Nature Chemical Biology

Spatiotemporal resolution of the Ntla transcriptome in axial mesoderm development.

Publication Date: 2012 Jan 29 PMID: 22286130
Authors: Shestopalov, I. A. - Pitt, C. L. - Chen, J. K.
Journal: Nat Chem Biol

Transcription factors have diverse roles during embryonic development, combinatorially controlling cellular states in a spatially and temporally defined manner. Resolving the dynamic transcriptional profiles that underlie these patterning processes is essential for understanding embryogenesis at the molecular level. Here we show how temporal, tissue-specific changes in embryonic transcription factor function can be discerned by integrating caged morpholino oligonucleotides with photoactivatable fluorophores, fluorescence-activated cell sorting and microarray technologies. As a proof of principle, we have dynamically profiled No tail a (Ntla)-dependent genes at different stages of axial mesoderm development in zebrafish, discovering discrete sets of transcripts that are coincident with either notochord cell fate commitment or differentiation. Our studies reveal new regulators of notochord development and the sequential activation of distinct transcriptomes within a cell lineage by a single transcriptional factor and demonstrate how optically controlled chemical tools can dissect developmental processes with spatiotemporal precision.

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BCR-ABL uncouples canonical JAK2-STAT5 signaling in chronic myeloid leukemia.

Publication Date: 2012 Jan 29 PMID: 22286129
Authors: Hantschel, O. - Warsch, W. - Eckelhart, E. - Kaupe, I. - Grebien, F. - Wagner, K. U. - Superti-Furga, G. - Sexl, V.
Journal: Nat Chem Biol

Constitutive activation of STAT5 is critical for the maintenance of chronic myeloid leukemia (CML) characterized by the BCR-ABL oncoprotein. Tyrosine kinase inhibitors (TKIs) for the STAT5-activating kinase JAK2 have been discussed as a treatment option for CML patients. Using murine leukemia models combined with inducible ablation of JAK2, we show JAK2 dependence for initial lymphoid transformation, which is lost once leukemia is established. In contrast, initial myeloid transformation and leukemia maintenance were independent of JAK2. Nevertheless, several JAK2 TKIs induced apoptosis in BCR-ABL(+) cells irrespective of the presence of JAK2. This is caused by the previously unknown direct 'off-target' inhibition of BCR-ABL. Cellular and enzymatic analyses suggest that BCR-ABL phosphorylates STAT5 directly. Our findings suggest uncoupling of the canonical JAK2-STAT5 module upon BCR-ABL expression, thereby making JAK2 targeting dispensable. Thus, attempts to pharmacologically target STAT5 in BCR-ABL(+) diseases need to focus on STAT5 itself.

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Menin-MLL inhibitors reverse oncogenic activity of MLL fusion proteins in leukemia.

Publication Date: 2012 Jan 29 PMID: 22286128
Authors: Grembecka, J. - He, S. - Shi, A. - Purohit, T. - Muntean, A. G. - Sorenson, R. J. - Showalter, H. D. - Murai, M. J. - Belcher, A. M. - Hartley, T. - Hess, J. L. - Cierpicki, T.
Journal: Nat Chem Biol

Translocations involving the mixed lineage leukemia (MLL) gene result in human acute leukemias with very poor prognosis. The leukemogenic activity of MLL fusion proteins is critically dependent on their direct interaction with menin, a product of the multiple endocrine neoplasia (MEN1) gene. Here we present what are to our knowledge the first small-molecule inhibitors of the menin-MLL fusion protein interaction that specifically bind menin with nanomolar affinities. These compounds effectively reverse MLL fusion protein-mediated leukemic transformation by downregulating the expression of target genes required for MLL fusion protein oncogenic activity. They also selectively block proliferation and induce both apoptosis and differentiation of leukemia cells harboring MLL translocations. Identification of these compounds provides a new tool for better understanding MLL-mediated leukemogenesis and represents a new approach for studying the role of menin as an oncogenic cofactor of MLL fusion proteins. Our findings also highlight a new therapeutic strategy for aggressive leukemias with MLL rearrangements.

