Nature Chemical Biology

Tah18 transfers electrons to Dre2 in cytosolic iron-sulfur protein biogenesis.

Publication Date: 2010 Aug 29 PMID: 20802492
Authors: Netz, D. J. - Stumpfig, M. - Dore, C. - Muhlenhoff, U. - Pierik, A. J. - Lill, R.
Journal: Nat Chem Biol

Cytosolic and nuclear iron-sulfur (Fe-S) proteins play key roles in processes such as ribosome maturation, transcription and DNA repair-replication. For biosynthesis of their Fe-S clusters, a dedicated cytosolic Fe-S protein assembly (CIA) machinery is required. Here, we identify the essential flavoprotein Tah18 as a previously unrecognized CIA component and show by cell biological, biochemical and spectroscopic approaches that the complex of Tah18 and the CIA protein Dre2 is part of an electron transfer chain functioning in an early step of cytosolic Fe-S protein biogenesis. Electrons are transferred from NADPH via the FAD- and FMN-containing Tah18 to the Fe-S clusters of Dre2. This electron transfer chain is required for assembly of target but not scaffold Fe-S proteins, suggesting a need for reduction in the generation of stably inserted Fe-S clusters. The pathway is conserved in eukaryotes, as human Ndor1-Ciapin1 proteins can functionally replace yeast Tah18-Dre2.

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Engineered diubiquitin synthesis reveals Lys29-isopeptide specificity of an OTU deubiquitinase.

Publication Date: 2010 Aug 29 PMID: 20802491
Authors: Virdee, S. - Ye, Y. - Nguyen, D. P. - Komander, D. - Chin, J. W.
Journal: Nat Chem Biol

Ubiquitination is a reversible post-translational modification that regulates a myriad of eukaryotic functions. Our ability to study the effects of ubiquitination is often limited by the inaccessibility of homogeneously ubiquitinated proteins. In particular, elucidating the roles of the so-called 'atypical' ubiquitin chains (chains other than Lys48- or Lys63-linked ubiquitin), which account for a large fraction of ubiquitin polymers, is challenging because the enzymes for their biosynthesis are unknown. Here we combine genetic code expansion, intein chemistry and chemoselective ligations to synthesize 'atypical' ubiquitin chains. We solve the crystal structure of Lys6-linked diubiquitin, which is distinct from that of structurally characterized ubiquitin chains, providing a molecular basis for the different biological functions this linkage may regulate. Moreover, we profile a panel containing 10% of the known human deubiquitinases on Lys6- and Lys29-linked ubiquitin and discover that TRABID cleaves the Lys29 linkage 40-fold more efficiently than the Lys63 linkage.

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Methylation of FEN1 suppresses nearby phosphorylation and facilitates PCNA binding.

Publication Date: 2010 Aug 22 PMID: 20729856
Authors: Guo, Z. - Zheng, L. - Xu, H. - Dai, H. - Zhou, M. - Pascua, M. R. - Chen, Q. M. - Shen, B.
Journal: Nat Chem Biol

Flap endonuclease 1 (FEN1), a structure-specific endo- and exonuclease, has multiple functions that determine essential biological processes, such as cell proliferation and cell death. As such, the enzyme must be precisely regulated to execute each of its functions with the right timing and in a specific subcellular location. Here we report that FEN1 is methylated at arginine residues, primarily at Arg192. The methylation suppresses FEN1 phosphorylation at Ser187. The methylated form, but not the phosphorylated form, of FEN1 strongly interacts with proliferating cell nuclear antigen (PCNA), ensuring the 'on' and 'off' timing of its reaction. Mutations of FEN1 disrupting arginine methylation and PCNA interaction result in unscheduled phosphorylation and a failure to localize to DNA replication or repair foci. This consequently leads to a defect in Okazaki fragment maturation, a delay in cell cycle progression, impairment of DNA repair and a high frequency of genome-wide mutations.

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Genome instability due to ribonucleotide incorporation into DNA.

Publication Date: 2010 Aug 22 PMID: 20729855
Authors: McElhinny, S. A. - Kumar, D. - Clark, A. B. - Watt, D. L. - Watts, B. E. - Lundstrom, E. B. - Johansson, E. - Chabes, A. - Kunkel, T. A.
Journal: Nat Chem Biol

Maintaining the chemical identity of DNA depends on ribonucleotide exclusion by DNA polymerases. However, ribonucleotide exclusion during DNA synthesis in vitro is imperfect. To determine whether ribonucleotides are incorporated during DNA replication in vivo, we substituted leucine or glycine for an active-site methionine in yeast DNA polymerase (Pol ). Ribonucleotide incorporation in vitro was three-fold lower for M644L and 11-fold higher for M644G Pol compared to wild-type Pol . This hierarchy was recapitulated in vivo in yeast strains lacking RNase H2. Moreover, the pol2-M644G rnh201Delta strain progressed more slowly through S phase, had elevated dNTP pools and generated 2-5-base-pair deletions in repetitive sequences at a high rate and in a gene orientation-dependent manner. The data indicate that ribonucleotides are incorporated during replication in vivo, that they are removed by RNase H2-dependent repair and that defective repair results in replicative stress and genome instability via DNA strand misalignment.

