Nature Chemical Biology

Disulfide-directed histone ubiquitylation reveals plasticity in hDot1L activation.

Publication Date: 2010 Mar 7 PMID: 20208522
Authors: Chatterjee, C. - McGinty, R. K. - Fierz, B. - Muir, T. W.
Journal: Nat Chem Biol

We have developed a readily accessible disulfide-directed methodology for the site-specific modification of histones by ubiquitin and ubiquitin-like proteins. The disulfide-linked analog of mono-ubiquitylated H2B stimulated the H3K79 methyltransferase activity of hDot1L to a similar extent as the native isopeptide linkage. This permitted structure-activity studies of ubiquitylated mononucleosomes that revealed plasticity in the mechanism of hDot1L stimulation and identified surfaces of ubiquitin important for activation.

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Chemically ubiquitylated PCNA as a probe for eukaryotic translesion DNA synthesis.

Publication Date: 2010 Mar 7 PMID: 20208521
Authors: Chen, J. - Ai, Y. - Wang, J. - Haracska, L. - Zhuang, Z.
Journal: Nat Chem Biol

The rapid growth in ubiquitin biology requires facile chemical approaches for protein ubiquitylation that can overcome the common problem of low yield faced by the enzymatic reaction catalyzed by ubiquitin ligases. We report a chemical approach for monoubiquitylation and SUMOylation of PCNA through disulfide exchange and intein chemistry. We used the chemically ubiquitylated and SUMOylated PCNAs in studying translesion DNA synthesis and revealed a surprising degree of flexibility of the ubiquitin modification.

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A chemical and phosphoproteomic characterization of dasatinib action in lung cancer.

Publication Date: 2010 Feb 28 PMID: 20190765
Authors: Li, J. - Rix, U. - Fang, B. - Bai, Y. - Edwards, A. - Colinge, J. - Bennett, K. L. - Gao, J. - Song, L. - Eschrich, S. - Superti-Furga, G. - Koomen, J. - Haura, E. B.
Journal: Nat Chem Biol

We describe a strategy for comprehending signaling pathways that are active in lung cancer cells and that are targeted by dasatinib using chemical proteomics to identify direct interacting proteins combined with immunoaffinity purification of tyrosine-phosphorylated peptides corresponding to activated tyrosine kinases. We identified nearly 40 different kinase targets of dasatinib. These include SRC-family kinase (SFK) members (LYN, SRC, FYN, LCK and YES), nonreceptor tyrosine kinases (FRK, BRK and ACK) and receptor tyrosine kinases (Ephrin receptors, DDR1 and EGFR). Using quantitative phosphoproteomics, we identified peptides corresponding to autophosphorylation sites of these tyrosine kinases that are inhibited in a concentration-dependent manner by dasatinib. Using drug-resistant gatekeeper mutants, we show that SFKs (particularly SRC and FYN), as well as EGFR, are relevant targets for dasatinib action. The combined mass spectrometry-based approach described here provides a system-level view of dasatinib action in cancer cells and suggests both functional targets and a rationale for combinatorial therapeutic strategies.

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Combinatorial profiling of chromatin binding modules reveals multisite discrimination.

Publication Date: 2010 Feb 28 PMID: 20190764
Authors: Garske, A. L. - Oliver, S. S. - Wagner, E. K. - Musselman, C. A. - Leroy, G. - Garcia, B. A. - Kutateladze, T. G. - Denu, J. M.
Journal: Nat Chem Biol

Specific interactions between post-translational modifications (PTMs) and chromatin-binding proteins are central to the idea of a 'histone code'. Here, we used a 5,000-member, PTM-randomized, combinatorial peptide library based on the N terminus of histone H3 to interrogate the multisite specificity of six chromatin binding modules, which read the methylation status of Lys4. We found that Thr3 phosphorylation, Arg2 methylation and Thr6 phosphorylation are critical additional PTMs that modulate the ability to recognize and bind histone H3. Notably, phosphorylation of Thr6 yielded the most varied effect on protein binding, suggesting an important regulatory mechanism for readers of the H3 tail. Mass spectrometry and antibody-based evidence indicate that this previously uncharacterized modification exists on native H3, and NMR analysis of ING2 revealed the structural basis for discrimination. These investigations reveal a continuum of binding affinities in which multisite PTM recognition involves both switch- and rheostat-like properties, yielding graded effects that depend on the inherent 'reader' specificity.

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Symbiotic streptomycetes provide antibiotic combination prophylaxis for wasp offspring.