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Regulation of CK2 by phosphorylation and O-GlcNAcylation revealed by semisynthesis.

Publication Date: 2012 Jan 22 PMID: 22267120
Authors: Tarrant, M. K. - Rho, H. S. - Xie, Z. - Jiang, Y. L. - Gross, C. - Culhane, J. C. - Yan, G. - Qian, J. - Ichikawa, Y. - Matsuoka, T. - Zachara, N. - Etzkorn, F. A. - Hart, G. W. - Jeong, J. S. - Blackshaw, S. - Zhu, H. - Cole, P. A.
Journal: Nat Chem Biol

Protein serine-threonine kinase casein kinase II (CK2) is involved in a myriad of cellular processes including cell growth and proliferation through its phosphorylation of hundreds of substrates, yet how CK2 function is regulated is poorly understood. Here we report that the CK2 catalytic subunit CK2alpha is modified by O-linked beta-N-acetyl-glucosamine (O-GlcNAc) on Ser347, proximal to a cyclin-dependent kinase phosphorylation site (Thr344). We use protein semisynthesis to show that phosphorylation of Thr344 increases the cellular stability of CK2alpha by strengthening its interaction with Pin1, whereas glycosylation of Ser347 seems to be antagonistic to Thr344 phosphorylation and permissive to proteasomal degradation. By performing kinase assays with site-specifically phospho- and glyco-modified CK2alpha in combination with CK2beta and Pin1 binding partners on human protein microarrays, we show that the kinase substrate selectivity of CK2 is modulated by these specific post-translational modifications. This study suggests how a promiscuous protein kinase can be regulated at multiple levels to achieve particular biological outputs.

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Metabolomics implicates altered sphingolipids in chronic pain of neuropathic origin.

Publication Date: 2012 Jan 22 PMID: 22267119
Authors: Patti, G. J. - Yanes, O. - Shriver, L. P. - Courade, J. P. - Tautenhahn, R. - Manchester, M. - Siuzdak, G.
Journal: Nat Chem Biol

Neuropathic pain is a debilitating condition for which the development of effective treatments has been limited by an incomplete understanding of its chemical basis. We show by using untargeted metabolomics that sphingomyelin-ceramide metabolism is altered in the dorsal horn of rats with neuropathic pain and that the upregulated, endogenous metabolite N,N-dimethylsphingosine induces mechanical hypersensitivity in vivo. These results demonstrate the utility of metabolomics to implicate unexplored biochemical pathways in disease.

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Dynamic O-GlcNAc modification regulates CREB-mediated gene expression and memory formation.

Publication Date: 2012 Jan 22 PMID: 22267118
Authors: Rexach, J. E. - Clark, P. M. - Mason, D. E. - Neve, R. L. - Peters, E. C. - Hsieh-Wilson, L. C.
Journal: Nat Chem Biol

The transcription factor cyclic AMP-response element binding protein (CREB) is a key regulator of many neuronal processes, including brain development, circadian rhythm and long-term memory. Studies of CREB have focused on its phosphorylation, although the diversity of CREB functions in the brain suggests additional forms of regulation. Here we expand on a chemoenzymatic strategy for quantifying glycosylation stoichiometries to characterize the functional roles of CREB glycosylation in neurons. We show that CREB is dynamically modified with an O-linked beta-N-acetyl-D-glucosamine sugar in response to neuronal activity and that glycosylation represses CREB-dependent transcription by impairing its association with CREB-regulated transcription coactivator (CRTC; also known as transducer of regulated CREB activity). Blocking glycosylation of CREB alters cellular function and behavioral plasticity, enhancing both axonal and dendritic growth and long-term memory consolidation. Our findings demonstrate a new role for O-glycosylation in memory formation and provide a mechanistic understanding of how glycosylation contributes to critical neuronal functions. Moreover, we identify a previously unknown mechanism for the regulation of activity-dependent gene expression, neural development and memory.

