesis and Growth in Trypanosoma brucei ()
Publication date: 22 October 2015 Source:Chemistry & Biology, Volume 22, Issue 10 Author(s): David J. Leaver, Presheet Patkar, Ujjal K. Singha, Matthew B. Miller, Brad A. Haubrich, Minu Chaudhuri, W. David Nes Trypanosoma brucei, the causal agent for sleeping sickness, depends on ergosterol for growth. Here, we describe the effects of a mechanism-based inhibitor, 26-fluorolanosterol (26FL), which converts in vivo to a fluorinated substrate of the sterol C24-methyltransferase essential for sterol methylation and function of ergosterol, and missing from the human host. 26FL showed potent inhibition of ergosterol biosynthesis and growth of procyclic and bloodstream forms while having no effect on cholesterol biosynthesis or growth of human epithelial kidney cells. During exposure of cloned TbSMT to 26-fluorocholesta-5,7,24-trienol, the enzyme is gradually killed as a consequence of the covalent binding of the intermediate C25 cation to the active site (k cat/k inact = 0.26 min−1/0.24 min−1; partition ratio of 1.08), whereas 26FL is non-productively bound. These results demonstrate that poisoning of ergosterol biosynthesis by a 26-fluorinated Δ24-sterol is a promising strategy for developing a new treatment for trypanosomiasis. Graphical abstract image Teaser Leaver et al. used fluorinated steroids as suicide inhibitors of sterol C24 methyltransferase to inhibit ergosterol biosynthesis and growth of Trypanosoma brucei. This study demonstrates the potential of treating neglected tropical diseases with fluorinated analogs of a crucial enzyme in protozoan parasites absent from the human host.
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Engineered Domain Swapping as an On/Off Switch for Protein Function ()
Publication date: 22 October 2015 Source:Chemistry & Biology, Volume 22, Issue 10 Author(s): Jeung-Hoi Ha, Joshua M. Karchin, Nancy Walker-Kopp, Carlos A. Castañeda, Stewart N. Loh Domain swapping occurs when identical proteins exchange segments in reciprocal fashion. Natural swapping mechanisms remain poorly understood, and engineered swapping has the potential for creating self-assembling biomaterials that encode for emergent functions. We demonstrate that induced swapping can be used to regulate the function of a target protein. Swapping is triggered by inserting a “lever” protein (ubiquitin) into one of four loops of the ribose binding protein (RBP) target. The lever splits the target, forcing RBP to refold in trans to generate swapped oligomers. Identical RBP-ubiquitin fusions form homo-swapped complexes with the ubiquitin domain acting as the hinge. Surprisingly, some pairs of non-identical fusions swap more efficiently with each other than they do with themselves. Nuclear magnetic resonance experiments reveal that the hinge of these hetero-swapped complexes maps to a region of RBP distant from both ubiquitins. This design is expected to be applicable to other proteins to convert them into functional switches. Graphical abstract image Teaser Prevalent in nature yet poorly understood, domain swapping provides protein engineers with a heretofore unrecognized tool to manipulate protein structure, function, and self-assembly. Ha et al. introduce swapping into a target protein in a rational way, and show that by doing so the activity of that protein can be switched on and off.
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Mapping Proteome-Wide Targets of Environmental Chemicals Using Reactivity-Based Chemoproteomic Platforms ()
Publication date: 22 October 2015 Source:Chemistry & Biology, Volume 22, Issue 10 Author(s): Daniel Medina-Cleghorn, Leslie A. Bateman, Breanna Ford, Ann Heslin, Karl J. Fisher, Esha D. Dalvie, Daniel K. Nomura We are exposed to a growing number of chemicals in our environment, most of which have not been characterized in terms of their toxicological potential or mechanisms. Here, we employ a chemoproteomic platform to map the cysteine reactivity of environmental chemicals using reactivity-based probes to mine for hyper-reactive hotspots across the proteome. We show that environmental contaminants such as monomethylarsonous acid and widely used pesticides such as chlorothalonil and chloropicrin possess common reactivity with a distinct set of proteins. Many of these proteins are involved in key metabolic processes, suggesting that these targets may be particularly sensitive to environmental electrophiles. We show that the widely used fungicide chlorothalonil specifically inhibits several metabolic enzymes involved in fatty acid metabolism and energetics, leading to dysregulated lipid metabolism in mice. Our results underscore the utility of using reactivity-based chemoproteomic platforms to uncover novel mechanistic insights into the toxicity of environmental chemicals. Teaser Medina-Cleghorn et al. employ a chemoproteomic platform to map the direct protein targets of several environmental chemicals, and show that these chemicals possess common reactivity with enzymes involved in lipid metabolism.