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Screening: Your brain on drugs.

Publication Date: 2010 Sep PMID: 20720548
Authors: Saxe, J. P.
Journal: Nat Chem Biol

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Chemical ecology: Reprogramming a termite monarchy.

Publication Date: 2010 Sep PMID: 20720547
Authors: Bussell, J. J. - Vosshall, L. B.
Journal: Nat Chem Biol

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Metalloenzymes: Natural product nitrosation.

Publication Date: 2010 Sep PMID: 20720546
Authors: Kersten, R. D. - Dorrestein, P. C.
Journal: Nat Chem Biol

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Post-translational modifications: A shift for the O-GlcNAc paradigm.

Publication Date: 2010 Sep PMID: 20720545
Authors: Kohler, J. J.
Journal: Nat Chem Biol

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Postgenomic chemical biology.

Publication Date: 2010 Sep PMID: 20720543
Authors:
Journal: Nat Chem Biol

Chemical biology contributions will become increasingly important as we enter the second decade of the postgenomic era.

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Allosteric non-bisphosphonate FPPS inhibitors identified by fragment-based discovery.

Publication Date: 2010 Sep PMID: 20711197
Authors: Jahnke, W. - Rondeau, J. M. - Cotesta, S. - Marzinzik, A. - Pelle, X. - Geiser, M. - Strauss, A. - Gotte, M. - Bitsch, F. - Hemmig, R. - Henry, C. - Lehmann, S. - Glickman, J. F. - Roddy, T. P. - Stout, S. J. - Green, J. R.
Journal: Nat Chem Biol

Bisphosphonates are potent inhibitors of farnesyl pyrophosphate synthase (FPPS) and are highly efficacious in the treatment of bone diseases such as osteoporosis, Paget's disease and tumor-induced osteolysis. In addition, the potential for direct antitumor effects has been postulated on the basis of in vitro and in vivo studies and has recently been demonstrated clinically in early breast cancer patients treated with the potent bisphosphonate zoledronic acid. However, the high affinity of bisphosphonates for bone mineral seems suboptimal for the direct treatment of soft-tissue tumors. Here we report the discovery of the first potent non-bisphosphonate FPPS inhibitors. These new inhibitors bind to a previously unknown allosteric site on FPPS, which was identified by fragment-based approaches using NMR and X-ray crystallography. This allosteric and druggable pocket allows the development of a new generation of FPPS inhibitors that are optimized for direct antitumor effects in soft tissue.

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A small-molecule inhibitor shows that pirin regulates migration of melanoma cells.

Publication Date: 2010 Sep PMID: 20711196
Authors: Miyazaki, I. - Simizu, S. - Okumura, H. - Takagi, S. - Osada, H.
Journal: Nat Chem Biol

The discovery of small molecules that bind to a specific target and disrupt the function of proteins is an important step in chemical biology, especially for poorly characterized proteins. Human pirin is a nuclear protein of unknown function that is widely expressed in punctate subnuclear structures in human tissues. Here, we report the discovery of a small molecule that binds to pirin. We determined how the small molecule bound to pirin by solving the cocrystal structure. Either knockdown of pirin or treatment with the small molecule inhibited melanoma cell migration. Thus, inhibition of pirin by the small molecule has led to a greater understanding of the function of pirin and represents a new method of studying pirin-mediated signaling pathways.

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Biotin synthesis begins by hijacking the fatty acid synthetic pathway.

Publication Date: 2010 Sep PMID: 20693992
Authors: Lin, S. - Hanson, R. E. - Cronan, J. E.
Journal: Nat Chem Biol

Although biotin is an essential enzyme cofactor found in all three domains of life, our knowledge of its biosynthesis remains fragmentary. Most of the carbon atoms of biotin are derived from pimelic acid, a seven-carbon dicarboxylic acid, but the mechanism whereby this intermediate is assembled remains unknown. Genetic analysis in Escherichia coli identified only two genes of unknown function required for pimelate synthesis, bioC and bioH. We report in vivo and in vitro evidence that the pimeloyl moiety is synthesized by a modified fatty acid synthetic pathway in which the omega-carboxyl group of a malonyl-thioester is methylated by BioC, which allows recognition of this atypical substrate by the fatty acid synthetic enzymes. The malonyl-thioester methyl ester enters fatty acid synthesis as the primer and undergoes two reiterations of the fatty acid elongation cycle to give pimeloyl-acyl carrier protein (ACP) methyl ester, which is hydrolyzed to pimeloyl-ACP and methanol by BioH.