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Signaling: An oxysterol ligand for Smoothened.

Publication Date: 2012 PMID: 22257852
Authors: Sharpe, H. J. - de Sauvage, F. J.
Journal: Nat Chem Biol

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Glycobiology: Enzyme deficiencies deciphered.

Publication Date: 2012 PMID: 22257851
Authors: Kjellen, L.
Journal: Nat Chem Biol

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Ion channels: Cooperativity in twin gatings.

Publication Date: 2012 PMID: 22257850
Authors: Zhou, L.
Journal: Nat Chem Biol

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Molecular switches: The pore of the matter.

Publication Date: 2012 PMID: 22257849
Authors: Bucci, M.
Journal: Nat Chem Biol

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Translation: ribosome 3.0.

Publication Date: 2012 PMID: 22257848
Authors: Donner, A.
Journal: Nat Chem Biol

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Chemical genetics: Bumping into AMPK substrates.

Publication Date: 2012 PMID: 22257847
Authors: Sheppard, T. L.
Journal: Nat Chem Biol

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Metabolism: Closing the loop.

Publication Date: 2012 PMID: 22257846
Authors: Goodman, C.
Journal: Nat Chem Biol

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Enzyme engineering: Just a jump to the left.

Publication Date: 2012 PMID: 22257845
Authors: Finkelstein, J. M.
Journal: Nat Chem Biol

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Protein-protein interactions: In the TANK.

Publication Date: 2012 PMID: 22257844
Authors: Donner, A.
Journal: Nat Chem Biol

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Methods: Zebrafish click.

Publication Date: 2012 PMID: 22257843
Authors: Bucci, M.
Journal: Nat Chem Biol

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Allostery: A systematic search.

Publication Date: 2012 PMID: 22257842
Authors: Goodman, C.
Journal: Nat Chem Biol

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Chemical chaperones assist intracellular folding to buffer mutational variations.

Publication Date: 2012 Jan 15 PMID: 22246401
Authors: Bandyopadhyay, A. - Saxena, K. - Kasturia, N. - Dalal, V. - Bhatt, N. - Rajkumar, A. - Maity, S. - Sengupta, S. - Chakraborty, K.
Journal: Nat Chem Biol

Hidden genetic variations have the potential to lead to the evolution of new traits. Molecular chaperones, which assist protein folding, may conceal genetic variations in protein-coding regions. Here we investigate whether the chemical milieu of cells has the potential to alleviate intracellular protein folding, a possibility that could implicate osmolytes in concealing genetic variations. We found that the model osmolyte trimethylamine N-oxide (TMAO) can buffer mutations that impose kinetic traps in the folding pathways of two model proteins. Using this information, we rationally designed TMAO-dependent mutants in vivo, starting from a TMAO-independent protein. We show that different osmolytes buffer a unique spectrum of mutations. Consequently, the chemical milieu of cells may alter the folding pathways of unique mutant variants in polymorphic populations and lead to unanticipated spectra of genetic buffering.

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Evidence for dynamics in proteins as a mechanism for ligand dissociation.

Publication Date: 2012 Jan 15 PMID: 22246400
Authors: Carroll, M. J. - Mauldin, R. V. - Gromova, A. V. - Singleton, S. F. - Collins, E. J. - Lee, A. L.
Journal: Nat Chem Biol

Signal transduction, regulatory processes and pharmaceutical responses are highly dependent upon ligand residence times. Gaining insight into how physical factors influence residence times (1/k(off)) should enhance our ability to manipulate biological interactions. We report experiments that yield structural insight into k(off) involving a series of eight 2,4-diaminopyrimidine inhibitors of dihydrofolate reductase whose binding affinities vary by six orders of magnitude. NMR relaxation-dispersion experiments revealed a common set of residues near the binding site that undergo a concerted millisecond-timescale switching event to a previously unidentified conformation. The rate of switching from ground to excited conformations correlates exponentially with the binding affinity K(i) and k(off), suggesting that protein dynamics serves as a mechanical initiator of ligand dissociation within this series and potentially for other macromolecule-ligand systems. Although the forward rate of conformational exchange, k(conf,forward), is faster than k(off), the use of the ligand series allowed for connections to be drawn between kinetic events on different timescales.