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In-Depth High-Throughput Screening of Protein Engineering Libraries by Split-GFP Direct Crude Cell Extract Data Normalization ()
Publication date: 22 October 2015 Source:Chemistry & Biology, Volume 22, Issue 10 Author(s): Javier Santos-Aberturas, Mark Dörr, Geoffrey S. Waldo, Uwe T. Bornscheuer Here, we report a widely and generally applicable strategy to obtain reliable information in high-throughput protein screenings of enzyme mutant libraries. The method is based on the usage of the split-GFP technology for the normalization of the expression level of each individual protein variant combined with activity measurements, thus resolving the important problems associated with the different solubility of each mutant and allowing the detection of previously invisible variants. The small size of the employed protein tag (16 amino acids) required for the reconstitution of the GFP fluorescence reduces possible interferences such as enzyme activity variations or solubility disturbances to a minimum. Specific enzyme activity measurements without purification, in situ soluble protein expression monitoring, and data normalization are the powerful outputs of this methodology, thus enabling the accurate identification of improved protein variants during high-throughput screening by substantially reducing the occurrence of false negatives and false positives. Graphical abstract image Teaser Split-GFP protein content quantification developed by Santos et al. allows the rescue of valuable but previously undetectable hits during the screening of protein engineering libraries, thus reducing the experimental effort required for the discovery of interesting mutants during directed evolution campaigns.
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In This Issue ()
Publication date: 17 September 2015 Source:Chemistry & Biology, Volume 22, Issue 9
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Pushing Cancer Cells over the Edge, Following Transcription Where Tin Leads, and Why Bugs Make Antibiotics ()
Publication date: 17 September 2015 Source:Chemistry & Biology, Volume 22, Issue 9 Each month, Chemistry & Biology Select highlights a selection of research reports from the recent literature. These highlights are a snapshot of interesting research done across the field of chemical biology. Our September 2015 selection includes a way to indulge cancer cell oncogene addiction and push cancer cells to self-destroy; a tin-containing, small-molecule tool compound that targets an intrinsically disordered region of eukaryotic transcription factor TFIID; and evidence that bacteria produce antibiotics for one purpose only—as deadly weapons.
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Identification and Characterization of an Irreversible Inhibitor of CDK2 ()
Publication date: 17 September 2015 Source:Chemistry & Biology, Volume 22, Issue 9 Author(s): Elizabeth Anscombe, Elisa Meschini, Regina Mora-Vidal, Mathew P. Martin, David Staunton, Matthis Geitmann, U. Helena Danielson, Will A. Stanley, Lan Z. Wang, Tristan Reuillon, Bernard T. Golding, Celine Cano, David R. Newell, Martin E.M. Noble, Stephen R. Wedge, Jane A. Endicott, Roger J. Griffin Irreversible inhibitors that modify cysteine or lysine residues within a protein kinase ATP binding site offer, through their distinctive mode of action, an alternative to ATP-competitive agents. 4-((6-(Cyclohexylmethoxy)-9H-purin-2-yl)amino)benzenesulfonamide (NU6102) is a potent and selective ATP-competitive inhibitor of CDK2 in which the sulfonamide moiety is positioned close to a pair of lysine residues. Guided by the CDK2/NU6102 structure, we designed 6-(cyclohexylmethoxy)-N-(4-(vinylsulfonyl)phenyl)-9H-purin-2-amine (NU6300), which binds covalently to CDK2 as shown by a co-complex crystal structure. Acute incubation with NU6300 produced a durable inhibition of Rb phosphorylation in SKUT-1B cells, consistent with it acting as an irreversible CDK2 inhibitor. NU6300 is the first covalent CDK2 inhibitor to be described, and illustrates the potential of vinyl sulfones for the design of more potent and selective compounds. Graphical abstract image Teaser Irreversible inhibitors have a distinctive mode of action and offer an alternative route to competitive ATP inhibitors to target protein kinases. Anscombe et al. describe NU6300, a covalent CDK2 inhibitor that illustrates the potential of using vinyl sulfones to mediate irreversible inhibition.