Publication Date: 2010 Feb 28 PMID: 20190763
Authors: Kroiss, J. - Kaltenpoth, M. - Schneider, B. - Schwinger, M. G. - Hertweck, C. - Maddula, R. K. - Strohm, E. - Svatos, A.
Journal: Nat Chem Biol

Beewolf digger wasps cultivate specific symbiotic bacteria (Streptomyces spp.) that are incorporated into the larval cocoon for protection against pathogens. We identified the molecular basis of this protective symbiosis in the natural context and demonstrate that the bacteria produce a 'cocktail' of nine antibiotic substances. The complementary action of all symbiont-produced antibiotics confers a potent antimicrobial defense for the wasp larvae that parallels the 'combination prophylaxis' known from human medicine.

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A combined method for producing homogeneous glycoproteins with eukaryotic N-glycosylation.

Publication Date: 2010 Feb 28 PMID: 20190762
Authors: Schwarz, F. - Huang, W. - Li, C. - Schulz, B. L. - Lizak, C. - Palumbo, A. - Numao, S. - Neri, D. - Aebi, M. - Wang, L. X.
Journal: Nat Chem Biol

We describe a new method for producing homogeneous eukaryotic N-glycoproteins. The method involves the engineering and functional transfer of the Campylobacter jejuni glycosylation machinery in Escherichia coli to express glycosylated proteins with the key GlcNAc-Asn linkage. The bacterial glycans were then trimmed and remodeled in vitro by enzymatic transglycosylation to fulfill a eukaryotic N-glycosylation. It provides a potentially general platform for producing eukaryotic N-glycoproteins.

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Two-photon uncaging of gamma-aminobutyric acid in intact brain tissue.

Publication Date: 2010 Feb 21 PMID: 20173751
Authors: Matsuzaki, M. - Hayama, T. - Kasai, H. - Ellis-Davies, G. C.
Journal: Nat Chem Biol

We have synthesized a photosensitive (or caged) 4-carboxymethoxy-5,7-dinitroindolinyl (CDNI) derivative of gamma-aminobutyric acid (GABA). Two-photon excitation of CDNI-GABA produced rapid activation of GABAergic currents in neurons in brain slices with an axial resolution of approximately 2 mum and enabled high-resolution functional mapping of GABA-A receptors. Two-photon uncaging of GABA, the main inhibitory neurotransmitter, should allow detailed studies of receptor function and synaptic integration with subcellular precision.

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Chemical decay of an antibiotic inverts selection for resistance.

Publication Date: 2010 Mar PMID: 20154670
Authors: Palmer, A. C. - Angelino, E. - Kishony, R.
Journal: Nat Chem Biol

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Aminoglycoside activity observed on single pre-translocation ribosome complexes.

Publication Date: 2010 Mar PMID: 20154669
Authors: Feldman, M. B. - Terry, D. S. - Altman, R. B. - Blanchard, S. C.
Journal: Nat Chem Biol

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The p110delta structure: mechanisms for selectivity and potency of new PI(3)K inhibitors.

Publication Date: 2010 Mar PMID: 20154668
Authors: Berndt, A. - Miller, S. - Williams, O. - Le, D. D. - Houseman, B. T. - Pacold, J. I. - Gorrec, F. - Hon, W. C. - Liu, Y. - Rommel, C. - Gaillard, P. - Ruckle, T. - Schwarz, M. K. - Shokat, K. M. - Shaw, J. P. - Williams, R. L.
Journal: Nat Chem Biol

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Garbled messages and corrupted translations.

Publication Date: 2010 Mar PMID: 20154667
Authors: Schneider-Poetsch, T. - Usui, T. - Kaida, D. - Yoshida, M.
Journal: Nat Chem Biol

Following transcription, genomic information begins a long journey toward translation of its nucleotide sequence into the amino acids of a protein. In eukaryotes, synthesized pre-mRNAs become processed to mature mRNAs by 5'-end capping, splicing, 3'-end cleavage and polyadenylation in the nucleus, before being scrutinized for premature stop codons. Each step requires high precision and control to ensure that an intact and readable message is exported to the cytoplasm before finally becoming translated. Two important aspects of these processes are accurately managed by ribonucleoprotein machineries-the spliceosome and the ribosome. Recently, several natural products targeting these macromolecular assemblies have been reported. For the first time in eukaryotes, these molecules allow chemical disruption and dissection of the sophisticated machinery that regulates post-transcriptional events. Beyond their great potential as bioprobes for investigating mRNA regulation and protein synthesis, these compounds also show promise in opening new therapeutic approaches.

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Turning enzymes ON with small molecules.

Publication Date: 2010 Mar PMID: 20154666
Authors: Zorn, J. A. - Wells, J. A.
Journal: Nat Chem Biol

Drug discovery and chemical genetic efforts typically focus on the identification and design of inhibitors or loss-of-function probes as a means to perturb enzyme function. These tools are effective in determining the physiological consequence of ablating the activity of a specific enzyme. Remarkably, nearly a dozen examples of non-natural small molecules that activate enzyme catalysis have been identified within the past decade. In aggregate, these studies delineate four unique activation mechanisms that the small molecules exploit. These complementary gain-of-function probes offer a way to address the sufficiency of an enzyme to drive a particular cellular phenotype, and they also provide new opportunities for drug discovery. This review covers the identification and characterization of these unique small-molecule activators.