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Oxysterols are allosteric activators of the oncoprotein Smoothened.

Publication Date: 2012 PMID: 22231273
Authors: Nachtergaele, S. - Mydock, L. K. - Krishnan, K. - Rammohan, J. - Schlesinger, P. H. - Covey, D. F. - Rohatgi, R.
Journal: Nat Chem Biol

Oxysterols are a class of endogenous signaling molecules that can activate the Hedgehog pathway, which has critical roles in development, regeneration and cancer. However, it has been unclear how oxysterols influence Hedgehog signaling, including whether their effects are mediated through a protein target or indirectly through effects on membrane properties. To answer this question, we synthesized the enantiomer and an epimer of the most potent oxysterol, 20(S)-hydroxycholesterol. Using these molecules, we show that the effects of oxysterols on Hedgehog signaling are exquisitely stereoselective, consistent with the hypothesis that they function through a specific protein target. We present several lines of evidence that this protein target is the seven-pass transmembrane protein Smoothened, a major drug target in oncology. Our work suggests that these enigmatic sterols, which have multiple effects on cell physiology, may act as ligands for signaling receptors and provides a generally applicable framework for probing sterol signaling mechanisms.

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Timing facilitated site transfer of an enzyme on DNA.

Publication Date: 2012 PMID: 22231272
Authors: Schonhoft, J. D. - Stivers, J. T.
Journal: Nat Chem Biol

Many enzymes that react with specific sites in DNA have the property of facilitated diffusion, in which the DNA chain is used as a conduit to accelerate site location. Despite the importance of such mechanisms in gene regulation and DNA repair, there have been few viable approaches to elucidate the microscopic process of facilitated diffusion. Here we describe a new method in which a small-molecule trap (uracil) is used to clock a DNA repair enzyme as it hops and slides between damaged sites in DNA. The 'molecular clock' provides unprecedented information: the mean length for DNA sliding, the one-dimensional diffusion constant, the maximum hopping radius and the time frame for DNA hopping events. In addition, the data establish that the DNA phosphate backbone is a sufficient requirement for DNA sliding.

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Disease-specific non-reducing end carbohydrate biomarkers for mucopolysaccharidoses.

Publication Date: 2012 PMID: 22231271
Authors: Lawrence, R. - Brown, J. R. - Al-Mafraji, K. - Lamanna, W. C. - Beitel, J. R. - Boons, G. J. - Esko, J. D. - Crawford, B. E.
Journal: Nat Chem Biol

A considerable need exists for improved biomarkers for differential diagnosis, prognosis and monitoring of therapeutic interventions for mucopolysaccharidoses (MPS), inherited metabolic disorders that involve lysosomal storage of glycosaminoglycans. Here we report a simple, reliable method based on the detection of abundant nonreducing ends of the glycosaminoglycans that accumulate in cells, blood and urine of individuals with MPS. In this method, glycosaminoglycans are enzymatically depolymerized, releasing unique mono-, di- or trisaccharides from the nonreducing ends of the chains. The composition of the released mono- and oligosaccharides depends on the nature of the lysosomal enzyme deficiency, and therefore they serve as diagnostic biomarkers. Analysis by LC/MS allowed qualitative and quantitative assessment of the biomarkers in biological samples. We provide a simple conceptual scheme for diagnosing MPS in uncharacterized samples and a method to monitor efficacy of enzyme replacement therapy or other forms of treatment.

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