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Adaptive Assembly: Maximizing the Potential of a Given Functional Peptide with a Tailor-Made Protein Scaffold ()
Publication date: 17 September 2015 Source:Chemistry & Biology, Volume 22, Issue 9 Author(s): Hideki Watanabe, Shinya Honda Protein engineering that exploits known functional peptides holds great promise for generating novel functional proteins. Here we propose a combinatorial approach, termed adaptive assembly, which provides a tailor-made protein scaffold for a given functional peptide. A combinatorial library was designed to create a tailor-made scaffold, which was generated from β hairpins derived from a 10-residue minimal protein “chignolin” and randomized amino acid sequences. We applied adaptive assembly to a peptide with low affinity for the Fc region of human immunoglobulin G, generating a 54-residue protein AF.p17 with a 40,600-fold enhanced affinity. The crystal structure of AF.p17 complexed with the Fc region revealed that the scaffold fixed the active conformation with a unique structure composed of a short α helix, β hairpins, and a loop-like structure. Adaptive assembly can take full advantage of known peptides as assets for generating novel functional proteins. Graphical abstract image Teaser Watanabe et al. propose a segment-based combinatorial approach termed adaptive assembly that generates a tailor-made protein scaffold for a given functional peptide. Adaptive assembly can achieve significant functional enhancement without relying on known protein structures.
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Inflammatory Signaling by NOD-RIPK2 Is Inhibited by Clinically Relevant Type II Kinase Inhibitors ()
Publication date: 17 September 2015 Source:Chemistry & Biology, Volume 22, Issue 9 Author(s): Peter Canning, Qui Ruan, Tobias Schwerd, Matous Hrdinka, Jenny L. Maki, Danish Saleh, Chalada Suebsuwong, Soumya Ray, Paul E. Brennan, Gregory D. Cuny, Holm H. Uhlig, Mads Gyrd-Hansen, Alexei Degterev, Alex N. Bullock RIPK2 mediates pro-inflammatory signaling from the bacterial sensors NOD1 and NOD2, and is an emerging therapeutic target in autoimmune and inflammatory diseases. We observed that cellular RIPK2 can be potently inhibited by type II inhibitors that displace the kinase activation segment, whereas ATP-competitive type I inhibition was only poorly effective. The most potent RIPK2 inhibitors were the US Food and Drug Administration-approved drugs ponatinib and regorafenib. Their mechanism of action was independent of NOD2 interaction and involved loss of downstream kinase activation as evidenced by lack of RIPK2 autophosphorylation. Notably, these molecules also blocked RIPK2 ubiquitination and, consequently, inflammatory nuclear factor κB signaling. In monocytes, the inhibitors selectively blocked NOD-dependent tumor necrosis factor production without affecting lipopolysaccharide-dependent pathways. We also determined the first crystal structure of RIPK2 bound to ponatinib, and identified an allosteric site for inhibitor development. These results highlight the potential for type II inhibitors to treat indications of RIPK2 activation as well as inflammation-associated cancers. Graphical abstract image Teaser Canning et al. report the structure of the diverse kinase RIPK2 and characterize its inhibition by the FDA-approved drugs ponatinib and regorafenib. The inhibitors prevent the autophosphorylation and ubiquitination of RIPK2 upon NOD2 stimulation, and block downstream NF-κB activation and inflammatory signaling.
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KDM4/JMJD2 Histone Demethylase Inhibitors Block Prostate Tumor Growth by Suppressing the Expression of AR and BMYB-Regulated Genes ()
Publication date: 17 September 2015 Source:Chemistry & Biology, Volume 22, Issue 9 Author(s): Lingling Duan, Ganesha Rai, Carlos Roggero, Qing-Jun Zhang, Qun Wei, Shi Hong Ma, Yunyun Zhou, John Santoyo, Elisabeth D. Martinez, Guanghua Xiao, Ganesh V. Raj, Ajit Jadhav, Anton Simeonov, David J. Maloney, Josep Rizo, Jer-Tsong Hsieh, Zhi-Ping Liu Histone lysine demethylase KDM4/JMJD2s are overexpressed in many human tumors including prostate cancer (PCa). KDM4s are co-activators of androgen receptor (AR) and are thus potential therapeutic targets. Yet to date few KDM4 inhibitors that have anti-prostate tumor activity in vivo have been developed. Here, we report the anti-tumor growth effect and molecular mechanisms of three novel KDM4 inhibitors (A1, I9, and B3). These inhibitors repressed the transcription of both AR and BMYB-regulated genes. Compound B3 is highly selective for a variety of cancer cell lines including PC3 cells that lack AR. B3 inhibited the in vivo growth of tumors derived from PC3 cells and ex vivo human PCa explants. We identified a novel mechanism by which KDM4B activates the transcription of Polo-like kinase 1 (PLK1). B3 blocked the binding of KDM4B to the PLK1 promoter. Our studies suggest a potential mechanism-based therapeutic strategy for PCa and tumors with elevated KDM4B/PLK1 expression. Graphical abstract image Teaser KDM4 proteins are co-activators of androgen receptor and may play a role in castration-resistant prostate cancer development. Duan et al. identify several novel inhibitors of KDM4 and describe how they inhibit expression of genes critical for cell-cycle progression and tumor growth in vivo.
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