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Stem cells: Metabolism regulates differentiation.

Publication Date: 2010 Mar PMID: 20154665
Authors: McGraw, T. E. - Mittal, V.
Journal: Nat Chem Biol

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Synthetic chemistry: An upfront investment.

Publication Date: 2010 Mar PMID: 20154664
Authors: Young, D. W.
Journal: Nat Chem Biol

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Phenotypic screening: Fishing for neuroactive compounds.

Publication Date: 2010 Mar PMID: 20154663
Authors: Jenkins, J. L. - Urban, L.
Journal: Nat Chem Biol

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Our choices from the recent literature.

Publication Date: 2010 Mar PMID: 20154662
Authors:
Journal: Nat Chem Biol

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The (un)targeted cancer kinome.

Publication Date: 2010 Mar PMID: 20154661
Authors: Fedorov, O. - Muller, S. - Knapp, S.
Journal: Nat Chem Biol

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Rethinking screening.

Publication Date: 2010 Mar PMID: 20154660
Authors: Kodadek, T.
Journal: Nat Chem Biol

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The art of the chemical probe.

Publication Date: 2010 Mar PMID: 20154659
Authors: Frye, S. V.
Journal: Nat Chem Biol

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Retooling chemical probes.

Publication Date: 2010 Mar PMID: 20154658
Authors:
Journal: Nat Chem Biol

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Chemical phylogenetics of histone deacetylases.

Publication Date: 2010 Mar PMID: 20139990
Authors: Bradner, J. E. - West, N. - Grachan, M. L. - Greenberg, E. F. - Haggarty, S. J. - Warnow, T. - Mazitschek, R.
Journal: Nat Chem Biol

The broad study of histone deacetylases in chemistry, biology and medicine relies on tool compounds to derive mechanistic insights. A phylogenetic analysis of class I and II histone deacetylases (HDACs) as targets of a comprehensive, structurally diverse panel of inhibitors revealed unexpected isoform selectivity even among compounds widely perceived as nonselective. The synthesis and study of a focused library of cinnamic hydroxamates allowed the identification of, to our knowledge, the first nonselective HDAC inhibitor. These data will guide a more informed use of HDAC inhibitors as chemical probes and therapeutic agents.

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Inhibition of eukaryotic translation elongation by cycloheximide and lactimidomycin.

Publication Date: 2010 Mar PMID: 20118940
Authors: Schneider-Poetsch, T. - Ju, J. - Eyler, D. E. - Dang, Y. - Bhat, S. - Merrick, W. C. - Green, R. - Shen, B. - Liu, J. O.
Journal: Nat Chem Biol

Although the protein synthesis inhibitor cycloheximide (CHX) has been known for decades, its precise mechanism of action remains incompletely understood. The glutarimide portion of CHX is seen in a family of structurally related natural products including migrastatin, isomigrastatin and lactimidomycin (LTM). We found that LTM, isomigrastatin and analogs have a potent antiproliferative effect on tumor cell lines and selectively inhibit translation. A systematic comparative study of the effects of CHX and LTM on protein synthesis revealed both similarities and differences between the two inhibitors. Both LTM and CHX were found to block the translocation step in elongation. Footprinting experiments revealed protection of a single cytidine nucleotide (C3993) in the E-site of the 60S ribosomal subunit, thus defining a common binding pocket for the two inhibitors in the ribosome. These results shed new light on the molecular mechanism of inhibition of translation elongation by both CHX and LTM.

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The 2'-OH group at the group II intron terminus acts as a proton shuttle.

Publication Date: 2010 Mar PMID: 20118939
Authors: Roitzsch, M. - Fedorova, O. - Pyle, A. M.
Journal: Nat Chem Biol

Group II introns are self-splicing ribozymes that excise themselves from precursor RNAs and catalyze the joining of flanking exons. Excised introns can behave as parasitic RNA molecules: they can catalyze their own insertion into DNA and RNA via a reverse splicing reaction. Previous studies have identified mechanistic roles for various functional groups located in the catalytic core of the intron and within target molecules. Here we introduce a new method for synthesizing long RNA molecules with a modified nucleotide at the 3' terminus. This modification allows us to examine the mechanistic role of functional groups adjacent to the reaction nucleophile. During reverse splicing, the 3'-OH group of the intron terminus attacks the phosphodiester linkage of spliced exon sequences. Here we show that the adjacent 2'-OH group on the intron terminus plays an essential role in activating the nucleophile by stripping away a proton from the 3'-OH and then shuttling it from the active site.