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Zinc availability regulates exit from meiosis in maturing mammalian oocytes.

Publication Date: 2010 Sep PMID: 20693991
Authors: Kim, A. M. - Vogt, S. - O'Halloran, T. V. - Woodruff, T. K.
Journal: Nat Chem Biol

Cellular metal ion fluxes are known in alkali and alkaline earth metals but are not well documented in transition metals. Here we describe major changes in the zinc physiology of the mammalian oocyte as it matures and initiates embryonic development. Single-cell elemental analysis of mouse oocytes by synchrotron-based X-ray fluorescence microscopy (XFM) revealed a 50% increase in total zinc content within the 12-14-h period of meiotic maturation. Perturbation of zinc homeostasis with a cell-permeable small-molecule chelator blocked meiotic progression past telophase I. Zinc supplementation rescued this phenotype when administered before this meiotic block. However, after telophase arrest, zinc triggered parthenogenesis, suggesting that exit from this meiotic step is tightly regulated by the availability of a zinc-dependent signal. These results implicate the zinc bolus acquired during meiotic maturation as an important part of the maternal legacy to the embryo.

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Replication-dependent instability at (CTG) x (CAG) repeat hairpins in human cells.

Publication Date: 2010 Sep PMID: 20676085
Authors: Liu, G. - Chen, X. - Bissler, J. J. - Sinden, R. R. - Leffak, M.
Journal: Nat Chem Biol

Instability of (CTG) x (CAG) microsatellite trinucleotide repeat (TNR) sequences is responsible for more than a dozen neurological or neuromuscular diseases. TNR instability during DNA synthesis is thought to involve slipped-strand or hairpin structures in template or nascent DNA strands, although direct evidence for hairpin formation in human cells is lacking. We have used targeted recombination to create a series of isogenic HeLa cell lines in which (CTG) x (CAG) repeats are replicated from an ectopic copy of the Myc (also known as c-myc) replication origin. In this system, the tendency of chromosomal (CTG) x (CAG) tracts to expand or contract was affected by origin location and the leading or lagging strand replication orientation of the repeats, and instability was enhanced by prolonged cell culture, increased TNR length and replication inhibition. Hairpin cleavage by synthetic zinc finger nucleases in these cells has provided the first direct evidence for the formation of hairpin structures during replication in vivo.

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A copper-containing oxidase catalyzes C-nitrosation in nitrosobenzamide biosynthesis.

Publication Date: 2010 Sep PMID: 20676084
Authors: Noguchi, A. - Kitamura, T. - Onaka, H. - Horinouchi, S. - Ohnishi, Y.
Journal: Nat Chem Biol

Although C-nitroso aromatic compounds have several bioactivities of medicinal interest, the biosynthetic enzymes involved in C-nitrosation have remained unknown until now. Here, we report the entire biosynthesis pathway of 4-hydroxy-3-nitrosobenzamide in Streptomyces murayamaensis, in which a tyrosinase-like copper-containing monooxygenase is responsible for the C-nitrosation. This finding indicates diverse catalytic functions of tyrosinase-like copper-containing monooxygenases in nature.

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Quantification of O-glycosylation stoichiometry and dynamics using resolvable mass tags.

Publication Date: 2010 Sep PMID: 20657584
Authors: Rexach, J. E. - Rogers, C. J. - Yu, S. H. - Tao, J. - Sun, Y. E. - Hsieh-Wilson, L. C.
Journal: Nat Chem Biol

Mechanistic studies of O-GlcNAc glycosylation have been limited by an inability to monitor the glycosylation stoichiometries of proteins obtained from cells. Here we describe a powerful method to visualize the O-GlcNAc-modified protein subpopulation using resolvable polyethylene glycol mass tags. This approach enables rapid quantification of in vivo glycosylation levels on endogenous proteins without the need for protein purification, advanced instrumentation or expensive radiolabels. In addition, it establishes the glycosylation state (for example, mono-, di-, tri-) of proteins, providing information regarding overall O-GlcNAc site occupancy that cannot be obtained using mass spectrometry. Finally, we apply this strategy to rapidly assess the complex interplay between glycosylation and phosphorylation and discover an unexpected reverse 'yin-yang' relationship on the transcriptional repressor MeCP2 that was undetectable by traditional methods. We anticipate that this mass-tagging strategy will advance our understanding of O-GlcNAc glycosylation, as well as other post-translational modifications and poorly understood glycosylation motifs.

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