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Capture and release of alkyne-derivatized glycerophospholipids using cobalt chemistry.

Publication Date: 2010 Mar PMID: 20098428
Authors: Milne, S. B. - Tallman, K. A. - Serwa, R. - Rouzer, C. A. - Armstrong, M. D. - Marnett, L. J. - Lukehart, C. M. - Porter, N. A. - Brown, H. A.
Journal: Nat Chem Biol

Alkyne-modified phospholipids can be unambiguously identified and differentiated from native species in complex mixtures by formation of dicobalthexacarbonyl complexes. This reaction is specific for alkynes and is unaffected by other glycerophospholipid-related moieties. Enrichment of cells with alkyne-derivatized fatty acids or glycerophospholipids followed by solid-phase sequestration and release is a promising new method for unequivocally monitoring individual glycerophospholipids following incorporation into cells. This technique also facilitates lipidomic analysis of substrates and products.

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Domino access to highly substituted chromans and isochromans from carbohydrates.

Publication Date: 2010 Mar PMID: 20098427
Authors: Leibeling, M. - Koester, D. C. - Pawliczek, M. - Schild, S. C. - Werz, D. B.
Journal: Nat Chem Biol

Herein we describe the synthesis of highly substituted chromans and isochromans using carbohydrates as starting materials. Our approach makes use of a Pd-catalyzed domino reaction consisting of oxidative addition, followed by two carbopalladation steps and completed by a cyclization to annelate the benzene moiety. The versatility of this route has been demonstrated by a small library of highly substituted chromans and isochromans.

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Carbon metabolism-mediated myogenic differentiation.

Publication Date: 2010 Mar PMID: 20081855
Authors: Bracha, A. L. - Ramanathan, A. - Huang, S. - Ingber, D. E. - Schreiber, S. L.
Journal: Nat Chem Biol

The role of nutrients and metabolism in cellular differentiation is poorly understood. Using RNAi screening, metabolic profiling and small-molecule probes, we discovered that the knockdown of three metabolic enzymes-phosphoglycerate kinase (Pgk1), hexose-6-phosphate dehydrogenase (H6pd) and ATP citrate lyase (Acl)-induces differentiation of mouse C2C12 myoblasts even in the presence of mitogens. These enzymes and the pathways they regulate provide new targets for the control of myogenic differentiation in myoblasts and rhabdomyosarcoma cells.

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Rapid behavior-based identification of neuroactive small molecules in the zebrafish.

Publication Date: 2010 Mar PMID: 20081854
Authors: Kokel, D. - Bryan, J. - Laggner, C. - White, R. - Cheung, C. Y. - Mateus, R. - Healey, D. - Kim, S. - Werdich, A. A. - Haggarty, S. J. - Macrae, C. A. - Shoichet, B. - Peterson, R. T.
Journal: Nat Chem Biol

Neuroactive small molecules are indispensable tools for treating mental illnesses and dissecting nervous system function. However, it has been difficult to discover novel neuroactive drugs. Here, we describe a high-throughput, behavior-based approach to neuroactive small molecule discovery in the zebrafish. We used automated screening assays to evaluate thousands of chemical compounds and found that diverse classes of neuroactive molecules caused distinct patterns of behavior. These 'behavioral barcodes' can be used to rapidly identify new psychotropic chemicals and to predict their molecular targets. For example, we identified new acetylcholinesterase and monoamine oxidase inhibitors using phenotypic comparisons and computational techniques. By combining high-throughput screening technologies with behavioral phenotyping in vivo, behavior-based chemical screens can accelerate the pace of neuroactive drug discovery and provide small-molecule tools for understanding vertebrate behavior.

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Differences in prion strain conformations result from non-native interactions in a nucleus.

Publication Date: 2010 Mar PMID: 20081853
Authors: Ohhashi, Y. - Ito, K. - Toyama, B. H. - Weissman, J. S. - Tanaka, M.
Journal: Nat Chem Biol

Aggregation-prone proteins often misfold into multiple distinct amyloid conformations that dictate different physiological impacts. Although amyloid formation is triggered by a transient nucleus, the mechanism by which an initial nucleus is formed and allows the protein to form a specific amyloid conformation has been unclear. Here we show that, before fiber formation, the prion domain (Sup35NM, consisting of residues 1-254) of yeast prion Sup35, the [PSI(+)] protein determinant, forms oligomers in a temperature-dependent, reversible manner. Mutational and biophysical analyses revealed that 'non-native' aromatic interactions outside the amyloid core drive oligomer formation by bringing together different Sup35NM monomers, which specifically leads to the formation of highly infectious strain conformations with more limited amyloid cores. Thus, transient non-native interactions in the initial nucleus are pivotal in determining the diversity of amyloid conformations and resulting prion strain phenotypes.

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